U.S. patent application number 16/300442 was filed with the patent office on 2019-08-08 for expression inhibitor of inflammation promoting factor, screening method for active ingredient thereof, expression cassette usefu.
This patent application is currently assigned to National University Corporation Tokyo Medical and Dental University. The applicant listed for this patent is National University Corporation Tokyo Medical and Dental University, Nippon Zoki Pharmaceutical Co., Ltd.. Invention is credited to Kentaro ABE, Hiroshi ASAHARA, Tomoki CHIBA.
Application Number | 20190242875 16/300442 |
Document ID | / |
Family ID | 60267239 |
Filed Date | 2019-08-08 |
![](/patent/app/20190242875/US20190242875A1-20190808-D00000.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00001.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00002.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00003.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00004.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00005.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00006.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00007.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00008.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00009.png)
![](/patent/app/20190242875/US20190242875A1-20190808-D00010.png)
View All Diagrams
United States Patent
Application |
20190242875 |
Kind Code |
A1 |
ASAHARA; Hiroshi ; et
al. |
August 8, 2019 |
Expression Inhibitor of Inflammation Promoting Factor, Screening
Method for Active Ingredient Thereof, Expression Cassette Useful
for Said Method, Diagnostic Agent and Diagnosis Method
Abstract
Provided are an expression inhibitor of an inflammation
promoting factor based on the discovery of a new factor influencing
the expression amount/level of an inflammation promoting factor,
and a development tool therefor. Provided are also a diagnostic
agent and a diagnosis method for immune diseases, inflammatory
diseases, painful conditions and similar. More specifically
provided are: an expression inhibitor of an inflammation promoting
factor containing at least one kind of inhibitor selected from the
group consisting of RBMS2 expression inhibitor and RBMS2 function
inhibitor; a screening method using as an indicator the expression
or the function of RBMS2; an expression cassette useful for said
method; as well as a diagnostic agent containing a product
detection agent for RBMS2 gene expression and disease detection
method using as an indicator RBMS2 gene expression
amount/level.
Inventors: |
ASAHARA; Hiroshi; (Tokyo,
JP) ; CHIBA; Tomoki; (Tokyo, JP) ; ABE;
Kentaro; (Osaka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National University Corporation Tokyo Medical and Dental
University
Nippon Zoki Pharmaceutical Co., Ltd. |
Tokyo
Osaka |
|
JP
JP |
|
|
Assignee: |
National University Corporation
Tokyo Medical and Dental University
Tokyo
JP
Nippon Zoki Pharmaceutical Co., Ltd.
Osaka
JP
|
Family ID: |
60267239 |
Appl. No.: |
16/300442 |
Filed: |
May 10, 2017 |
PCT Filed: |
May 10, 2017 |
PCT NO: |
PCT/JP2017/017640 |
371 Date: |
November 9, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/53 20130101;
C12Q 1/68 20130101; C12N 15/1086 20130101; C12N 15/09 20130101;
C12N 15/63 20130101; C12N 15/113 20130101; C12N 2310/18 20130101;
G01N 33/5023 20130101; C12N 2320/10 20130101; C12N 2320/50
20130101; G01N 2500/02 20130101; G01N 2500/10 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12N 15/113 20060101 C12N015/113; C12N 15/63 20060101
C12N015/63; C12N 15/10 20060101 C12N015/10 |
Foreign Application Data
Date |
Code |
Application Number |
May 10, 2016 |
JP |
2016-094931 |
Aug 22, 2016 |
JP |
2016-162120 |
Claims
1. A reagent for screening for an active ingredient for an
inhibitor of the expression of an inflammation promoting factor,
containing at least one component selected from the group
consisting of an expression cassette containing a RBMS2 gene
expression regulation region and a gene arranged in such a manner
that the expression thereof can be regulated by the region, a
vector carrying the expression cassette and a cell harboring the
vector.
2. A method for screening for an active ingredient for an inhibitor
of the expression of an inflammation promoting factor by employing
at least one item selected from the group consisting of items (i)
to (iii) as an index in the presence of a test substance: (i) the
expression amount/level of a gene of which the expression can be
regulated by a RBMS2 gene expression regulation region; (ii) the
binding amount/level of RBMS2 to RNA containing an AU-rich element;
and (iii) the amount/level of mRNA containing an AU-rich element in
a 3'-UTR thereof or the amount/level of a protein derived from the
mRNA in a RBMS2-overexpressing cell.
3. The screening method according to claim 2, wherein, when the
value of the index in the presence of a test substance is smaller
than the value of the index in the absence of the test substance,
the test substance is selected as an active ingredient for an
inhibitor of the expression of an inflammation promoting
factor.
4. The screening method according to claim 2, wherein the AU-rich
element is an AU-rich element derived from at least one type of
mRNA selected from the group consisting of IL-6 mRNA, COX-2 mRNA,
IL-8 mRNA, IL-1.beta. mRNA, TNF-.alpha. mRNA, MMP1 mRNA, IL-24 mRNA
and c-Myc mRNA.
5. The screening method according to claim 2, wherein the method
includes steps (a1) to (c1): (a1) bringing an expression system
which contains an expression cassette containing a RBMS2 gene
expression regulation region and a gene arranged in such a manner
that the expression thereof can be regulated by the region into
contact with a test substance; (b1) measuring, as an expression
amount/level of interest, the amount/level of the gene expressed in
the expression system that has been contacted with the test
substance and then comparing the expression amount/level of
interest with a control expression amount/level that is the
expression amount/level of the gene in an expression system that
has not been contacted with the test substance; and (c1) selecting
the test substance as an active ingredient for an inhibitor of the
expression of an inflammation promoting factor when the expression
amount/level of interest is smaller than the control expression
amount/level.
6. The screening method according to claim 5, wherein the
expression system is a cell.
7. The screening method according to claim 5, wherein the gene is a
reporter gene.
8. The screening method according to, wherein the method includes
steps (a2) to (c2): (a2) bringing RNA containing an AU-rich element
into contact with RBMS2 in the presence of a test substance; (b2)
measuring the binding amount/level between the RNA and the RBMS2
which are contacted with each other in the presence of a test
substance as a binding amount/level of interest, and then comparing
the binding amount/level of interest with a control binding
amount/level that is the binding amount/level between the RNA and
the RBMS2 which are contacted with each other in the absence of the
test substance; and (c2) selecting the test substance as an active
ingredient for an inhibitor of the expression of an inflammation
promoting factor when the binding amount/level of interest is
smaller than the control binding amount/level.
9. The screening method according to claim 2, wherein the method
includes steps (a3) to (c3): (a3) bringing a cell which contains
mRNA containing an AU-rich element in a 3'-UTR thereof and in which
RBMS2 is overexpressed into contact with a test substance; (b3)
measuring the amount/level of the mRNA or a protein derived from
the mRNA in the cell that has been contacted with the test
substance as an amount/level of interest, and then comparing the
amount/level of interest with a control amount/level that is the
amount/level of the mRNA or a protein derived from the mRNA in a
cell that is not contacted with the test substance; and (c3)
selecting the test substance as an active ingredient for an
inhibitor of the expression of an inflammation promoting factor
when the amount/level of interest is smaller than the control
amount/level.
10. The screening method according to claim 9, wherein the mRNA
contains an ORF of a reporter protein.
11. An inhibitor of the expression of an inflammation promoting
factor containing at least one component selected from the group
consisting of a RBMS2 expression inhibitor and a RBMS2 function
inhibitor.
12. The inhibitor of the expression of an inflammation promoting
factor according to claim 11, wherein the RBMS2 expression
inhibitor contains at least one RBMS2 expression inhibitor selected
from the group consisting of RBMS2-specific siRNA, RBMS2-specific
miRNA, a RBMS2-specific antisense nucleic acid, expression vectors
for these components and IL-10.
13. The inhibitor of the expression of an inflammation promoting
factor according to claim 11, wherein the inflammation promoting
factor of which the expression is to be inhibited is at least one
component selected from the group consisting of IL-6, COX-2, IL-8,
IL-1.beta., TNF-.alpha., MMP1, IL-24 and c-Myc.
14. The inhibitor of the expression of an inflammation promoting
factor according to claim 11, wherein the inhibitor is used as a
prophylactic or therapeutic agent for at least one disease selected
from the group consisting of immune diseases, inflammatory diseases
and painful diseases.
15. A diagnostic agent for a disease which can be developed or
worsened by an inflammation promoting factor, which contains a
RBMS2 gene expression product detecting agent.
16. The diagnostic agent according to claim 15, wherein the disease
is at least one disease selected from the group consisting of
immune diseases, inflammatory diseases and painful diseases.
17. The diagnostic agent according to claim 15, wherein the
inflammation promoting factor is at least one component selected
from the group consisting of IL-6, COX-2, IL-1.beta., IL-8,
TNF-.alpha., MMP1, IL-24 and c-Myc.
18-20. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a national phase entry pursuant to 35
U.S.C. .sctn. 371 of International Application No.
PCT/JP2017/017640, filed May 10, 2017, which claims the benefit of
priority of JP Application Nos. 2016-094931, filed May 10, 2016,
and 2016-162120, filed Aug. 22, 2016, each of which is incorporated
by reference herein in its entirety for any purpose.
SEQUENCE LISTING
[0002] The present application is filed with a Sequence Listing in
electronic format. The Sequence Listing is provided as a file
entitled "2018-11-09_01178-0003-00US_Seq_List_ST25 txt" created on
Nov. 9, 2018, which is 56,062 bytes in size. The information in the
electronic format of the sequence listing is incorporated herein by
reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates to an inhibitor of the
expression of an inflammation promoting factor; a method for
screening for an active ingredient of the inhibitor; an expression
cassette useful for the method; a diagnostic agent and a diagnosis
method both for immune diseases, inflammatory diseases, painful
diseases and the like; and others.
BACKGROUND ART
[0004] There are great many patients who are suffering from
diseases of which the symptoms can be developed or worsened by an
inflammation promoting factor (e.g., IL-6 and COX-2), such as an
immune disease, an inflammatory disease and a painful disease. For
example, with respect to rheumatoid arthritis which is a major
immune disease, it is estimated that there are about 700,000
patients in Japan and there are about 70,000,000 patients
worldwide. With respect to arthrosis which is one of inflammatory
diseases, it is estimated that there are about 15,600,000 patients
even in Japan. With respect to osteoarthritis of the knee which is
considered to be major arthrosis, it is estimated that there are
about 10,000,000 patients in Japan. With respect to low back pain
which is a major painful disease, it is estimated that there are
about 24,000,000 patients even in Japan. The number of patients of
these diseases tends to increase year by year as the population
ages. It is believed that this phenomenon would be caused in Japan
where the population aging rate is highest in the world, as well as
in all of countries where the population aging would advance in the
future due to the improvement in living environments, dietary
habits and nutrition conditions, the advance in medical
technologies and the like. By detecting these diseases in earlier
stages, it becomes possible to start a proper treatment, prevent
the increase in severity of the diseases and, as a result, keep the
QOL of patients at higher levels. Therefore, it has been a socially
important demand to study about therapeutic agents for the diseases
and tools for developing the therapeutic agents and to develop
diagnostic agents and diagnosis methods for the diseases.
[0005] In these situations, the relationship between the
post-transcriptional regulation of inflammation promoting factor
mRNA and the above-mentioned diseases has been obvious recently. It
is suggested that the regulation of the degradation of mRNA for an
inflammation promoting factor can be a factor that can determine
the sustainability of an inflammatory response. Therefore, it is
believed that a factor associated with the degradation or
stabilization of an inflammation promoting factor at mRNA level is
involved in the onset or chronicity of an immune disease, an
inflammatory disease and a painful disease. For example, Non-Patent
Document 1 discloses that Tristetraprolin (TTP) and Regnase-1 are
involved in the degradation of IL-6 mRNA. Non-Patent Document 2
discloses that Arid5a can regulate the stability of IL-6 mRNA. It
has been elucidated that the abnormalities of these factors play an
important role in the mechanism of onset or worsening of the
diseases, including the acceleration of inflammations. For these
reasons, a factor that affects the expression amount/level of an
inflammation promoting factor can become a target of therapeutic
agents for the diseases, and a novel discovery about the factor has
been demanded. On the other hand, a diagnostic agent and a
diagnosis method whereby it becomes possible to diagnose as to the
presence or absence of the above-mentioned diseases or the degree
of progression of the diseases by measuring the amount/level of a
substance capable of affecting the expression amount/level of an
inflammation promoting factor in a cell, blood or the like from a
patient are very useful. Therefore, a novel discovery about the
factor has been demanded.
[0006] RNA-binding motif, single-stranded-interacting protein 2
(RBMS2) is a protein that is believed to have two RNA-binding
domains on the N-terminal side. However, it is not reported yet
that the functions of the protein are actually analyzed, and the
functions of the protein are not elucidated yet.
PRIOR ART DOCUMENTS
Non-Patent Documents
[0007] Non-Patent Document 1: Inflammation and Regeneration, Vol.
33, No. 1, January 2013, pp 54-65. [0008] Non-Patent Document 2:
PNAS, Jun. 4, 2013, vol. 110, no. 23, pp 9409-9414
SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0009] The present invention addresses the problem of: discovering
a novel factor that can affect the expression amount/level of an
inflammation promoting factor; providing an inhibitor of the
expression of an inflammation promoting factor and a tool for
developing the inhibitor on the basis of the discovering the
factor; and providing a diagnostic agent and a diagnosis method
both for immune diseases, inflammatory diseases, painful diseases
and other diseases.
Means for Solving the Problems
[0010] The present inventors have made extensive and intensive
studies for the purpose of solving the problem. As a result, it is
found that RBMS2 is involved in the post-transcriptional regulation
of mRNA molecules of various inflammation promoting factors. It is
also found that there is a positive correlation between the
expression of RBMS2 and the expression of an inflammation promoting
factor in diseases which can be developed or worsened by an
inflammation promoting factor. On the basis of these findings, it
is also found that an inflammation promoting factor can be
inhibited and the above-mentioned diseases can be prevented or
treated by inhibiting the expression or function of RBMS2, and that
these diseases can be diagnosed by employing the expression
amount/level of RBMS2 as an index. The studies are further pursued
on the basis of these findings. As a result, the present invention
has been accomplished. That is, the present invention includes the
following aspects.
[0011] 1. An expression cassette containing a RBMS2 gene expression
regulation region and a gene arranged in such a manner that the
expression thereof can be regulated by the region.
[0012] 2. The expression cassette according to item 1, wherein the
gene is a reporter gene.
[0013] 3. A vector carrying an expression cassette as recited in
item 1 or 2.
[0014] 4. A cell harboring a vector as recited in item 3.
[0015] 5. A reagent for screening for an active ingredient for an
inhibitor of the expression of an inflammation promoting factor,
containing at least one component selected from the group
consisting of an expression cassette containing a RBMS2 gene
expression regulation region and a gene arranged in such a manner
that the expression thereof can be regulated by the region, a
vector carrying the expression cassette and a cell harboring the
vector.
[0016] 6. A method for screening for a substance capable of
inhibiting the expression or function of RBMS2 by employing at
least one item selected from the group consisting of items (i) to
(iii) as an index in the presence of a test substance:
[0017] (i) the expression amount/level of a gene of which the
expression can be regulated by a RBMS2 gene expression regulation
region;
[0018] (ii) the binding amount/level of RBMS2 to RNA containing an
AU-rich element; and
[0019] (iii) the amount/level of mRNA containing an AU-rich element
in a 3'-UTR thereof or the amount/level of a protein derived from
the mRNA in a RBMS2-overexpressing cell.
[0020] 7. The screening method according to item 6, wherein, when
the value of the index in the presence of a test substance is
smaller than the value of the index in the absence of the test
substance, the test substance is selected as a substance capable of
inhibiting the expression or function of RBMS2.
[0021] 8. The screening method according to item 6 or 7, wherein
the AU-rich element is an AU-rich element derived from at least one
member of mRNA selected from the group consisting of IL-6 mRNA,
COX-2 mRNA, IL-8 mRNA, IL-1.beta. mRNA, TNF-.alpha. mRNA, MMP1
mRNA, IL-24 mRNA and c-Myc mRNA.
[0022] 9. The screening method according to any one of items 6 to
8, wherein a substance capable of inhibiting the expression or
function of RBMS2 is selected as an active ingredient for an
inhibitor of the expression of an inflammation promoting factor or
a candidate for the active ingredient.
[0023] 10. The screening method according to any one of items 6 to
9, wherein the method includes steps (a1) to (c1):
[0024] (a1) bringing an expression system which contains an
expression cassette containing a RBMS2 gene expression regulation
region and a gene arranged in such a manner that the expression
thereof can be regulated by the region into contact with a test
substance;
[0025] (b1) measuring, as an expression amount/level of interest,
the amount/level of the gene expressed in the expression system
that has been contacted with the test substance and then comparing
the expression amount/level of interest with a control expression
amount/level that is the expression amount/level of the gene in an
expression system that has not been contacted with the test
substance; and
[0026] (c1) selecting the test substance as a substance capable of
inhibiting the expression of RBMS2 when the expression amount/level
of interest is smaller than the control expression
amount/level.
[0027] 11. The screening method according to item 10, wherein the
expression system is a cell.
[0028] 12. The screening method according to item 10 or 11, wherein
the gene is a reporter gene.
[0029] 13. The screening method according to any one of items 6 to
9, wherein the method includes steps (a2) to (c2):
[0030] (a2) bringing RNA containing an AU-rich element into contact
with RBMS2 in the presence of a test substance;
[0031] (b2) measuring the binding amount/level between the RNA and
the RBMS2 which are contacted with each other in the presence of a
test substance as a binding amount/level of interest, and then
comparing the binding amount/level of interest with a control
binding amount/level that is the binding amount/level between the
RNA and the RBMS2 which are contacted with each other in the
absence of the test substance; and
[0032] (c2) selecting the test substance as a substance capable of
inhibiting the function of RBMS2 when the binding amount/level of
interest is smaller than the control binding amount/level.
[0033] 14. The screening method according to item 13, wherein the
method for measuring the binding amount/level is an
immunoprecipitation method or a gel shift method.
[0034] 15. The screening method according to any one of items 6 to
9, wherein the method includes steps (a3) to (c3):
[0035] (a3) bringing a cell which contains mRNA containing an
AU-rich element in a 3'-UTR thereof and in which RBMS2 is
overexpressed into contact with a test substance;
[0036] (b3) measuring the amount/level of the mRNA or a protein
derived from the mRNA in the cell that has been contacted with the
test substance as an amount/level of interest, and then comparing
the amount/level of interest with a control amount/level that is
the amount/level of the mRNA or a protein derived from the mRNA in
a cell that is not contacted with the test substance; and
[0037] (c3) selecting the test substance as a substance capable of
inhibiting the function of RBMS2 when the amount/level of interest
is smaller than the control amount/level.
[0038] 16. The screening method according to item 15, wherein the
mRNA contains an ORF of a reporter protein.
[0039] 17. An inhibitor of the expression of an inflammation
promoting factor containing at least one component selected from
the group consisting of a RBMS2 expression inhibitor and a RBMS2
function inhibitor.
[0040] 18. The inhibitor of the expression of an inflammation
promoting factor according to item 17, wherein the inhibitor
contains a RBMS2 expression inhibitor.
[0041] 19. The inhibitor of the expression of an inflammation
promoting factor according to item 18, wherein the RBMS2 expression
inhibitor contains at least one RBMS2 expression inhibitor selected
from the group consisting of RBMS2-specific siRNA, RBMS2-specific
miRNA, a RBMS2-specific antisense nucleic acid and expression
vectors for these components.
[0042] 20. The inhibitor of the expression of an inflammation
promoting factor according to item 18, wherein the RBMS2 expression
inhibitor is IL-10.
[0043] 21. The inhibitor of the expression of an inflammation
promoting factor according to any one of items 17 to 20, wherein
the inflammation promoting factor of which the expression is to be
inhibited is at least one component selected from the group
consisting of IL-6, COX-2, IL-8, IL-1.beta., TNF-.alpha., MMP1,
IL-24 and c-Myc.
[0044] 22. The inhibitor of the expression of an inflammation
promoting factor according to any one of items 17 to 21, wherein
the inhibitor is used as a prophylactic or therapeutic agent for at
least one disease selected from the group consisting of immune
diseases, inflammatory diseases and painful diseases.
[0045] 23. The inhibitor of the expression of an inflammation
promoting factor according to any one of items 17 to 22, wherein
the inhibitor is used as a prophylactic or therapeutic agent for at
least one disease selected from the group consisting of autoimmune
diseases, rheumatic diseases, rheumatoid arthritis, degenerative
arthritis disease, arthritis, sepsis, lymphoproliferative diseases,
demyelinating diseases in the central nervous system,
spondyloarthropathy, inflammatory pain, postoperative pain and
allergic diseases.
[0046] 24. A diagnostic agent for a disease which can be developed
or worsened by an inflammation promoting factor, which contains a
RBMS2 gene expression product detecting agent.
[0047] 25. The diagnostic agent according to item 24, wherein the
disease is a disease which can be developed or worsened by an
inflammation promoting factor.
[0048] 26. The diagnostic agent according to item 24 or 25, wherein
the disease is at least one disease selected from the group
consisting of immune diseases, inflammatory diseases and painful
diseases.
[0049] 27. The diagnostic agent according to item 25 or 26, wherein
the inflammation promoting factor is at least one component
selected from the group consisting of IL-6, COX-2, IL-1.beta.,
IL-8, TNF-.alpha., MMP1, IL-24 and c-Myc.
[0050] 28. The diagnostic agent according to any one of items 24 to
27, wherein the RBMS2 gene expression product detecting agent is a
probe or a primer for RBMS2 mRNA or a nucleic acid derived from the
RBMS2 mRNA or an antibody capable of recognizing RBMS2 protein.
[0051] 29. The diagnostic agent according to any one of items 24 to
28, wherein the disease that is a disease to be diagnosed is at
least one disease selected from the group consisting of autoimmune
diseases, rheumatic disease, rheumatoid arthritis, degenerative
arthritis diseases, arthritis, sepsis, lymphoproliferative
diseases, demyelinating diseases in the central nervous system,
spondyloarthropathy, inflammatory pain, postoperative pain and
allergic diseases.
[0052] 30. The diagnostic agent according to item 29, wherein the
disease that is a disease to be diagnosed is rheumatoid
arthritis.
[0053] 31. The diagnostic agent according to any one of items 24 to
30, wherein the item to be diagnosed is the presence or absence of
the disease.
[0054] 32. The diagnostic agent according to any one of items 24 to
30, wherein the item to be diagnosed is the degree of progression
of the disease.
[0055] 33. A method for detecting an immune disease, an
inflammatory disease or a painful disease, comprising the steps
of:
[0056] (a1) measuring the expression amount/level of interest of a
RBMS2 gene expression product in a sample collected from a subject;
and
[0057] (b1) comparing the expression amount/level of interest
measured in step (a1) with a control expression amount/level of the
RBMS2 gene expression product in a sample collected from a control
subject which does not suffer from any one of the immune disease,
the inflammatory disease and the painful disease,
[0058] wherein
[0059] (c1) the matter that the expression amount/level of interest
is larger than the control expression amount/level is employed as
an index for the determination that the subject has the immune
disease, the inflammatory disease or the painful disease.
[0060] 34. A method for determining the degree of progression of an
immune disease, an inflammatory disease or a painful disease,
comprising the steps of:
[0061] (a2) measuring the expression amount/level of interest of a
RBMS2 gene expression product in a sample collected from a subject
suffering from the immune disease, the inflammatory disease or the
painful disease; and
[0062] (b2) comparing the expression amount/level of interest
measured in step (a2) with a control expression amount/level of a
RBMS2 gene expression product in a sample collected from a control
subject suffering from the immune disease, the inflammatory disease
or the painful disease,
[0063] wherein
[0064] (c3) the matter that the expression amount/level of interest
is larger than the control expression amount/level is employed as
an index for the determination that the subject has a higher degree
of progression of the immune disease, the inflammatory disease or
the painful disease than that of the control subject.
[0065] 35. The method according to item 33 or 34, wherein the
sample to be used in each of step (a1) and step (a2) is a blood
sample.
[0066] 36. The method according to any one of items 33 to 35,
wherein the sample to be used in each of step (a1) and step (a2) is
a blood cell component in a blood sample.
[0067] 37. The method according to any one of items 33 to 36,
wherein the immune disease, the inflammatory disease and the
painful disease can be developed or can be worsened by at least one
component selected from the group consisting of IL-6, COX-2,
IL-1.beta., IL-8, TNF-.alpha., MMP1, IL-24 and c-Myc.
[0068] 38. The method according to any one of items 33 to 37,
wherein the expression amount/level of interest of the RBMS2 gene
expression product and the control expression amount/level are
measured using a probe or primer directed against RBMS2 mRNA or a
nucleic acid derived from the RBMS2 mRNA or an antibody capable of
recognizing RBMS2 protein.
Advantages of the Invention
[0069] According to the present invention, it becomes possible to
provide a novel inhibitor of the expression of an inflammation
promoting factor, a novel prophylactic or therapeutic agent for an
immune disease, an inflammatory disease, a painful disease or the
like, and a tool for developing the inhibitor or the prophylactic
or therapeutic agent (e.g., a method for screening for an active
ingredient, an expression cassette useful for the method), all of
which utilize a novel target factor that affects the expression
amount/level of an inflammation promoting factor. According to the
present invention, it also becomes possible to provide a diagnostic
agent and a diagnosis method for an immune disease, an inflammatory
disease, a painful disease or the like, which rely on a novel
mechanism.
BRIEF DESCRIPTION OF DRAWINGS
[0070] FIG. 1A illustrates a schema of screening of Example 1A.
[0071] FIG. 1B illustrates the results of Example 1B. In the left
side of the graph, an upper schematic diagram illustrates a partial
structure of a control reporter vector (without an IL-6 3'UTR)
used, and a lower schematic diagram illustrates a partial structure
of a reporter vector (with an IL-6 3'UTR) used. A white column
represents a case where an empty vector (pcDNA3.1) is introduced,
and a black column represents a case where a RBMS2 expression
vector (pcDNA3.1 FLAG-RBMS2) is introduced. The transverse axis
represents a relative value of a measured luciferase activity.
[0072] FIG. 1C illustrates the results of Example 1C. The vertical
axis on the left side in the drawings represents a relative value
of the amount/level of RBMS2 mRNA relative to the amount/level of
GAPDH mRNA, the vertical axis on the right side in the drawings
represents a relative value of the amount/level of IL-6 mRNA
relative to the amount/level of GAPDH mRNA. In the transverse axis,
"N.C." represents a case where control siRNA is introduced, and
"RBMS2-1" and "RBMS2-2" respectively represent cases where siRNA
for different region of RBMS2 is introduced. A white column
represents a case where IL-1.beta. is not added, and a black column
represents a case where IL-1.beta. is added.
[0073] FIG. 1D illustrates the results of Example 1D. The vertical
axis represents the concentration of IL-6 protein in a supernatant.
In the transverse axis, "N.C." represents a case where control
siRNA is introduced, and "RBMS2-1" and "RBMS2-2" respectively
represent cases where siRNA for different region of RBMS2 is
introduced. A white column represents a case where IL-1.beta. is
not added, and a black column represents a case where IL-1.beta. is
added.
[0074] FIG. 1E illustrates the results of Example 1E. The vertical
axis on the left side of the drawings represents a relative value
of a amount/level of RBMS2 mRNA relative to the amount/level of
GAPDH mRNA, and the vertical axis on the right side of the drawings
represents a relative value of a amount/level of IL-6 mRNA relative
to a amount/level of GAPDH mRNA. In each of the transverse axes,
"Empty" represents a case where a control retrovirus is allowed to
infect, and "RBMS2" represents a case where a retrovirus capable of
expressing RBMS2 is allowed to infect. A white column represents a
case where IL-1.beta. is not added, and a black column represents a
case where IL-1.beta. is added.
[0075] FIG. 1F illustrates the results of Example 1F. The vertical
axis represents a relative value of a measured luciferase activity.
In the transverse axes, "Empty" represents a case where an empty
vector is introduced, and "RBMS2" represents a case where a RBMS2
expression vector is introduced.
[0076] FIG. 2A illustrates the results of Example 2A. The vertical
axis represents a relative value of a amount/level of IL-6 mRNA
relative to a amount/level of .beta.-actin (Actb) mRNA. In the
transverse axis, "-" represents a cell sample collected 48 hours
after the introduction of an expression vector, "LPS" represents a
cell sample collected 5 hours after the addition of LPS, and "ActD"
represents a cell sample collected 3 hours after the addition of
actinomycin D. The black column represents a case where an empty
vector is introduced, ".circle-solid." represents a case where a
RBMS2 expression vector is introduced.
[0077] FIG. 2B illustrates the results of Example 2B. In the
transverse axis, "0" represents a cell sample collected 24 hours
after the introduction of a vector (during which doxycycline is not
added yet), and "2" represents a cell sample collected 2 hours
after the addition of doxycycline. The vertical axis represents a
relative ratio of a amount/level of luciferase mRNA (when 0, the
relative ratio is 100%). The black column represents a case where
an empty vector is introduced, and ".circle-solid." represents a
case where a RBMS2 expression vector is introduced.
[0078] FIG. 2C illustrates the results of Example 2C. The schematic
diagrams on the left side of the graph show the partial structures
of a mutant 3'UTR reporter vector. The transverse axis represents a
relative value of a luciferase activity. A white column represents
a case where an empty vector is introduced, and a black column
represents a case where a RBMS2 expression vector is introduced.
"*" represents the fact that a p value was 0.05 or less as the
result of a t-test.
[0079] FIG. 3A illustrates the results of Example 3A. The vertical
axis represents a relative value of a amount/level of luciferase
mRNA in an immunoprecipitation product, wherein the amount/level of
luciferase mRNA in a cell lysate before immunoprecipitation is
100%. In the transverse axis, "ARE WT" represents a case where a
reporter vector having, linked thereto, a wild-type 3'UTR of IL-6
downstream from a luciferase ORF (Example 1A), and "ARE Mut"
represents a case where a mutant 3'UTR reporter vector (Example 2C:
ARE mutant) is introduced. A black column represents a case where
immunoprecipitation is performed with an anti-FLAG antibody, and a
white column represents a case where immunoprecipitation is
performed by a non-specific IgG.
[0080] FIG. 3B illustrates the results of Example 3B. The vertical
axis represents a relative value of a amount/level of luciferase
mRNA in an immunoprecipitation product, wherein the amount/level of
luciferase mRNA in a cell lysate before immunoprecipitation is
100%. In the transverse axis, "PBMS2 WT" represents a case where an
expression vector for FLAG-tagged wild-type RBMS2 is introduced,
and "RBMS2 .DELTA.RRM1" represents a case where an expression
vector for FLAG-tagged RRM1 domain-deleted RBMS2 is introduced. A
black column represents a case where immunoprecipitation is
performed with an anti-FLAG antibody, and a white column represents
a case where immunoprecipitation is performed by a non-specific
IgG.
[0081] FIG. 3C illustrates the results of Example 3C. The schematic
diagrams on the left side of the graph show the structures of RBMS2
which can be expressed with the used expression vectors. The
transverse axis represents a relative value of a luciferase
activity.
[0082] FIG. 4A illustrates the results of Example 4A. The vertical
axis in each drawing represents a relative value of a amount/level
of mRNA of each of genes indicated in upper parts of the drawings
relative to a amount/level of GAPDH mRNA. In each of the transverse
axes, "N.C." represents a case where control siRNA is introduced,
and "RBMS2" represents a case where siRNA for RBMS2 is introduced.
A white column represents a case where IL-1.beta. is not added, and
a black column represents a case where IL-1.beta. is added.
[0083] FIG. 4B illustrates the results of Example 4B. The upper row
represents cases where LPS is used, the middle row represents cases
where Pam3CSK4 is used, and the lower row represents cases where
IL-1.beta. is used. The vertical axis in each drawing represents a
relative value of a amount/level of mRNA of each of genes indicated
in upper parts of the drawings relative to a amount/level of GAPDH
mRNA. In each of the transverse axes, "N.C." represents a case
where control siRNA is introduced, and "RBMS2" represents a case
where siRNA for RBMS2 is introduced. A white column represents a
case where no one of LPS, Pam3CSK4 and IL-1.beta. is added, and a
black column represents a case where LPS, Pam3CSK4 or IL-1.beta. is
added.
[0084] FIG. 4C illustrates the results of Example 4C. The vertical
axis represents a relative value of a luciferase activity in the
case where a reporter vector in which a wild-type 3'-UTR (or an ARE
mutant 3'-UTR) of a gene indicated in the upper part of each
drawing is located downstream from a luciferase ORF is used. In
each of the transverse axes, "WT" represents a case where a
wild-type 3'UTR is used, and "Mut" represents a case where an ARE
mutant 3'UTR is used. A white column represents a case where an
empty vector is introduced, and a black column represents a case
where a RBMS2 expression vector is introduced.
[0085] FIG. 5A illustrates the scheme of the production of a
RBMS2-deficient mouse in Example 5A.
[0086] FIG. 5B illustrates the results of Example 5B. The vertical
axis in each drawing represents an expression amount/level of IL-6
when each of factors indicated in the upper parts of the drawings
is added to a culture medium (a relative value relative to a
amount/level of GAPDH mRNA). In each of the transverse axes, "0"
represents the fact that the factor indicated in each of the upper
parts of the drawings is not added, and each of "1", "3", "5" and
"9" represents the time lapsed after the addition of the factor
indicated in each of the upper parts of the drawings. A white
column represents a case where a cell derived from a wild-type
mouse is used, a gray column represents a case where a cell derived
from a RBMS2 heterozygously deficient mouse is used, and a black
column represents a case where a cell derived from a RBMS2
homozygously deficient mouse is used.
[0087] FIG. 5C illustrates the results of Example 5C. The vertical
axis in each drawing represents an expression amount/level of mRNA
for a gene indicated in each of the upper parts of the drawings (a
relative value relative to a amount/level of GAPDH mRNA). In each
of the transverse axes, "0" represents the fact that Pam3CSK4 is
not added yet, and each of "1", "3", "5," and "9" represents a time
lapsed after the addition of Pam3CSK4. A white column represents a
case where a cell derived from a wild-type mouse is used, a gray
column represents a case where a cell derived from a RBMS2
heterozygously deficient mouse is used, and a black column
represents a case where a cell derived from a RBMS2 homozygously
deficient mouse is used.
[0088] FIG. 6A illustrates the results of the measurement of
survival times in Example 6. The vertical axis represents a
survival rate, and the transverse axis represents a time lapsed
after the administration of LPS. "WT" represents a survival rate of
a wild-type mouse, and "KO" represents a survival rate of a RBMS2
homozygously deficient mouse.
[0089] FIG. 6B illustrates the results of the measurement of the
concentration of IL-6 in Example 6. The vertical axis represents a
concentration of IL-6 in serum. In the transverse axis, "WT"
represents a wild-type mouse, and "KO" represents a RBMS2
homozygously deficient mouse. In the graph, each plot represents a
concentration of IL-6 in serum from each individual, and each of
the bars represents an average value.
[0090] FIG. 7 illustrates the results of Example 7. The vertical
axis represents a relative value of an expression amount/level of
RBMS2. The transverse axis represents a relative value of an
expression amount/level of IL-6.
[0091] FIG. 8 illustrates the results of Example 8. The vertical
axis represents a relative value of an expression amount/level of
RBMS2 mRNA relative to an expression amount/level of HPRT mRNA. The
transverse axis represents a time lapsed after the addition of
IL-10 protein or TGF.beta. protein. A white column represents a
case where IL-10 protein is added, and a black column represents a
case where TGF.beta. protein is added.
[0092] FIG. 9 illustrates the results of Example 9. The vertical
axis on the left side of the drawings represents a luciferase
activity obtained in the case where an IL-6 3'UTR is linked
downstream from a luciferase ORF, and the vertical axis on the
right side of the drawings represents a luciferase activity
obtained in the case where a PTGS2(COX2) 3'UTR is linked downstream
from a luciferase ORF. In each of the transverse axes, "Empty"
represents a case where an empty vector is introduced, and "RBMS2"
represents a case where a RBMS2 expression vector is introduced,
and "HuR" represents a case where a HuR expression vector is
introduced.
[0093] FIG. 10A illustrates the results of Example 10A. The
vertical axis represents a relative value of a luciferase activity.
In each of the transverse axes, "Empty" represents a case where an
empty vector is introduced, "FL" represents a case where a
wild-type RBMS2 expression vector is introduced, "dR1" represents a
case where a RRM1 domain deficient expression vector is introduced,
and "F101A F104A" represents a case where an expression vector in
which F101A and F104A are double-mutated is introduced. "**"
represents the fact that a P value <0.01.
[0094] FIG. 10B illustrates the results of Example 10B. The left
drawing represents the results of the measurement of a amount/level
of IL-6 mRNA, and the right drawing represents the results of the
measurement of a amount/level of IL-8 mRNA. The vertical axis in
each of the drawings represents a relative value of a amount/level
of IL-6 mRNA or IL-8 mRNA relative to a amount/level of GAPDH mRNA.
In each of the transverse axes, "empty" represents a case where a
control lentivirus is allowed to infect, "FL" represents a case
where a lentivirus capable of expressing wild-type RBMS2 is allowed
to infect, "RRM1" represents a case where a lentivirus capable of
expressing a RRM1 domain-deficient body is allowed to infect, and
"FF" represents a case where a lentivirus capable of expressing a
F101A/F104A double mutant is allowed to infect.
[0095] FIG. 10C illustrates the results of Example 10C. The
vertical axis represents a relative value of a amount/level of
luciferase mRNA in an immunoprecipitation product, wherein the
amount/level of luciferase mRNA in a cell lysate before
immunoprecipitation is 100%. In the transverse axis, "WT FL"
represents a case where an expression vector for FLAG-tagged
wild-type RBMS2 is introduced, "WT dR1" represents a case where an
expression vector for FLAG-tagged RRM1 domain-deleted RBMS2 is
introduced, and "WT F101/F104A" represents a case where an
expression vector for a F101A/F104A double mutant is introduced. A
black column represents a case where immunoprecipitation is
performed with an anti-FLAG antibody, and a white column represents
a case where immunoprecipitation is performed by a non-specific
IgG.
[0096] FIG. 11 illustrates the results of Example 11. The vertical
axis represents a value produced by subtracting a value of the
thickness of an auricle before the test from a value of the
thickness of the auricle after the test (i.e., an index indicating
the swelling of the auricle). In the transverse axis, "Het"
represents a RBMS2 gene heterozygously deficient mouse, and "KO"
represents a RBMS2 gene homozygously deficient mouse.
[0097] FIG. 12 illustrates the results of Example 12. The vertical
axis represents an expression amount/level of mRNA in the case
where siRNA for RBMS2 is allowed to transfect, and the transverse
axis represents an expression amount/level of mRNA in the case
where non-specific siRNA (negative control) is allowed to
transfect. Each plot represents a gene of which the expression
amount/level is 2-fold or more increased or is 2-fold or more
decreased by the knockdown of RBMS2.
MODE FOR CARRYING OUT THE INVENTION
1. Definitions
[0098] The wordings "contain" and "comprise" as used herein include
all of the concepts of "contain", "comprise", "substantially
consisting of" and "consisting only of".
[0099] The amino acid sequence "identity" refers to the degree of
amino acid sequence agreement between at least two amino acid
sequences that can be compared with each other. Therefore, the
identity or similarity of two amino acid sequences becomes higher
with the increase in the degree of amino acid sequence agreement
between the amino acid sequences. The level of amino acid sequence
identity can be determined using FASTA as a sequence analysis tool
and employing a default parameter. Alternatively, the level of
amino acid sequence identity can also be determined using an
algorithm BLAST established by Karlin and Altschul (Karlin S,
Altschul SF." Methods for assessing the statistical significance of
molecular sequence features by using general scoring schemes" Proc
Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF."
Applications and statistics for multiple high-scoring segments in
molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A
program called "BLASTX" which relies on the algorithm BLAST has
been developed. Specific techniques for these analysis methods are
known, and see a web site of National Center of Biotechnology
Information (NCBI) (ncbi.nlm.nih.gov/). The nucleotide sequence
"identity" can also be defined accordingly as mentioned above.
[0100] The term "conservative substitution" as used herein refers
to the matter that an amino acid residue is substituted by an amino
acid residue having a similar side chain. For example, the
substitution between amino acid residues each having a basic side
chain, e.g., lysine, arginine, histidine, is included within the
scope of the conservative substitution. In addition, the
substitution between amino acid residues each having an acidic side
chain, e.g., aspartic acid, glutamic acid, the substitution between
amino acid residues each having an uncharged polar side chain,
e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine
and cysteine, the substitution between amino acid residues each
having a non-polar side chain, e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine and tryptophan, the
substitution between amino acid residues each having a
.beta.-branched side chain, e.g., threonine, valine and isoleucine,
and the substitution between amino acid residues each having an
aromatic side chain, e.g., tyrosine, phenylalanine, tryptophan and
histidine, are included within the scope of the conservative
substitution.
[0101] In the description, the expression "RBMS2" means RBMS2
protein.
[0102] In the description, each of "a nucleotide", "an
oligonucleotide" and "a polynucleotide" has the same meaning as "a
nucleic acid", and both of DNA and RNA are included. Each of these
substances may be in a double-stranded form or a single-stranded
form. The term a "nucleotide" (or an "oligonucleotide" or a
"polynucleotide") having a given sequence also includes a
"nucleotide" (or an "oligonucleotide" or a "polynucleotide") having
a sequence complementary to the sequence therefor comprehensively,
unless otherwise stated. In addition, in the case where the
"nucleotide" (or an "oligonucleotide" or a "polynucleotide") is
RNA, the nucleotide symbol "T" shown in a sequence for the
nucleotide is to be read "U".
2. Expression Cassette
[0103] The present invention relates to an expression cassette
(also sometimes referred to as an "expression cassette of the
present invention", hereinafter) which contains a RBMS2 gene
expression regulation region and a gene arranged in such a manner
that the expression thereof can be regulated by the region.
Hereinbelow, the expression cassette will be described.
[0104] In the present application, the term an "expression
cassette" refers to a polynucleotide having such a function that a
gene contained in the expression cassette can be expressed in a
cell (e.g., a eukaryotic cell, preferably an animal cell, more
preferably a mammalian cell).
[0105] In the present application, the "RBMS2 gene expression
regulation region" is not particularly limited, as long as the
region is a DNA region capable of regulating the expression of
endogenous RBMS2 gene in a cell or a DNA region having the same
regulation ability as that of the aforementioned DNA region. An
example of the region is a promoter that contains a transcription
initiation site for RBMS2 gene, a sequence located upstream (5'
side) from the transcription initiation site, and optionally a
sequence located downstream (3' side) from the transcription
initiation site. A specific example of the promoter is a DNA region
lying between -10000 to +2000, preferably -5000 to +1000, more
preferably -2000 to +500, further preferably -1000 to +200, still
further preferably -500 to +100, especially preferably -200 to +50,
wherein a nucleotide corresponding to the transcription initiation
site for RBMS2 gene is expressed as "+1", a nucleotide located
downstream (3' side) from the aforementioned nucleotide is
expressed by a positive value, and a nucleotide located upstream
from the aforementioned nucleotide is expressed by 0 or a negative
value. The promoter may have a mutation, as long as the promoter
has the same level of expression regulation ability as that of a
promoter capable of regulating the expression of endogenous RBMS2
gene in a cell. In this case, the nucleotide sequence for the
promoter having a mutation has, for example, a 70% or more,
preferably 80% or more, more preferably 90% or more, further
preferably 95% or more, still further preferably 97% or more,
especially preferably 99% or more identity to a nucleotide sequence
for a promoter capable of regulating the expression of endogenous
RBMS2 gene in a cell. It is desirable that the position of the
mutation is a position other than the position of a known
expression regulation element (e.g., a basic transcription factor
binding region, any one of various activator binding regions). The
consensus sequence for the expression regulation element is already
known, and can be searched easily on various data base.
[0106] In the present application, "RBMS2 gene" is not particularly
limited, and examples of the gene include those from animals
including various mammals such as human, monkey, mouse, rat, dog,
cat, rabbit, pig, horse, cow, sheep, goat and deer.
[0107] RBMS2 genes derived from various animals are known. Specific
examples of RBMS2 mRNA and RBMS2 protein which are expression
products of the RBMS2 genes include human RBMS2 mRNA such as mRNA
consisting of the nucleotide sequence represented by SEQ ID NO: 3
(NCBI Reference Sequence: NM_002898.3); murine RBMS2 mRNA such as
mRNA consisting of the nucleotide sequence represented by SEQ ID
NO: 4 (NCBI Reference Sequence: NM_019711.2); human RBMS2 protein
such as a protein consisting of the amino acid sequence represented
by SEQ ID NO: 2 (NCBI Reference Sequence: NP_002889.1); and murine
RBMS2 protein such as a protein consisting of the amino acid
sequence represented by SEQ ID NO: 5 (NCBI Reference Sequence:
NP_062685.2). The RBMS2 protein includes, within the scope thereof,
a type in which the N-terminal is deleted. Specific examples of the
RBMS2 protein of this type include a mouse-derived protein, which
is consisting of the amino acid sequence represented by SEQ ID NO:
6 (NCBI Reference Sequence: NP_001034169.1) (whose mRNA is
consisting of the nucleotide sequence represented by SEQ ID NO: 7
(NCBI Reference Sequence: NM_001039080.1)).
[0108] RBMS2 protein, which is an expression product of RBMS2 gene,
may have an amino acid mutation such as substitution, deletion,
addition and insertion, as long as the RBMS2 protein can have an
activity to promote the expression of mRNA having a 3'UTR derived
from inflammation promoting factor mRNA (e.g., IL-6 mRNA, COX-2
mRNA, IL-8 mRNA, IL-1.beta. mRNA, TNF-.alpha. mRNA, MMP1 mRNA,
IL-24 mRNA, c-Myc mRNA) or a protein translated from the mRNA
(wherein the activity is also referred to as an "inflammation
promoting factor expression promoting activity", hereinafter). From
the viewpoint that the inflammation promoting factor expression
promoting activity is less likely to be deteriorated, the type of
the mutation is preferably substitution, more preferably
conservative substitution.
[0109] RBMS2 mRNA, which is a transcription product of RBMS2 gene,
may have a nucleotide mutation, such as substitution, deletion,
addition and insertion, as long as a protein translated from the
mRNA can have an inflammation promoting factor expression promoting
activity. The type of the mutation is preferably a mutation by
which an amino acid substitution does not occur in a protein
translated from the mRNA or a mutation by which a conservative
substitution of an amino acid residue can occur.
[0110] The presence or absence of an inflammation promoting factor
expression promoting activity can be determined by or in accordance
with a known method. For example, this presence or absence can be
determined by or in accordance with the methods described in the
section "Examples". A specific example is as follows: in Example
1B, when an expression vector for a test protein is used as the
expression vector, it is determined that the test protein has an
inflammation promoting factor expression promoting activity when
the luciferase activity is higher than that achieved when an empty
vector is used as the expression vector.
[0111] A preferred specific example of RBMS2 protein which is an
expression product of RBMS2 gene is at least one protein selected
from the group consisting of a protein mentioned in (a) and a
protein mentioned in (b):
[0112] (a) a protein which is consisting of an amino acid sequence
represented by SEQ ID NO: 2, 5 or 6; and
[0113] (b) a protein which has 85% or more identity to an amino
acid sequence represented by SEQ ID NO: 2, 5 or 6 and has an
inflammation promoting factor expression promoting activity.
[0114] In (b), the degree of identity is more preferably 90% or
more, further preferably 95% or more, still further preferably 98%
or more.
[0115] An example of the protein mentioned in (b) is:
[0116] (b') a protein which is consisting of an amino acid sequence
having the substitution, deletion, addition or insertion of one or
several amino acid residues in an amino acid sequence represented
by SEQ ID NO: 2, 5 or 6 and has an inflammation promoting factor
expression promoting activity.
[0117] In (b'), the wording "several amino acid residues" refers
to, for example, 2 to 30 amino acid residues, preferably 2 to 10
amino acid residues, more preferably 2 to 5 amino acid residues,
still further preferably 2 or 3 amino acid residues.
[0118] A preferred specific example of RBMS2 mRNA which is a
transcription product of RBMS2 gene is at least one component
selected from the group consisting of mRNA mentioned in (c) and
mRNA mentioned in (d):
[0119] (c) mRNA consisting of a nucleotide sequence represented by
SEQ ID NO: 3, 4 or 7; and
[0120] (d) mRNA which has 85% or more identity to a nucleotide
sequence represented by SEQ ID NO: 3, 4 or 7 and encodes a protein
having an inflammation promoting factor expression promoting
activity.
[0121] In (d), the degree of identity is more preferably 90% or
more, further preferably 95% or more, still further preferably 98%
or more.
[0122] An example of the protein mentioned in (d) is:
[0123] (d') a protein which is consisting of a nucleotide sequence
having the substitution, deletion, addition or insertion of one or
several nucleotides in a nucleotide sequence represented by SEQ ID
NO: 3, 4 or 7 and has an inflammation promoting factor expression
promoting activity.
[0124] In (d'), the wording "several nucleotides" refers to, for
example, 2 to 500 nucleotides, preferably 2 to 100 nucleotides,
more preferably 2 to 50 nucleotides, still further preferably 2 to
10 nucleotides.
[0125] In the present application, the term "gene" which is
arranged so that the expression of the gene can be regulated by a
RBMS2 gene expression regulation region is not particularly
limited, as long as an expression product of the gene can be
detected. In this regard, the term "gene" has a concept that a
sequence encoding a protein that is an expression product of the
gene is included, other sequence in the gene (e.g., an intron
sequence) may be included, but a promoter is not contained.
Examples of the gene include a reporter gene, a drug-resistant
gene, an enzyme gene, a structural gene, a transport gene, a
storage gene, a contractile gene, a defense gene, a regulatory
gene, and modified genes thereof. Examples of the modified gene
include a gene produced by mutating a nucleotide so that an amino
acid mutation, e.g., substitution, deletion, addition and
insertion, can occur in a part of a protein that is an expression
product of the above-mentioned gene; and a gene which can express a
protein that is a fusion of expression products of some of the
above-mentioned genes. Among these genes, a reporter gene, a
drug-resistant gene or the like is preferred, and a reporter gene
is more preferred.
[0126] In the present application, the "reporter gene" is not
particularly limited, as long as the gene is, for example, a gene
encoding a light-emitting (color-developing) protein capable of
reacting with a specific substrate to emit light (develop a color)
or a fluorescent protein capable of emitting fluorescence by the
action of excited light. Examples of the light-emitting
(color-developing) protein include luciferase,
.beta.-galactosidase, chloramphenicol acetyltransferase and
.beta.-glucuronidase. Examples of the fluorescent protein include
GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP,
Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana,
KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed,
Cherry, HcRed and mPlum.
[0127] In the present application, the "drug-resistant gene" is not
particularly limited, as long as the gene is a gene capable of
imparting resistance to a drug, e.g., an antibacterial drug, to a
cell in which the gene is expressed. Specific examples of the
drug-resistant gene include a chloramphenicol-resistant gene, a
tetracycline-resistant gene, a neomycin-resistant gene, an
erythromycin-resistant gene, a spectinomycin-resistant gene, a
kanamycin-resistant gene, a hygromycin-resistant gene and a
puromycin-resistant gene.
[0128] With respect to the above-mentioned "gene", the wording
"(being) arranged so that the expression of the gene can be
regulated" means that the gene is arranged so that a protein
encoded by the gene can be expressed. A specific example of the
arrangement is an aspect in which the gene expression regulating
region and the gene are arranged in this order as observed from the
5'-side.
[0129] If necessary, the expression cassette of the present
invention may contain other element (e.g., a multi-cloning site
(MCS)). For example, in the case where a RBMS2 gene expression
regulation region and the above-mentioned "gene" are arranged in
this order as observed from the 5'-side, an aspect can be mentioned
in which a MCS is arranged on the 5'-side of the RBMS2 gene
expression regulation region (preferably adjacent to the region),
or between the RBMS2 gene expression regulation region and the
above-mentioned "gene" (preferably adjacent to one or both of the
region and the gene), or on the 3'-side of the above-mentioned
"gene" (preferably adjacent to the gene). The MCS is not
particularly limited, as long as multiple (e.g., 2 to 50,
preferably 2 to 20, more preferably 2 to 10) restriction enzyme
sites are contained.
[0130] The expression cassette of the present invention may
constitute a vector by itself or in conjunction with other
sequence. The vector (also referred to as a "vector of the present
invention", hereinafter) is also included within the scope of the
present invention. The "other sequence" is not particularly
limited, and any one of various known sequences that can be
contained in an expression vector can be used. Examples of the
sequence include a replication origin and a drug-resistant gene.
With respect to the type of the drug-resistant gene, the
above-mentioned types can be mentioned. The type of the vector is
not particularly limited, and examples of the vector include a
plasmid vector such as an animal cell expressing plasmid; and a
virus vector such as a retrovirus, a lentivirus, an adenovirus, an
adeno-associated virus, a herpesvirus and a Sendai virus.
[0131] The vector according to the present invention may be
contained in a cell. The cell (also referred to as "a cell of the
present invention", hereinafter) is also included within the scope
of the present invention. In the cell of the present invention, the
vector of the present invention may be present outside of the
genome or may be present in such a form that the vector is
integrated in the genome. The organism species from which the cell
is derived is not particularly limited, and examples of the species
include various mammals such as human, monkey, mouse, rat, dog,
cat, rabbit, pig, horse, cow, sheep, goat and deer. The type of the
cell is not particularly limited, and cells derived from various
tissues or having various properties can be mentioned, such as a
blood cell, a hematopoietic stem cell/progenitor cell, a gamete (a
sperm, an ovum), a fibroblast, an epithelial cell, a vascular
endothelial cell, a nerve cell, a liver cell, a keratin generating
cell, a muscle cell, an epidermal cell, an endocrine cell, an ES
cell, an iPS cell, a tissue stem cell and a cancer cell.
[0132] At least one component selected from the group consisting of
the expression cassette of the present invention, the vector of the
present invention and the cell of the present invention can be used
preferably in the below-mentioned screening method of the present
invention. From this viewpoint, the present invention also relates
to a reagent for use in the screening for a substance capable of
inhibiting the expression or function of RBMS2 (preferably an
active ingredient of an inhibitor of the expression of an
inflammation promoting factor) (wherein the reagent is also
referred to as a "reagent of the present invention", hereinafter),
which contains at least one component selected from the group
consisting of the expression cassette of the present invention, the
vector of the present invention and the cell of the present
invention.
[0133] The reagent of the present invention is not particularly
limited, as long as at least one component selected from the group
consisting of the expression cassette of the present invention, the
vector of the present invention and the cell of the present
invention is contained. In addition, the reagent may also contain,
for example, a substance necessary for the detection of an
expression product from the expression cassette of the present
invention. Specific examples of the substance include a reagent for
hybridization, a label for a probe, a labeled body detection agent,
a buffer solution and a device. The reagent of the present
invention may be in the form of a screening kit including these
components.
3. A Screening Method
[0134] The present invention relates to a method for screening for
a substance capable of inhibiting the expression or function of
RBMS2 by employing at least one item selected from the group
consisting of items (i) to (iii) as an index in the presence of a
test substance:
[0135] (i) the expression amount/level of a gene of which the
expression can be regulated by a RBMS2 gene expression regulation
region;
[0136] (ii) the binding amount/level of RBMS2 to RNA containing an
AU-rich element; and
[0137] (iii) the amount/level of mRNA containing an AU-rich element
in a 3'-UTR thereof or the amount/level of a protein derived from
the mRNA in a RBMS2-overexpressing cell. Hereinbelow, the
expression cassette will be described (wherein the reagent is also
referred to as a "screening method of the present invention",
hereinafter).
[0138] As the "test substance (i.e., substance to be tested)" to be
used in the present application, any one of wide varieties of
compounds can be used, regardless of the fact that the compound is
a naturally occurring compound or an artificially produced
compound. Alternatively, a purified compound, a composition
prepared by mixing many types of compounds together, or an extract
of an animal or plant origin may also be used. The compound
includes a low-molecular-weight compound as well as a
high-molecular-weight compound such as a protein, a nucleic acid
and a polysaccharide.
[0139] More specific examples of the screening method, when the
value of the index in the presence of a test substance is smaller
than the value of the index in the absence of the test substance,
the test substance can be selected as a substance capable of
inhibiting the expression or function of RBMS2.
[0140] The screened "substance capable of inhibiting the expression
or function of RBMS2" can be selected as an active ingredient for
an inhibitor of the expression of an inflammation promoting factor
or a candidate substance for the active ingredient.
[0141] Herein below, specific screening methods for the aspects
which utilize the indices (i) to (iii), respectively, will be
described.
[0142] 3-1. Screening Method Using Index (i)
[0143] A screening method using index (i) includes steps (a1) to
(c1):
[0144] (a1) bringing an expression system which contains an
expression cassette containing a RBMS2 gene expression regulation
region and a gene arranged in such a manner that the expression
thereof can be regulated by the region into contact with a test
substance;
[0145] (b1) measuring, as an expression amount/level of interest,
the amount/level of the gene expressed in the expression system
that has been contacted with the test substance and then comparing
the expression amount/level of interest with a control expression
amount/level that is the expression amount/level of the gene in an
expression system that has not been contacted with the test
substance; and
[0146] (c1) selecting the test substance as a substance capable of
inhibiting the expression of RBMS2 when the expression amount/level
of interest is smaller than the control expression
amount/level.
[0147] In step (a1), the "expression cassette containing a RBMS2
gene expression regulation region and a gene arranged in such a
manner that the expression thereof can be regulated by the region"
is as mentioned in the section "2. Expression cassette". However,
the expression cassette in step (a1) is different from the
expression cassette in the section "2. Expression cassette" in that
the expression cassette in step (a1) includes an expression
cassette containing an endogenous RBMS2 gene expression regulation
region in the genome of a cell and RBMS2 gene located downstream
from the region.
[0148] In step (a1), the "expression system" is not particularly
limited, as long as a component necessary for the expression of a
gene from the expression cassette is contained. Examples of the
expression system include a cell-free protein expression system and
a cell. The cell-free protein expression system is generally
consisting of a solution (e.g., a liquid extract from cells)
containing a factor necessary for transcription and translation
(e.g., an RNA polymerase, a ribosome, any one of various
ribonucleotides), and a commercially available product may be used.
The cell is not particularly limited, as long as a gene can be
expressed from the expression cassette in the cell. Examples of the
cell include a cell derived from any one of various tissues or a
cell having any one of various properties, such as a blood cell, a
hematopoietic stem cell/progenitor cell, a gamete (a sperm, an
ovum), a fibroblast, an epithelial cell, a vascular endothelial
cell, a nerve cell, a liver cell, a keratin generating cell, a
muscle cell, an epidermal cell, an endocrine cell, an ES cell, an
iPS cell, a tissue stem cell and a cancer cell. From the viewpoint
that the screening can be performed more easily, the expression
system is preferably a cell.
[0149] In step (a1), in the case where the expression system
containing the expression cassette is a cell-free protein
expression system, it is preferred that the expression cassette is
contained in the solution in the system. In the case where the
expression system is a cell, there are an aspect where the
expression cassette is integrated into the genome of the cell, an
aspect where the expression cassette is present outside of the
genome of the cell (e.g., in the form of a plasmid), and the
like.
[0150] In step (a1), the aspect of bringing the test substance into
contact is not particularly limited. In the case where the
expression system is a cell-free protein expression system, it is
preferred to add the test substance to the solution in the system,
for example. In the case where the expression system is a cell, it
is preferred to add the test substance to a cell culture medium,
for example.
[0151] In step (a1), the contacting time of the test substance is
not particularly limited, and can be set appropriately depending on
the type of the test substance, the type of the expression system
and others. The time is, for example, 5 minutes to 72 hours.
[0152] In step (b1), the measurement of the expression amount/level
of interest and the control expression amount/level can be carried
out in accordance with or based on a known method. It is preferred
to carry out the measurement using the diagnostic agent of the
present invention mentioned above. In the case where the substance
to be measured is a nucleic acid (RBMS2 mRNA or a nucleic acid
derived therefrom (e.g., cDNA)), the measurement can be carried out
by a northern blotting method, a RT-PCR method, a DNA chip analysis
method, an in situ hybridization analysis method or the like using
the nucleic acid as a probe or a primer, for example. In the case
where the substance to be measured is a protein, the measurement
can be carried out by a western blotting method, an ELISA method or
the like using a specific antibody. In the case where the substance
to be measured is a reporter protein, the measurement can be
carried out by a method capable of detecting a reporter signal
(e.g., a fluorescence, a developed color, emitted light) coming
from the reporter protein (e.g., the microscopic observation of
fluorescence, a luciferase assay). In the case where the substance
to be measured is a drug-resistant protein, the measurement can be
carried out indirectly by counting the number of cells surviving in
the presence of the drug.
[0153] In the case where a northern blotting method is employed,
concretely, a method can be exemplified, in which a probe is
labeled with a radioactive isotope (e.g., .sup.32P, .sup.33P: RI),
a fluorescent substance or the like, then the labeled probe is
hybridized with mRNA derived from the expression system and
transferred onto a nylon membrane or the like in the conventional
manner, and then a double strand formed by the diagnostic agent and
mRNA derived from the sample from the subject is subjected to the
detection and measurement of a signal coming from a labeled probe
(a labeling substance such as an RI or a fluorescent substance)
using a radioactive ray detector BAS-180011 (manufactured by
Fujifilm Corporation), a fluorescence detector or the like.
Alternatively, a method may also be employed, in which the
diagnostic agent is labeled using AlkPhos Direct Labelling and
Detection System (manufactured by Amersham Pharmacia Biotech) in
accordance with the protocol, then the labeled diagnostic agent is
hybridized with mRNA derived from the expression system, and then a
signal coming from a labeled product of the diagnostic agent is
detected and measured using a multibioimager STORM860 (manufactured
by Amersham Pharmacia Biotech).
[0154] In the case where a RT-PCR method is employed, concretely, a
method can be exemplified, in which cDNA is prepared from RNA
derived from the expression system in the conventional manner, then
a pair of primers prepared from the diagnostic agent of the present
invention (i.e., a positive strand capable of binding to the cDNA
(- strand), a negative strand capable of binding to + strand) are
hybridized with the cDNA to perform a PCR method so that a target
region can be amplified using the cDNA as a template, and then
amplified double-stranded DNA thus produced is detected. For the
detection of the amplified double-stranded DNA, a method in which
the above-mentioned PCR is carried out using a primer that is
labeled with an RI or a fluorescent substance in advance to detect
labeled double-stranded DNA produced; a method in which
double-stranded DNA thus produced is transferred onto a nylon
membrane or the like in the conventional manner, then the labeled
probe is used as a probe and is hybridized with the double-stranded
DNA, and then a hybridized product is detected; and other method
can be employed. In this regard, a labeled double-stranded DNA
product thus produced can be measured using an arrangement 2100
bioanalyzer (manufactured by Yokogawa Analytical Systems, Inc.) or
the like. Alternatively, it is also possible to prepare a RT-PCR
reaction solution using SYBR Green RT-PCR Reagents (manufactured by
Applied Biosystems) in accordance with the protocol, then react the
reaction solution using ABI PRISM 7700 Sequence Detection System
(manufactured by Applied Biosystems), and then detect a reaction
product.
[0155] In the case where DNA chip analysis is utilized, a method
can be mentioned, in which a DNA chip to which a (single-stranded
or double-stranded) DNA probe is attached is provided, then the DNA
chip is hybridized with cRNA prepared from RNA derived from the
expression system in the conventional manner to produce a
double-stranded product formed from the DNA and the cRNA, then the
double-stranded product is bonded to a labeling probe prepared from
the diagnostic agent of the present invention, and then the bonded
product is detected.
[0156] As the western blotting method, a method can be exemplified,
in which a primary antibody is used, then a labeled antibody (an
antibody capable of binding to the primary antibody) that is
labeled with a radioactive isotope such as .sup.125I, a fluorescent
substance, an enzyme such as horseradish peroxidase (HRP) or the
like is used as a secondary antibody, and a signal coming from the
labeling substance, e.g., the radioactive isotope, the fluorescent
substance or the like, in the labeled compound is detected using a
radioactive ray measurement device BAS-1800II (manufactured by
Fujifilm Corporation), a fluorescence detector or the like.
Alternatively, it is also possible to use a primary antibody, then
the signal is detected using ECL Plus Western Blotting Detection
System (manufactured by Amersham Pharmacia Biotech) in accordance
with the protocol and is then measured using multibioimager
STORM860 (manufactured by Amersham Pharmacia Biotech).
[0157] In step (c1), for example, in the case where the expression
amount/level of interest is smaller than the control expression
amount/level, the test substance can be selected as a substance
capable of inhibiting the expression of RBMS2 when the expression
amount/level of interest is smaller by 1/2, 1/5, 1/10, 1/20, 1/50
1/100 than the control expression amount/level.
[0158] 3-2. Screening Method Using Index (ii)
[0159] A screening method using index (ii) includes steps (a2) to
(c2):
[0160] (a2) bringing RNA containing an AU-rich element into contact
with RBMS2 in the presence of a test substance;
[0161] (b2) measuring the binding amount/level between the RNA and
the RBMS2 which are contacted with each other in the presence of a
test substance as a binding amount/level of interest, and then
comparing the binding amount/level of interest with a control
binding amount/level that is the binding amount/level between the
RNA and the RBMS2 which are contacted with each other in the
absence of the test substance; and
[0162] (c2) selecting the test substance as a substance capable of
inhibiting the function of RBMS2 when the binding amount/level of
interest is smaller than the control binding amount/level.
[0163] In step (a2), the "AU-rich element" refers to an element in
which a nucleotide sequence represented by general formula:
(U).sub.nW.sup.1(U).sub.mW.sup.2(U).sub.o [wherein U represents an
uracil; W.sup.1 and W.sup.2 may be the same as or different from
each other and independently represent an adenine or uracil
(provided that a case where each of W.sup.1 and W.sup.2 represents
an uracil is excluded); n represents an integer of 0 to 3; o
represents an integer of 0 to 3; and m represents an integer of 3
to 5 (preferably 3)] typified by a sequence AUUUA is a consensus
sequence. The AU-rich element is preferably an AU-rich element
derived from mRNA of an inflammation promoting factor (e.g., at
least one type of mRNA selected from the group consisting of IL-6
mRNA, COX-2 mRNA, IL-8 mRNA, IL-1.beta. mRNA, TNF-.alpha. mRNA,
MMP1 mRNA, IL-24 mRNA and c-Myc mRNA). In other words, the term "an
AU-rich element derived from . . . " refers to an AU-rich element
contained in each of these mRNA.
[0164] In step (a2), the RNA containing an AU-rich element is not
particularly limited, as long as the RNA contains the AU-rich
element. The number of the AU-rich elements in the RNA is, for
example, 1 to 20, preferably 2 to 15, more preferably 3 to 12,
further preferably 4 to 10, still further preferably 6 to 9. When
the number of the AU-rich elements in the RNA is multiple, it is
desirable that the AU-rich elements are present in a relatively
narrow region (e.g., 20 to 400 bp, preferably 40 to 200 bp, more
preferably 60 to 150 bp, further preferably 80 to 120 bp). It is
preferred that the region is U-rich. The degree of U-richness is as
follows: the rate of the number of U residues relative to the total
number of nucleotides in the region is, for example, 20% or more,
preferably 30% or more, more preferably 50% or more. The upper
limit of the ratio is not particularly limited, and examples of the
upper limit include 90%, 80%, 70% and the like.
[0165] In step (a2), the RBMS2 is the same as that in the RBMS2
protein mentioned in the section "2. Expression cassette".
[0166] In step (a2), the aspect of bringing the test substance into
contact is not particularly limited, as long as the three
components, i.e., the RNA containing an AU-rich element, RBMS2 and
the test substance, can be contacted with one another. For example,
an aspect where the three components are mixed together in a proper
solvent, an aspect where the three components are allowed to
co-exist in a cell, and the like can be mentioned.
[0167] In step (a2), the contacting time of the test substance is
not particularly limited, and can be set appropriately depending on
the type of the test substance, whether the contact is achieved in
a test tube or in a cell, or the like. The time is, for example, 5
minutes to 72 hours.
[0168] In step (b2), the measurement of the binding amount/level of
interest and the control binding amount/level can be carried out by
or in accordance with a known method. For example, the measurement
can be carried out by an immunoprecipitation method, a gel shift
method or the like.
[0169] The immunoprecipitation method can be carried out typically
in the following manner. A cell lysate each containing RNA
containing an AU-rich element and RBMS2 (and being in contact with
the test substance or being not in contact with the test substance)
is prepared, then the lysate is immunoprecipitated with an antibody
directed against RBMS2 or an antibody directed against a tag in the
case where the tag is attached to the RBMS2, and then the
amount/level of "RNA containing an AU-rich element" contained in a
precipitate is measured by a PCR. It is demonstrated that the
binding amount/level of interest or the control binding
amount/level becomes larger with the increase in the measured
amount/level.
[0170] A gel shift method can be carried out typically in the
following manner. A solution containing the RNA containing an
AU-rich element and RBMS2 (and further containing the test
substance or not containing the test substance) is electrophoresed
using a proper gel (e.g., an acrylamide gel), and then a signal of
a band that indicates a complex of the RNA containing an AU-rich
element and RBMS2 which are bonded together is measured. It is
demonstrated that the binding amount/level of interest or the
control binding amount/level becomes larger with the increase in
the measured amount/level.
[0171] In step (c2), for example, in the case where the binding
amount/level of interest is smaller than the control binding
amount/level, the test substance can be selected as a substance
capable of inhibiting the expression of RBMS2 when the binding
amount/level of interest is smaller by 1/2, 1/5, 1/10, 1/20, 1/50
1/100 than the control binding amount/level.
[0172] 3-3. Screening Method Using Index (iii)
[0173] A screening method using index (iii) includes steps (a3) to
(c3):
[0174] (a3) bringing a cell which contains mRNA containing an
AU-rich element in a 3'-UTR thereof and in which RBMS2 is
overexpressed into contact with a test substance;
[0175] (b3) measuring the amount/level of the mRNA or a protein
derived from the mRNA in the cell that has been contacted with the
test substance as an amount/level of interest, and then comparing
the amount/level of interest with a control amount/level that is
the amount/level of the mRNA or a protein derived from the mRNA in
a cell that is not contacted with the test substance; and
[0176] (c3) selecting the test substance as a substance capable of
inhibiting the function of RBMS2 when the amount/level of interest
is smaller than the control amount/level.
[0177] In step (a3), the AU-rich element is the same as that
mentioned in the section "3-2. Screening method using index
(ii)".
[0178] In step (a3), the mRNA containing an AU-rich element in a
3'-UTR thereof is not particularly limited, as long as the mRNA
contains an AU-rich element in a 3'-UTR thereof. The number of
AU-rich elements in the 3'-UTR of the mRNA is, for example, 1 to
20, preferably 2 to 15, more preferably 3 to 12, further preferably
4 to 10, and still further preferably 6 to 9. In the case where the
number if AU-rich elements in the mRNA is multiple, it is desirable
that the AU-rich elements are present in a relatively narrow region
(e.g., 20 to 400, preferably 40 to 200, more preferably 60 to 150,
further preferably 80 to 120).
[0179] In step (a3), the mRNA containing an AU-rich element in the
3'-UTR thereof is preferably mRNA for an inflammation promoting
factor, more preferably IL-6 mRNA, COX-2 mRNA, IL-8 mRNA,
IL-1.beta. mRNA, TNF-.alpha. mRNA, MMP1 mRNA, IL-24 mRNA, c-Myc
mRNA or the like, or a variant of any one of these mRNA molecules.
As the variant, mRNA in which one or several (e.g., 2 to 50,
preferably 2 to 20, more preferably 2 to 10, further preferably 2
to 5, still further preferably 2 or 3) nucleotides are substituted,
deleted, added or inserted preferably in a sequence other than the
AU-rich element or a part of the AU-rich element can be
mentioned.
[0180] In step (a3), the RBMS2 is the same as the RBMS2 protein in
the section "2. Expression cassette".
[0181] In step (a3), the cell is the same as the cell in the
section "3-1. Screening method using index (i)".
[0182] In step (a3), the aspect of bringing the test substance into
contact is not particularly limited. For example, an aspect where
the test substance is added to the cell culture medium can be
mentioned.
[0183] In step (a3), the contacting time of the test substance is
not particularly limited, and can be set appropriately depending on
the type of the test substance and the like. The time is, for
example, 5 minutes to 72 hours.
[0184] In step (b3), the measurement of the amount/level of
interest and the control amount/level is carried out in the same
manner as in the measurement of the expression amount/level of
interest and the control expression amount/level in the section
"3-1. Screening method using index (i)".
[0185] In step (c3), in the case where the amount/level of interest
is smaller than the control amount/level, the test substance can be
selected as a substance capable of inhibiting the expression of
RBMS2 when the amount/level of interest is smaller by 1/2, 1/5,
1/10, 1/20, 1/50 or 1/100 than the control amount/level.
[0186] 4. Inhibitor of Expression of Inflammation Promoting
Factor
[0187] The present invention relates to an inhibitor of the
expression of an inflammation promoting factor (wherein the
inhibitor is also referred to as "the agent of the present
invention" in the description) which contains at least one
component selected from the group consisting of a RBMS2 expression
inhibitor and a RBMS2 function inhibitor. Hereinbelow, the
inhibitor will be described.
[0188] The RBMS2 of which the expression and function are to be
inhibited is the same as the RBMS2 protein in the section "2.
Expression cassette" above.
[0189] The RBMS2 expression inhibitor is not particularly limited,
as long as the inhibitor can reduce the expression amount/level of
RBMS2 protein. Specific examples of the inhibitor include
RBMS2-specific small interfering RNA (siRNA), RBMS2-specific
microRNA (miRNA), a RBMS2-specific antisense nucleic acid, and
expression vectors therefore. In addition, it is mentioned in the
section "Examples" that IL-10 protein can act as a RBMS2 expression
inhibitor.
[0190] The RBMS2-specific siRNA is not particularly limited, as
long as the siRNA is a double-stranded RNA molecule capable of
specifically inhibiting the expression of a gene encoding RBMS2. In
one embodiment, the siRNA preferably has a length of 18 nucleotides
or more, 19 nucleotides or more, 20 nucleotides or more, or 21
nucleotides or more. The siRNA preferably has a length of, for
example, 25 nucleotides or less, 24 nucleotides or less, 23
nucleotides or less, or 22 nucleotides or less. It is conceived
that the value of the upper limit and the value of the lower limit
of the length of the siRNA which are mentioned herein can be
combined arbitrarily. For example, the following combinations of
the length can be conceived: a length in which the lower limit is
18 nucleotides and the upper limit is 25 nucleotides, 24
nucleotides, 23 nucleotides, or 22 nucleotides; a length in which
the lower limit is 19 nucleotides and the upper limit is 25
nucleotides, 24 nucleotides, 23 nucleotides, or 22 nucleotides; a
length in which the lower limit is 20 nucleotides and the upper
limit is 25 nucleotides, 24 nucleotides, 23 nucleotides, or 22
nucleotides; and a length in which the lower limit is 21
nucleotides and the upper limit is 25 nucleotides, 24 nucleotides,
23 nucleotides, or 22 nucleotides.
[0191] The siRNA may be shRNA (small hairpin RNA). The shRNA can be
designed so that a part thereof forms a stem loop structure. For
example, when a sequence lying in a specific region is named
"sequence a" and a complementary strand to the sequence a is named
"sequence b", the shRNA can be designed in such a manner that the
sequence a, a spacer and the sequence b are arranged in this order
on a single RNA strand and the whole length becomes 45 to 60
nucleotides. The sequence a is a sequence for a partial region of
the nucleotide sequence encoding the target RBMS2, and the target
region is not limited particularly, and an arbitrary region can be
employed as a candidate. The length of the sequence a is 19 to 25
nucleotides, preferably 19 to 21 nucleotides.
[0192] The RBMS2-specific siRNA may have additional nucleotides at
the 5'- or 3'-terminal thereof. The length of the additional
nucleotides is generally about 2 to 4 nucleotides. The additional
nucleotides may be in the form of DNA or RNA. When additional
nucleotides are in the form of DNA, the stability of the nucleic
acid may be improved. Examples of the sequence for the additional
nucleotides include, but are not limited to, ug-3', uu-3', tg-3',
tt-3', ggg-3', guuu-3', gttt-3', ttttt-3' and uuuuu-3'.
[0193] The siRNA may have an overhang at the 3'-terminal thereof,
and a specific example of the overhang is dTdT (wherein dT
represents a deoxythymidine). Alternatively, the siRNA may have a
blunt end without the addition of a terminal. In the siRNA, the
number of nucleotides in a sense strand may be different from that
in an antisense strand. For example, the siRNA may be "asymmetrical
interfering RNA (aiRNA)" in which the antisense strand has an
overhang at each of the 3'-terminal and the 5'-terminal. Typical
aiRNA is one in which the antisense strand is consisting of 21
nucleotides, the sense strand is consisting of 15 nucleotides, and
an overhang structure consisting of 3 nucleotides is formed at each
terminal of the antisense strand.
[0194] The position of the target sequence in the RBMS2-specific
siRNA is not particularly limited. In one embodiment, it is
desirable to select the target sequence from a 5'-UTR and a
sequence lying between the initiation codon and about 50th
nucleotide from the initiation codon and not to select the target
sequence from a 3'-UTR. It is preferred that candidates for the
selected target sequence are determined with respect to the matter
that there is no homology in a sequence consisting of contiguous 16
to 17 nucleotides in mRNA other than the target sequence using a
homology search software such as BLAST (ncbi.nlm.nih.gov/BLAST/),
and the specificity of the selected target sequence is confirmed.
With respect to a target sequence of which the specificity has been
confirmed, double-stranded RNA which is consisting of a sense
strand having a 3'-terminal TT or UU overhang in a sequence lying
between 19st nucleotide and 21st nucleotide following AA (or NA)
and an antisense strand which is consisting of a sequence
complementary to the sequence lying between 19st nucleotide and
21st nucleotide and a 3'-terminal TT or UU overhang may be designed
as the siRNA. shRNA, which is a precursor of the siRNA, can be
designed by appropriately selecting an arbitrary linker sequence
(e.g., about 5 to 25 nucleotides) that can form a loop structure
and then connecting the sense strand to the antisense strand with
the linker sequence interposed therebetween.
[0195] The sequence for the siRNA and/or the shRNA can be searched
using various search software which is provided for free on web
sites. Examples of the sites are as follows. siRNA Target Finder
(ambion.com/jp/techlib/misc/siRNA_finder.html) provided by Ambion,
pSilencer (registered trademark) Insert design tool for Expression
Vectors (ambion.com/jp/techlib/misc/psilencer_converter.html)
GeneSeer (codex.cshl.edu/scripts/newsearchhairpin.cgi) provided by
RNAi Codex.
[0196] The siRNA can be prepared by separately synthesizing an
sense strand and an antisense strand in the target sequenced on
mRNA using a DNA/RNA automatic synthesizer, then denaturing these
strands in a proper annealing buffer solution at about 90 to
95.degree. C. for about 1 minute and then annealing the resultant
product at about 30 to about 70.degree. C. for about 1 to about 8
hours. Alternatively, the siRNA may also be prepared by
synthesizing shRNA that is a precursor of the siRNA and then
cutting the shRNA with a RNA cutting protein dicer.
[0197] The RBMS2-specific miRNA may be any one, as long as the
miRNA can inhibit the translation of a gene encoding RBMS2. For
example, the miRNA may inhibit the translation of target mRNA by
forming a pair with a 3'-untranslated region (UTR) in the target
rather than by cutting the target mRNA like siRNA. The miRNA may be
any one of pri-miRNA (primary miRNA), pre-miRNA (precursor miRNA)
and mature miRNA. The length of the miRNA is not particularly
limited, and the length of the pri-miRNA is generally several
hundreds to several thousands of nucleotides, the length of the
pre-miRNA is generally 50 to 80 nucleotides, and the length of the
mature miRNA is generally 18 to 30 nucleotides. In one embodiment,
the RBMS2-specific miRNA is preferably pre-miRNA or mature miRNA,
more preferably mature miRNA. The RBMS2-specific miRNA may be
synthesized by a known procedure, or may be purchased from a
synthetic RNA supplier company.
[0198] The RBMS2-specific antisense nucleic acid contains a
nucleotide sequence complementary or substantially complementary to
the nucleotide sequence for mRNA of a gene encoding RBMS2 or a part
of the nucleotide sequence, and has a function to form a specific
and stable double strand with the mRNA and bind to the mRNA so as
to inhibit the synthesis of RBMS2 protein. The antisense nucleic
acid may be any one selected from DNA, RNA and DNA/RNA chimera. In
the case where the antisense nucleic acid is DNA, an RNA:DNA hybrid
formed from a target RNA and the antisense DNA can be recognized by
endogenous ribonuclease H (RNase H) to cause the selective
degradation of the target RNA. Therefore, in the case of antisense
DNA that directs the degradation with RNase H, the target sequence
may be contained in mRNA as well as a sequence for an intron region
in an RBMS2 initial translation product. The intron sequence can be
determined by comparing the genome sequence with cDNA nucleotide
sequence in RBMS2 gene using a homology search program such as
BLAST and FASTA.
[0199] The length of the target region in the RBMS2-specific
antisense nucleic acid is not particularly limited, as long as the
antisense nucleic acid can hybridize with the target region so as
to inhibit the translation into RBMS2 protein. The RBMS2-specific
antisense nucleic acid may be the full length or a partial sequence
of mRNA encoding RBMS2. From the viewpoint of easiness of the
synthesis and the problems of antigenicity and intracellular
migration and the like, an oligonucleotide having a length of about
10 to about 40 nucleotides, particularly about 15 to about 30
nucleotides, is preferred, but the oligonucleotide is not limited
thereto. More concretely, a preferred target region for the
antisense nucleic acid can be selected from a 5'-terminal hairpin
loop, a 5'-terminal untranslated region, a translation initiation
codon, a protein coding region, an ORF translation stop codon, a
3'-terminal untranslated region, a 3'-terminal palindrome region
and a 3'-terminal hairpin loop of RBMS2 gene, but is not limited
thereto.
[0200] The RBMS2-specific antisense nucleic acid may also be one
which can hybridize with mRNA for RBMS2 gene or an initial
transcription product of the mRNA to inhibit the translation into a
protein and can also bind to these genes, each of which is
double-stranded DNA, to form a triplex so as to inhibit the
transcription into RNA (i.e., an antigene).
[0201] Each of nucleotide molecules that constitute the
RBMS2-specific siRNA, the RBMS2-specific miRNA and the
RBMS2-specific antisense nucleic acid may contain various chemical
modification for the purpose of improving (chemical and/or
enzymatical) stability or specific activity (affinity for RNA). For
example, in order to prevent the degradation caused by a hydrolysis
enzyme such as a nuclease, a phosphate residue (phosphate) in each
of the nucleotides constituting the antisense nucleic acid may be
substituted by, for example, a chemically modified phosphate
residue such as phosphorothioate (PS), methylphosphonate and
phosphorodithioate. Alternatively, the hydroxyl group at
2'-position in a ribose in each nucleotide may be substituted by
--OR (R.dbd.CH.sub.3(2'-O-Me), CH.sub.2CH.sub.2OCH.sub.3(2'-O-MOE),
CH.sub.2CH.sub.2NHC(NH)NH.sub.2, CH.sub.2CONHCH.sub.3, or
CH.sub.2CH.sub.2CN, etc.). In addition, the base moiety
(pyrimidine, purine) may be chemically modified. For example, the
introduction of a methyl group or a cationic functional group to
the 5'-position in a pyrimidine base, the substitution of a
carbonyl group at 2'-position by a thiocarbonyl group, or the like
may be applied. In addition, a part of each of the nucleotide
molecules that constitute siRNA and miRNA may be substituted by
naturally occurring DNA.
[0202] The RBMS2-specific siRNA, the RBMS2-specific miRNA, the
RBMS2-specific antisense nucleic acid and the like can be prepared
by determining mRNA or a target sequence for an initial
transcription product on the basis of the cDNA sequence or the
genomic DNA sequence for RBMS2 gene and then synthesizing a
sequence complementary to the sequence using a commercially
available DNA/RNA automatic synthesizer. The antisense nucleic acid
containing the above-mentioned modifications can also be chemically
synthesized by a known technique.
[0203] The expression vector for the RBMS2-specific siRNA, the
RBMS2-specific miRNA or the RBMS2-specific antisense nucleic acid
is not particularly limited, as long as the RBMS2-specific siRNA,
the RBMS2-specific miRNA or the RBMS2-specific antisense nucleic
acid is integrated in an expressible state. Typically, the
expression vector contains a promoter sequence, a polynucleotide
containing a sequence encoding the RBMS2-specific siRNA, the
RBMS2-specific miRNA or the RBMS2-specific antisense nucleic acid
(optionally also containing a transcription stop signal sequence)
and optionally other sequence. The promoter is not particularly
limited, and examples of the promoter include an RNA polymerase II
(polII)-type promoter, such as a CMV promoter, an EF1 promoter, an
SV40 promoter, a MSCV promoter, a hTERT promoter, a .beta.-actin
promoter and a CAG promoter; and a RNA polymerase III (polIII)-type
promoter, such as a mouse or human U6-snRNA promoter, a human
H1-RNase P RNA promoter and a human valine-tRNA promoter. Among
these promoters, a polIII-type promoter is preferred from the
viewpoint of the correct transcription ability of short RNA. The
"other sequence" is not particularly limited, and any one of
various known sequences which can be contained in expression
vectors can be employed. Examples of the sequence include a
replication origin and a drug-resistant gene. As the types of the
drug-resistant gene and the vector, those mentioned above can be
exemplified.
[0204] Another example of the RBMS2 expression inhibitor is a
RBMS2-specific ribozyme. The term "ribozyme" refers to RNA having
an enzymatic activity to cleave a nucleic acid in the narrow sense.
In the present application, however, DNA can also be included, as
long as the DNA has a sequence-specific nucleic acid cleavage
activity. The ribozyme having the broadest utility as a ribozyme
nucleic acid is self-splicing RNA that is found in infectious RNA
such as a viroid and a virusoid, and a hammerhead-type,
hairpin-type and the like are known. A hammerhead-type ribozyme
nucleic acid can exhibit the enzymatic activity thereof when the
length is about 40 nucleotides, and can cleave only target mRNA
specifically by converting several nucleotides located in both ends
adjacent to a hammerhead structure-forming part (about 10
nucleotides in total) into sequences complementary to a desired
cleavage site in mRNA. The ribozyme nucleic acid of this type can
utilize only RNA as the substrate thereof, and therefore has such
an advantage that the ribozyme nucleic acid never attacks genomic
DNA. In the case where the mRNA for the RBMS2 gene forms a
double-stranded structure by itself, the target sequence can be
made into a single strand by using a hybrid ribozyme to which an
RNA motif derived from a viral nucleic acid and capable of binding
specifically to an RNA helicase is linked [Proc. Natl. Acad. Sci.
USA, 98(10): 5572-5577 (2001)]. In the case where it is intended to
use the ribozyme in the form of an expression vector containing DNA
encoding the ribozyme, the ribozyme may be used in the form of a
hybrid ribozyme to which a tRNA-modified sequence is linked in
order to accelerate the migration of a transcript into a cell
cytoplasm [Nucleic Acids Res., 29(13): 2780-2788 (2001)].
[0205] The RBMS2 function inhibitor is not limited particularly, as
long as the inhibitor can inhibit the function of RBMS2 protein.
The wording "inhibition of the function of RBMS2 protein" refers to
(x) the matter that the binding amount/level between RBMS2 and RNA
containing an AU-rich element is reduced and/or (y) the matter that
the amount/level of mRNA containing an AU-rich element in the
3'-UTR thereof or the amount/level of a protein derived from the
mRNA in an RBMS2-overexpressing cell is reduced. Whether or not the
function of RBMS2 protein is inhibited can be determined by, for
example, the methods mentioned in the sections "3-2. Screening
method using index (ii)" and "3-3. Screening method using index
(iii)" below.
[0206] The inflammation promoting factor of which the expression is
to be inhibited by the agent of the present invention is not
particularly limited, as long as the inflammation promoting factor
is a factor involved in the induction or worsening of an
inflammatory reaction in vivo. Examples of the factor include IL-6,
COX-2, IL-1.beta., IL-8, TNF-.alpha., MMP1, IL-24 and c-Myc.
[0207] The agent of the present invention can be used as a
prophylactic or therapeutic agent for a disease which can be
developed or worsened by an inflammation promoting factor (e.g.,
IL-6, COX-2, IL-1.beta., IL-8, TNF-.alpha., MMP1, IL-24, c-Myc).
Specific examples of the disease include an immune disease, an
inflammatory disease, a painful disease and the like. More specific
examples of the disease to be diagnosed include an autoimmune
disease, a rheumatic disease, rheumatoid arthritis, a degenerative
arthritis disease, arthritis, sepsis, a lymphoproliferative
disease, a demyelinating disease in the central nervous system,
spondyloarthropathy, inflammatory pain, postoperative pain,
allergic disease, preferably an autoimmune disease, rheumatoid
arthritis, a lymphoproliferative disease, a demyelinating disease
in the central nervous system, postoperative pain and an allergic
disease, more preferably rheumatoid arthritis.
[0208] Examples of the autoimmune disease in the present
application include, but are not limited to Guillain-Barre
syndrome, ulcerative colitis, Crohn's disease, Graves' disease,
rheumatoid arthritis, systemic lupus erythematosus, polymyositis,
scleroderma and Sjogren's syndrome.
[0209] Examples of the rheumatic disease in the present application
include, but are not limited to rheumatoid arthritis, juvenile
idiopathic arthritis, systemic lupus erythematosus, scleroderma,
polymyositis, vasculitis syndrome, Behcet's disease, Sjogren's
syndrome and psoriatic arthritis.
[0210] Examples of the degenerative arthritis disease in the
present application include, but are not limited to osteoarthritis
of the knee, osteoarthritis of the hip, osteoarthritis of the
elbow, osteoarthritis of the shoulder, osteoarthritis of the wrist
and osteoarthritis of the foot.
[0211] Examples of the lymphoproliferative disease in the present
application include, but are not limited to lymphedema,
lymphadenitis, malignant lymphoma and Castleman's disease.
[0212] Examples of the demyelinating disease in the central nervous
system in the present application include, but are not limited to
multiple sclerosis, acute disseminated encephalomyelitis and
inflammatory diffuse sclerosis.
[0213] Examples of the spondyloarthropathy in the present
application include, but are not limited to ankylosing spondylitis,
reactive arthritis and psoriatic arthritis.
[0214] Examples of the inflammatory pain in the present application
include, but are not limited to osteoarthritis, low back pain,
shoulder periarthritis, cervico-omo-brachial syndrome, flexor
tenosynovitis and dental pain.
[0215] Examples of the allergic disease in the present application
include, but are not limited to an allergic skin disease such as
contact dermatitis, eczema, hives, Quincke's edema, erythema
nodosum and atopic dermatitis, allergic rhinitis, bronchial asthma,
drug allergy and food allergy.
[0216] The agent of the present invention is not particularly
limited, as long as the agent contains at least one component
selected from the group consisting of a RBMS2 expression inhibitor
and a RBMS2 function inhibitor (wherein the component is also
simply referred to as an "active ingredient" in the description).
The agent may additionally contain other component, if necessary.
The "other component" is not particularly limited, as long as the
component is a pharmaceutically acceptable component. Examples of
the "other component" include a base material, a carrier, a
solvent, a dispersant, an emulsifying agent, a buffering agent, a
stabilizer, an excipient, a binder, a disintegrating agent, a
lubricant, a thickening agent, a moisturizing agent, a coloring
agent, a fragrance and a chelating agent.
[0217] The mode of the usage of the agent of the present invention
is not particularly limited, and a proper usage mode can be
selected depending on the type of the agent. The agent of the
present invention may be used in vitro (e.g., may be added to a
culture medium for cultured cells) or may be used in vivo (e.g.,
may be administered to an animal).
[0218] The subject to which the agent of the present invention is
to be applied is not particularly limited, and examples of the
subject include various mammals such as human, monkey, mouse, rat,
dog, cat, rabbit, pig, horse, cow, sheep, goat and deer; and animal
cells. The type of the cell is not particularly limited, either.
For example, a blood cell, a hematopoietic stem cell/progenitor
cell, a gamete (a sperm, an ovum), a fibroblast, an epithelial
cell, a vascular endothelial cell, a nerve cell, a liver cell, a
keratin generating cell, a muscle cell, an epidermal cell, an
endocrine cell, an ES cell, an iPS cell, a tissue stem cell, a
cancer cell and the like can be mentioned.
[0219] The dosage form of the agent of the present invention is not
particularly limited, and a proper dosage form may be selected
depending on the mode of the usage of the agent. For example, in
the case where it is intended to administer the agent to an animal,
examples of the dosage form include an oral preparation such as a
tablet, a capsule, a granule, a powder, a fine granule, a syrup, an
enteric preparation, a sustained release capsule, a chewable
tablet, a drop, a pill, a liquid or solution for oral application,
a lozenge, a sustained-release preparation and sustained-release
granule; and a preparation for cutaneous application such as a
nasal preparation, an inhalation, a suppository for rectal
application, a pessary, an enema and a jelly. The agent of the
present invention may have any dosage form selected from a solid
dosage form, a semi-solid dosage form and a liquid dosage form.
[0220] The content of the active ingredient in the agent of the
present invention is not limited and varies depending on the
intended use of the agent, a subject of the application of the
agent, the condition of a subject of the application of the agent,
and the like. The content is, for example, 0.0001 to 100% by
weight, preferably 0.001 to 50% by weight.
[0221] In the case where it is intended to administer the agent to
an animal, the amount/level of the agent to be administered is not
particularly limited, as long as the amount/level is an
amount/level effective for developing the pharmacological activity
of the agent. The amount/level is generally 0.1 to 1000 mg/kg body
weight per day, preferably 0.5 to 500 mg/kg body weight per day, in
terms of the weight of the active ingredient for oral
administration, and is generally 0.01 to 100 mg/kg body weight,
preferably 0.05 to 50 mg/kg body weight per day, in terms of the
weight of the active ingredient for parenteral administration. The
dose amount/level is preferably administered in 1, 2 or 3 divided
doses, per day, and may be increased or decreased appropriately
depending on the age, clinical condition and disease conditions of
a subject.
5. Diagnostic Agent
[0222] The present invention relates to a diagnostic agent for a
disease in an animal, which contains a RBMS2 gene expression
product detecting agent (wherein the diagnostic agent is also
referred to as a "diagnostic agent of the present invention" in the
present description). Hereinbelow, the diagnostic agent will be
described.
[0223] The RBMS2 gene expression product which is to be detected
with the RBMS2 gene expression product detecting agent is not
particularly limited, as long as the RBMS2 gene expression product
is one which is expressed in a living body of an organism to be
diagnosed. Examples of the RBMS2 gene expression product include
RBMS2 mRNA or a nucleic acid (e.g., cDNA) derived from the RBMS2
mRNA, RBMS2 protein and the like.
[0224] The animal to be diagnosed is not particularly limited, as
long as the animal can express RBMS2 gene in vivo. Examples of the
animal include various mammals such as human, monkey, mouse, rat,
dog, cat and rabbit.
[0225] A sequence of RBMS2 mRNA and RBMS2 protein derived from
various animals are known. Specific examples of RBMS2 mRNA and
RBMS2 protein which are expression products of the RBMS2 genes
include human RBMS2 mRNA such as mRNA consisting of the nucleotide
sequence represented by SEQ ID NO: 3 (NCBI Reference Sequence:
NM_002898.3); murine RBMS2 mRNA such as mRNA consisting of the
nucleotide sequence represented by SEQ ID NO: 4 (NCBI Reference
Sequence: NM_019711.2); human RBMS2 protein such as a protein
consisting of the amino acid sequence represented by SEQ ID NO: 2
(NCBI Reference Sequence: NP_002889.1); and murine RBMS2 protein
such as a protein consisting of the amino acid sequence represented
by SEQ ID NO: 5 (NCBI Reference Sequence: NP_062685.2). The RBMS2
protein includes, within the scope thereof, a type in which the
N-terminal is deleted. Specific examples of the RBMS2 protein of
this type include a mouse-derived protein, which is consisting of
the amino acid sequence represented by SEQ ID NO: 6 (NCBI Reference
Sequence: NP_001034169.1) (whose mRNA is consisting of the
nucleotide sequence represented by SEQ ID NO: 7 (NCBI Reference
Sequence: NM_001039080.1)).
[0226] RBMS2 protein to be detected may have an amino acid mutation
such as substitution, deletion, addition and insertion, as long as
the RBMS2 protein can have an activity to promote the expression of
mRNA having a 3'UTR derived from inflammation promoting factor mRNA
(e.g., IL-6 mRNA, COX-2 mRNA, IL-8 mRNA, IL-1.beta. mRNA,
TNF-.alpha. mRNA, MMP1 mRNA, IL-24 mRNA, c-Myc mRNA) or a protein
translated from the mRNA (inflammation promoting factor expression
promoting activity). From the viewpoint that the inflammation
promoting factor expression promoting activity is less likely to be
deteriorated, the type of the mutation is preferably substitution,
more preferably conservative substitution.
[0227] RBMS2 mRNA which is to be detected may have a nucleotide
mutation, such as substitution, deletion, addition and insertion,
as long as a protein translated from the mRNA can have an
inflammation promoting factor expression promoting activity. The
type of the mutation is preferably a mutation by which an amino
acid substitution does not occur in a protein translated from the
mRNA or a mutation by which a conservative substitution of an amino
acid residue can occur.
[0228] The presence or absence of an inflammation promoting factor
expression promoting activity can be determined by or in accordance
with a known method. For example, this presence or absence can be
determined by or in accordance with the methods described in the
section "Examples". A specific example is as follows: in Example
1B, when an expression vector for a test protein is used as the
expression vector, it is determined that the test protein has an
inflammation promoting factor expression promoting activity when
the luciferase activity is higher than that achieved when an empty
vector is used as the expression vector.
[0229] A preferred specific example of RBMS2 protein to be detected
is at least one protein selected from the group consisting of a
protein mentioned in (a) and a protein mentioned in (b):
[0230] (a) a protein which is consisting of an amino acid sequence
represented by SEQ ID NO: 2, 5 or 6; and
[0231] (b) a protein which has 85% or more identity to an amino
acid sequence represented by SEQ ID NO: 2, 5 or 6 and has an
inflammation promoting factor expression promoting activity.
[0232] In (b), the degree of identity is more preferably 90% or
more, further preferably 95% or more, still further preferably 98%
or more.
[0233] An example of the protein mentioned in (b) is:
[0234] (b') a protein which is consisting of an amino acid sequence
having the substitution, deletion, addition or insertion of one or
several amino acid residues in an amino acid sequence represented
by SEQ ID NO: 2, 5 or 6 and has an inflammation promoting factor
expression promoting activity.
[0235] In (b'), the wording "several amino acid residues" refers
to, for example, 2 to 30 amino acid residues, preferably 2 to 10
amino acid residues, more preferably 2 to 5 amino acid residues,
still further preferably 2 or 3 amino acid residues.
[0236] A preferred specific example of RBMS2 mRNA which is to be
detected is at least one component selected from the group
consisting of mRNA mentioned in (c) and mRNA mentioned in (d):
[0237] (c) mRNA consisting of a nucleotide sequence represented by
SEQ ID NO: 3, 4 or 7; and
[0238] (d) mRNA which has 85% or more identity to a nucleotide
sequence represented by SEQ ID NO: 3, 4 or 7 and encodes a protein
having an inflammation promoting factor expression promoting
activity.
[0239] In (d), the degree of identity is more preferably 90% or
more, further preferably 95% or more, still further preferably 98%
or more.
[0240] An example of the protein mentioned in (d) is:
[0241] (d') a protein which is consisting of a nucleotide sequence
having the substitution, deletion, addition or insertion of one or
several nucleotides in a nucleotide sequence represented by SEQ ID
NO: 3, 4 or 7 and has an inflammation promoting factor expression
promoting activity.
[0242] In (d'), the wording "several nucleotides" refers to, for
example, 2 to 500 nucleotides, preferably 2 to 100 nucleotides,
more preferably 2 to 50 nucleotides, still further preferably 2 to
10 nucleotides.
[0243] The RBMS2 gene expression product detecting agent is not
particularly limited, as long as the detection and the
quantification of the RBMS2 gene expression product as mentioned
above can be achieved. In the case where the RBMS2 gene expression
product is a nucleic acid (e.g., RBMS2 mRNA, a nucleic acid derived
therefrom (e.g., cDNA)), examples of the RBMS2 gene expression
product detecting agent include a primer and a probe. In the case
where the RBMS2 gene expression product is a protein, an example of
the RBMS2 gene expression product detecting agent is an
antibody.
[0244] The primer, the probe or the like is not particularly
limited, as long as RBMS2 mRNA, a nucleic acid derived therefrom or
the like can be recognized selectively (specifically). The wording
"recognize (recognizing) selectively (specifically)" as used herein
refers to, but is not limited to, the matter that RBMS2 mRNA can be
detected specifically in a northern blotting method, the matter
that RBMS2 mRNA or a nucleic acid derived therefrom (e.g., cDNA)
can be amplified specifically in a RT-PCR method, for example. The
primer, the probe or the like may be any one, as long as a person
skilled in the art can determine that the above-mentioned detected
substance or amplified product is derived from RBMS2 mRNA.
[0245] Specific examples of the primer or the probe include at
least one substance selected from the group consisting of a
polynucleotide mentioned in item (e) and a polynucleotide mentioned
in item (0:
[0246] (e) a polynucleotide having at least 15 contiguous
nucleotides in the nucleotide sequence for RBMS2 mRNA and/or a
polynucleotide complementary to the polynucleotide; and
[0247] (f) a polynucleotide which can hybridize with the nucleotide
sequence for RBMS2 mRNA or a nucleotide sequence complementary to
the nucleotide sequence for RBMS2 mRNA under stringent conditions
and has at least 15 nucleotides.
[0248] The term "a complementary polynucleotide" or "a
complementary nucleotide sequence (a complementary strand, a
negative strand) refers to a polynucleotide or a nucleotide
sequence which is complementary to the full length sequence of a
polynucleotide consisting of the nucleotide sequence for RBMS2 mRNA
or a partial sequence of the full length sequence which has a
nucleotide sequence consisting of at least 15 contiguous
nucleotides in the above-mentioned nucleotide sequence (wherein
each of the full length sequence and the partial sequence is
referred to as a "positive strand" for convenience) on the basis of
the nucleotide base pair relationship of A:T and G: C. In this
regard, the complementary strand is not limited to one that can
form a completely complementary sequence with the nucleotide
sequence for the positive strand, and may also be one which has a
complementary relationship with the positive strand of interest to
such an extent that the complementary strand can hybridize with the
positive strand under stringent conditions. The stringent
conditions can be determined on the basis of the melting
temperature (Tm) of a nucleic acid to which a complex or a probe is
to be bonded, as taught by Berger and Kimmel (1987, Guide to
Molecular Cloning Techniques Methods in Enzymology, Vol. 152,
Academic Press, San Diego Calif.). For example, as the washing
conditions after hybridization, the conditions around "1.times.SSC,
0.1% SDS, 37.degree. C." can be mentioned. It is preferred that the
complementary strand can be maintained in such a state that the
complementary strand is hybridized with the positive strand of
interest even when the complementary strand is washed under these
conditions. Examples of the washing conditions include, but are not
limited to around "0.5.times.SSC, 0.1% SDS, 42.degree. C." as more
stringent hybridization conditions, around "0.1.times.SSC, 0.1%
SDS, 65.degree. C." as still more stringent conditions. Concrete
examples of the complementary strand include a strand consisting of
a nucleotide sequence completely complementary to the nucleotide
sequence for the positive strand of interest, and a strand
consisting of a nucleotide sequence having at least 90%, preferably
95%, more preferably 98% or more, further preferably 99% or more
identity to the strand.
[0249] The primer, the probe or the like can be designed by
utilizing a vector NTI (manufactured by Infomax) on the basis of
the nucleotide sequence for RBMS2 mRNA, such as a nucleotide
sequence represented by SEQ ID NO: 3, 4, or 7. Concretely, the
probe or the like can be obtained by applying the nucleotide
sequence for RBMS2 mRNA to software of vector NTI. It is also
possible to use a candidate sequence for the primer or probe or a
sequence containing at least the sequence as a part thereof, as the
primer or probe.
[0250] The nucleotide length of the primer, probe or the like is
not particularly limited, as long as the primer, probe or the like
has a length consisting of at least contiguous 15 nucleotides as
mentioned above. The length of the primer, probe or the like can be
set appropriately depending on the intended use. For example, in
the case where it is intended to use as a primer, the nucleotide
length is, for example, 15 to 100 nucleotides, preferably 15 to 50
nucleotides, more preferably 15 to 35 nucleotides. In the case
where it is intended to use as a probe, the nucleotide length is,
for example, 15 nucleotides to total number of nucleotides in the
entire sequence, preferably 15 to 1000 nucleotides, more preferably
100 to 1000 nucleotides.
[0251] The primer, probe or the like may be modified, as long as
the function thereof cannot be impaired significantly. Examples of
the modification include the addition of a labeling substance, such
as a fluorescent dye, an enzyme, a protein, a radioactive isotope,
a chemiluminescent substance and biotin.
[0252] As the fluorescent dye to be used in the present invention,
one which can label a nucleotide and can be used for the detection
or quantification of a nucleic acid can be generally used. Specific
examples of the fluorescent dye include, but are not limited to HEX
(4,7,2',4',5',7'-hexachloro-6-carboxylfluorescein, a green
fluorescent dye), fluorescein, NED (product name, manufactured by
Applied Biosystems, a yellow fluorescent dye), 6-FAM (product name,
manufactured by Applied Biosystems, a greenish yellow fluorescent
dye), and rhodamine and a derivative thereof [e.g., tetramethyl
rhodamine (TMR)]. As the method for labeling the nucleotide with a
fluorescent dye, a proper method among known labeling methods can
be employed [see Nature Biotechnology, 14, 303-308 (1996)].
Alternatively, a commercially available fluorescent labeling kit
may also be used (e.g., an oligonucleotide ECL 3'-oligolabeling
system, manufactured by Amersham Pharmacia).
[0253] The primer may also be immobilized onto an arbitrary solid
phase upon use. Therefore, in the diagnostic agent of the present
invention, the probe (an oligo- or polynucleotide) can be provided
in the form of an immobilized probe (e.g., a DNA chip, a cDNA
microarray, an oligo DNA array, a membrane filter or the like, on
each of which the probe is immobilized; which are collectively
named "a DNA chip or the like", hereinafter).
[0254] The solid phase to be used for the immobilization is not
particularly limited, as long as an oligo- or polynucleotide can be
immobilized thereon. Specific examples of the solid phase include a
glass plate, a nylon membrane, microbeads, a silicon chip, a
capillary and other substrates. The immobilization of the oligo- or
polynucleotide onto the solid phase may be performed by a method in
which a previously synthesized oligo- or polynucleotide is applied
on a solid phase or by a method in which a desired oligo- or
polynucleotide is synthesized on a solid phase. The immobilization
method to be employed is well known in the art depending on the
type of the probe to be immobilized, such as a method in which a
commercially available spotter (manufactured by Amersham) is used
in a DNA microarray technique [e.g., in situ synthesis of an
oligonucleotide by a photolithographic technique (Affymetrix) or an
inkjet technique (Rosetta Inpharmatics)].
[0255] The antibody or the like is not particularly limited, as
long as the antibody or the like can recognize RBMS2 protein
selectively (specifically). The wording "recognize selectively
(specifically)" refers to the matter that RBMS2 protein can be
detected specifically in, for example, a western blotting method or
an ELISA method. The antibody or the like is not limited to the
above-mentioned one, and may be any one as long as a person skilled
in the art can determine that the substance to be detected is
derived from RBMS2 protein.
[0256] The antibody includes a polyclonal antibody, a monoclonal
antibody, a chimeric antibody, a single-stranded antibody, or a
part of the antibody which has an antigen-binding property, such as
a Fab fragment and a fragment produced by a Fab expression library.
An antibody having an antigen-binding property to a polypeptide
generally consisting of at least contiguous 8 amino acid residues,
preferably 15 amino acid residues, more preferably 20 amino acid
residues, contained in the amino acid sequence for RBMS2 protein
can also be included within the scope of the antibody of the
present invention.
[0257] The method for producing these antibodies is already known,
and the antibody of the present invention can be produced by this
conventional method (Current protocols in Molecular Biology,
Chapter 11.12 to 11.13(2000)). Concretely, in the case where the
antibody of the present invention is a polyclonal antibody, the
antibody can be obtained by using RBMS2 protein that is expressed
in Escherichia coli or the like and then purified in the
conventional manner, or by synthesizing an oligopeptide having a
partial amino acid sequence of the RBMS2 protein in the
conventional manner, then immunizing a non-human animal, e.g., a
domestic rabbit, with the RBMS2 protein or the oligopeptide and
then collecting the antibody from serum from the immunized animal
in the conventional manner. On the other hand, in the case where
the antibody is a monoclonal antibody, the antibody can be obtained
by immunizing a non-human animal, e.g., a mouse, with RBMS2 protein
that is expressed in Escherichia coli or the like and then purified
in the conventional manner or an oligopeptide having a partial
amino acid sequence of the RBMS2 protein to produce a spleen cell,
then performing cell fusion of the spleen cell to a myeloma cell to
prepare a hybridoma cell, and then collecting the antibody from the
hybridoma cell (Current protocols in Molecular Biology edit.
Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.4 to
11.11).
[0258] The RBMS2 protein to be used as an immunizing antigen in the
production of the antibody can be obtained on the basis of known
gene sequence information by procedures including the cloning of
DNA, the construction of plasmids, the transfection into a host
cell, the culture of a transformant and the collection of a protein
from a culture. These procedures can be carried out by a method
known to a person skilled in the art or a method disclosed in a
document (Molecular Cloning, T. Maniatis et al., CSH Laboratory
(1983), DNA Cloning, DM. Glover, IRL PRESS (1985)) or the like.
[0259] Concretely, a protein that can be used as an immunizing
antigen for the production of the antibody of the present invention
can be obtained by producing recombinant DNA (expression vector)
which enables the expression of a gene encoding RBMS2 to be
expressed in a desired host cell, then introducing the recombinant
DNA into a host cell to transform the host cell with the
recombinant DNA to produce a transformant, then culturing the
transformant to produce a culture, and then collecting the desired
protein from the culture. Alternatively, a partial peptide of RBMS2
protein can also be produced by a conventional chemical synthesis
method (peptide synthesis) in accordance with known gene sequence
information.
[0260] The antibody of the present invention may be prepared by
using an oligopeptide having a partial amino acid sequence of RBMS2
protein. The oligo(poly)peptide to be used for the production of
the antibody is not required to have a functional biological
activity but desirably has the same immunogenic property as that of
RBMS2 protein. An example of the antibody is an oligo(poly)peptide
that preferably has the immunogenic property and is consisting of
at least 8 contiguous amino acid residues, preferably 15 amino acid
residues, more preferably 20 amino acid residues, in the amino acid
sequence for the RBMS2 protein.
[0261] The production of the antibody against the
oligo(poly)peptide can be achieved by enhancing an immunological
reaction using any one of various adjuvants depending on a host to
be used. Examples of the adjuvant include, but are not limited to
Freund's adjuvant; a mineral gel such as aluminum hydroxide; a
surface-active substance such as lysolecithin, pluronic polyol, a
polyanion, a peptide, an oil emulsifying agent, keyhole limpet
hemocyanin and dinitrophenol; and a human adjuvant such as BCG
(Bacille Calmette-Guerin) and Corynebacterium parvum.
[0262] The diagnostic agent of the present invention is not
particularly limited, as long as the above-mentioned RBMS2 gene
expression product detecting agent is contained. The diagnostic
agent may be consisting of only the detection agent, or may
additionally contain a substance necessary for the detection of a
RBMS2 gene expression product in addition to the detection agent.
Specific examples of the substance include a hybridization reagent,
a label for a probe, a detection agent for a labeled substance, a
buffer solution and a device. The diagnostic agent of the present
invention may be in the form of a diagnostic agent kit including
the above-mentioned components.
[0263] The diseases to be diagnosed with diagnostic agent of the
present invention is not particularly limited, as long as a
diseases which can be developed or worsened by an inflammation
promoting factor (e.g., IL-6 mRNA, COX-2 mRNA, IL-8 mRNA,
IL-1.beta. mRNA, TNF-.alpha. mRNA, MMP1 mRNA, IL-24 mRNA, c-Myc
mRNA). Specific examples of the disease include an immune disease,
an inflammatory disease, a painful disease and the like. More
specific examples of the disease to be diagnosed include an
autoimmune disease, a rheumatic disease, rheumatoid arthritis, a
degenerative arthritis disease, arthritis, sepsis, a
lymphoproliferative disease, a demyelinating disease in the central
nervous system, spondyloarthropathy, inflammatory pain,
postoperative pain, allergic disease, preferably an autoimmune
disease, rheumatoid arthritis, a lymphoproliferative disease, a
demyelinating disease in the central nervous system, postoperative
pain and an allergic disease, more preferably rheumatoid
arthritis.
[0264] Examples of the autoimmune disease in the present
application include, but are not limited to Guillain-Barre
syndrome, ulcerative colitis, Crohn's disease, Graves' disease,
rheumatoid arthritis, systemic lupus erythematosus, polymyositis,
scleroderma and Sjogren's syndrome.
[0265] Examples of the rheumatic disease in the present application
include, but are not limited to rheumatoid arthritis, juvenile
idiopathic arthritis, systemic lupus erythematosus, scleroderma,
polymyositis, vasculitis syndrome, Behcet's disease, Sjogren's
syndrome and psoriatic arthritis.
[0266] Examples of the degenerative arthritis disease in the
present application include, but are not limited to osteoarthritis
of the knee, osteoarthritis of the hip, osteoarthritis of the
elbow, osteoarthritis of the shoulder, osteoarthritis of the wrist
and osteoarthritis of the foot.
[0267] Examples of the lymphoproliferative disease in the present
application include, but are not limited to lymphedema,
lymphadenitis, malignant lymphoma and Castleman's disease.
[0268] Examples of the demyelinating disease in the central nervous
system in the present application include, but are not limited to
multiple sclerosis, acute disseminated encephalomyelitis and
inflammatory diffuse sclerosis.
[0269] Examples of the spondyloarthropathy in the present
application include, but are not limited to ankylosing spondylitis,
reactive arthritis and psoriatic arthritis.
[0270] Examples of the inflammatory pain in the present application
include, but are not limited to osteoarthritis, low back pain,
shoulder periarthritis, cervico-omo-brachial syndrome, flexor
tenosynovitis and dental pain.
[0271] Examples of the allergic disease in the present application
include, but are not limited to an allergic skin disease such as
contact dermatitis, eczema, hives, Quincke's edema, erythema
nodosum and atopic dermatitis, allergic rhinitis, bronchial asthma,
drug allergy and food allergy.
[0272] The diagnostic agent of the present invention can be used
for the diagnosis about the occurrence of the above-mentioned
disease of interest (i.e., whether or not a subject is suffering
from the disease) and the diagnosis about the degree of progression
of the above-mentioned disease of interest, as mentioned in the
sections "2. Method for detection of disease" and "3. Method for
determination of degree of progression of disease" below.
2. Method for Detection of Disease
[0273] The present invention relates to a method for detecting an
immune disease, an inflammatory disease or a painful disease (which
is also referred to as the "disease detection method of the present
invention" in the description), using the expression amount/level
of RBMS2 as an index. Hereinbelow, the method will be
described.
[0274] As a specific aspect of the disease detection method of the
present invention, the following aspect can be mentioned:
[0275] (a1) measuring the expression amount/level of interest of a
RBMS2 gene expression product in a sample collected from a subject;
and
[0276] (b1) comparing the expression amount/level of interest
measured in step (a1) with a control expression amount/level of the
RBMS2 gene expression product in a sample collected from a control
subject which does not suffer from any one of the immune disease,
the inflammatory disease and the painful disease,
[0277] wherein
[0278] (c1) the matter that the expression amount/level of interest
is higher than the control expression amount/level is employed as
an index for the determination that the subject has the immune
disease, the inflammatory disease or the painful disease.
[0279] The subject is a target to which the disease detection
method of the present invention is to be applied, and the species
of the subject is not particularly limited. Examples of the subject
include various mammals including human, monkey, mouse, rat, dog,
cat, rabbit and the like.
[0280] The sample is not particularly limited, as long as a RBMS2
gene expression product is contained. The sample can be selected
appropriately depending on the type of the disease to be detected
and the like. Specific examples of the sample include a blood
sample, a urine sample, and various tissue grafts. As the sample, a
sample collected from a living organism may be used without any
modification, and a sample produced by purifying and concentrating
a RBMS2 gene expression product to be detected in the conventional
manner is preferred. In the case where the RBMS2 gene expression
product to be detected is a nucleic acid, it is possible to prepare
a nucleic acid (e.g., cDNA) that reflects the sequence information
of RBMS2 mRNA from the mRNA and use the resultant product as the
sample.
[0281] The measurement of the expression amount/level of interest
and the control expression amount/level of the RBMS2 gene
expression product can be carried out by or in accordance with a
known method. It is preferred to carry out the measurement using
the diagnostic agent of the present invention mentioned below. In
the case where the substance to be measured is a nucleic acid
(RBMS2 mRNA or a nucleic acid derived therefrom (e.g., cDNA)), the
measurement can be carried out by a northern blotting method, a
RT-PCR method, a DNA chip analysis method, an in situ hybridization
analysis method or the like using the nucleic acid as a probe or a
primer, for example. In the case where the substance to be measured
is a protein, the measurement can be carried out by a western
blotting method, an ELISA method or the like using a specific
antibody.
[0282] In the case where a northern blotting method is employed,
the presence or absence or the amount/level of RBMS2 mRNA or a
nucleic acid derived therefrom in a sample can be determined using
the diagnostic agent of the present invention as a probe.
Concretely, a method can be exemplified, in which the diagnostic
agent of the present invention (complementary strand) is labeled
with a radioactive isotope (e.g., .sup.32P, .sup.33P: RI), a
fluorescent substance or the like, then the labeled product is
hybridized with mRNA derived from a sample from a subject and
transferred onto a nylon membrane or the like in the conventional
manner, and then a double strand formed by the diagnostic agent and
mRNA derived from the sample from the subject is subjected to the
detection and measurement of a signal coming from a labeled product
of the diagnostic agent (a labeling substance such as an RI or a
fluorescent substance) using a radioactive ray detector BAS-1800II
(manufactured by Fujifilm Corporation), a fluorescence detector or
the like. Alternatively, a method may also be employed, in which
the diagnostic agent is labeled using AlkPhos Direct Labelling and
Detection System (manufactured by Amersham Pharmacia Biotech) in
accordance with the protocol, then the labeled diagnostic agent is
hybridized with mRNA derived from a sample from a subject, and then
a signal coming from a labeled product of the diagnostic agent is
detected and measured using a multibioimager STORM860 (manufactured
by Amersham Pharmacia Biotech).
[0283] In the case where a RT-PCR method is employed, the presence
or absence or the amount/level of RBMS2 mRNA or a nucleic acid
derived therefrom in a sample can be determined by using the
diagnostic agent of the present invention as a primer. Concretely,
a method can be exemplified, in which cDNA is prepared from RNA
derived from a biological tissue of a subject in the conventional
manner, then a pair of primers prepared from the diagnostic agent
of the present invention (i.e., a positive strand capable of
binding to the cDNA (-strand), a negative strand capable of binding
to + strand) are hybridized with the cDNA to perform a PCR method
so that a target region can be amplified using the cDNA as a
template, and then amplified double-stranded DNA thus produced is
detected. For the detection of the amplified double-stranded DNA, a
method in which the above-mentioned PCR is carried out using a
primer that is labeled with an RI or a fluorescent substance in
advance to detect labeled double-stranded DNA produced; a method in
which double-stranded DNA thus produced is transferred onto a nylon
membrane or the like in the conventional manner, then the labeled
diagnostic agent is used as a probe and is hybridized with the
double-stranded DNA, and then a hybridized product is detected; and
other method can be employed. In this regard, a labeled
double-stranded DNA product thus produced can be measured using an
arrangement 2100 bioanalyzer (manufactured by Yokogawa Analytical
Systems, Inc.) or the like. Alternatively, it is also possible to
prepare a RT-PCR reaction solution using SYBR Green RT-PCR Reagents
(manufactured by Applied Biosystems) in accordance with the
protocol, then react the reaction solution using ABI PRISM 7700
Sequence Detection System (manufactured by Applied Biosystems), and
then detect a reaction product.
[0284] In the case where DNA chip analysis is utilized, a method
can be mentioned, in which a DNA chip in which the diagnostic agent
of the present invention is attached as a (single-stranded or
double-stranded) DNA probe is provided, then the DNA chip is
hybridized with cRNA prepared from RNA derived from a biological
tissue from a subject in the conventional manner to produce a
double-stranded product formed from the DNA and the cRNA, then the
double-stranded product is bonded to a labeling probe prepared from
the diagnostic agent of the present invention, and then the bonded
product is detected.
[0285] As the western blotting method, a method can be exemplified,
in which the diagnostic agent of the present invention is used as a
primary antibody, a labeled antibody (an antibody capable of
binding to the primary antibody) that is labeled with a radioactive
isotope such as .sup.125I, a fluorescent substance, an enzyme such
as horseradish peroxidase (HRP) or the like is used as a secondary
antibody, and a signal coming from the labeling substance, e.g.,
the radioactive isotope, the fluorescent substance or the like, in
the labeled compound is detected using a radioactive ray
measurement device BAS-180011 (manufactured by Fujifilm
Corporation), a fluorescence detector or the like. Alternatively,
it is also possible to use the diagnostic agent of the present
invention as a primary antibody, then the signal is detected using
ECL Plus Western Blotting Detection System (manufactured by
Amersham Pharmacia Biotech) in accordance with the protocol and is
then measured using multibioimager STORM860 (manufactured by
Amersham Pharmacia Biotech).
[0286] The control expression amount/level, which is to be compared
with the expression amount/level of interest, may be a control
expression amount/level of a single sample. However, it is
preferred to employ an average value or a mean value of control
expression amount/levels of multiple samples.
[0287] The determination whether or not a subject is suffering from
an immune disease, an inflammatory disease or a painful disease is
carried out by employing the matter that an expression amount/level
of interest is larger compared with a control expression
amount/level as a criterion. Concretely, for example, the
determination can be carried out by employing, as an index, the
matter that an expression amount/level of interest is increased by
50% or more, preferably 100% or more, more preferably 200% or more,
relative to a control expression amount/level.
3. Method for Determination of Degree of Progression of Disease
[0288] The present invention relates to a method for determining
the degree of progression of an immune disease, an inflammatory
disease or a painful disease employing the expression amount/level
of RBMS2 as an index (wherein the method also referred to as "the
method for determining the degree of progression of the present
invention" in the description). Hereinbelow, the method will be
described.
[0289] As a specific aspect of the method for determining the
degree of progression of the present invention, the following
aspect can be mentioned:
[0290] (a2) measuring the expression amount/level of interest of a
RBMS2 gene expression product in a sample collected from a subject
suffering from the immune disease, the inflammatory disease or the
painful disease; and
[0291] (b2) comparing the expression amount/level of interest
measured in step (a2) with a control expression amount/level of a
RBMS2 gene expression product in a sample collected from a control
subject suffer from the immune disease, the inflammatory disease or
the painful disease,
[0292] wherein
[0293] (c3) the matter that the expression amount/level of interest
is larger than the control expression amount/level is employed as
an index for the determination that the subject has a higher degree
of progression of the immune disease, the inflammatory disease or
the painful disease than that of the control subject.
[0294] The subject, the sample, the measurement of the expression
amount/level of interest and the control expression amount/level,
the control expression amount/level to be compared with the
expression amount/level of interest, and the like are as described
in the section "2. Method for detection of disease" above.
[0295] The degree of progression of a disease can be defined as the
severity of a condition associated with the expression of an
inflammation promoting factor such as IL-6, COX-2, IL-1.beta.,
IL-8, TNF-.alpha., MMP1, IL-24 and c-Myc.
[0296] The determination as at whether or not the degree of
progression of an immune disease, an inflammatory disease or a
painful disease in a subject is higher than that in a control
subject can be determined by employing, as an index, the matter
that the expression amount/level of interest is higher compared
with the control expression amount/level. Concretely, the
determination can be carried out by employing, as an index, the
matter that the expression amount/level of interest is higher
compared with the control expression amount/level by 50% or more,
preferably 100% or more, more preferably 200% or more.
EXAMPLES
[0297] Hereinbelow, the present invention will be described in
detail with reference to examples. However, the present invention
is not limited to these examples.
Example 1: Identification of RBMS2 as IL-6 Post-Transcriptional
Regulatory Factor
Example 1A
[0298] For the purpose of discovering an IL-6 post-transcriptional
regulatory factor, screening was carried out. The schema is shown
in FIG. 1A. Concretely, the following procedures were carried out.
First, a reporter vector in which a luciferase ORF and an IL-6
3'UTR (SEQ ID NO: 1) were arranged downstream from a SV40 promoter
in this order as observed from the 5' side was produced. The
reporter vector and an expression vector for each of various genes
were transfected into a HEK293T cell on a 384-well plate using a
transfection reagent (Fugene HD, manufactured by Promega
Corporation). 48 hours after the transfection, the luciferase
activity in each well was measured. A measurement value thus
obtained was compared with a measurement value of a control (i.e.,
a sample in which an empty vector was introduced as an expression
vector), and an expression vector of which the measurement value
was changed compared with the control was screened (primary
screening). About 100 gene expression vectors which was correlated
with RNA was picked up from the selected expression vectors, and
the screening was carried out in the same manner as mentioned above
(secondary screening).
[0299] As a result of the secondary screening, RBMS2 (NP_002889.1,
SEQ ID NO: 2) was identified as an IL-6 post-transcriptional
regulatory factor.
Example 1B
[0300] The reporter vector produced in Example 1A or a control
reporter vector which was produced by removing a human IL-6 3'UTR
from the reporter vector was introduced into a HEK 293 T cell
together with a RBMS2 expression vector or an empty vector. 48
hours after the transfection, a luciferase activity was measured.
The results are shown in FIG. 1B.
[0301] As shown in FIG. 1B, RBMS2 increased a luciferase activity
in an IL-6 3'UTR-dependent manner.
Example 1C
[0302] siRNA for RBMS2 (Human RBMS2-1 (Thermo Fisher Scientific
(Ambion), Silencer Select, siRNA ID No. s11867, SEQ ID NO: 8)), or
Human RBMS2-2 (Thermo Fisher Scientific (Ambion), Silencer Select,
siRNA ID No. s11868, SEQ ID NO: 9), or control siRNA (Negative
control (Thermo Fisher Scientific (Ambion), Silencer Select
Negative Control No. 1 siRNA, Product number: 4390843)) was
introduced into a MDA-MB-231 cell. 48 hours after the introduction,
IL-1.beta. was added to a culture medium (final concentration: 20
ng/ml), and the cell was collected 3 hours after the addition. In
this regard, a MDA-MB-231 cell can constantly produce IL-6, and can
express IL-6 in a larger amount/level upon the stimulation with
IL-1.beta.. cDNA was prepared from the cell, and the expression
amount/levels of RBMS2 mRNA (PCR primer: Forward: SEQ ID NO: 12,
Reverse: SEQ ID NO: 13) and IL-6 mRNA (PCR primer: Forward: SEQ ID
NO: 14, Reverse: SEQ ID NO: 15) were measured by quantitative PCR.
The results are shown in FIG. 1C.
[0303] As shown in FIG. 1C, in a MDA-MB-231 cell, the expression
amount/level of IL-6 mRNA was significantly decreased by inhibiting
the expression of RBMS2.
Example 1D
[0304] The procedure as in Example 1C was carried out, except that
IL-1.beta. was added 36 hours after the introduction, a culture
supernatant was collected 16 hours after the addition, and the
expression amount/level of IL-6 protein in the supernatant was
measured by ELISA. The results are shown in FIG. 1D.
[0305] As shown in FIG. 1D, in a MDA-MB-231 cell, the expression
amount/level of IL-6 protein was decreased significantly by the
inhibition of the expression of RBMS2.
Example 1E
[0306] A MDA-MB-231 cell was transfected by a retrovirus capable of
expressing RBMS2 or a control retrovirus and was then cultured for
1 week in the presence of puromycin to produce a forcibly
RBMS2-expressing cell. The cell was stimulated with IL-1.beta. for
3 hours, then the cell was collected, and then cDNA was prepared
from the cell. The expression amount/level of IL-6 mRNA in the cell
was measured by quantitative PCR. The results are shown in FIG.
1E.
[0307] As shown in FIG. 1E, the expression amount/level of IL-6
mRNA was increased by the forcible expression of RBMS2.
Example 1F
[0308] A reporter vector in which an ORF of luciferase gene was
arranged downstream from a promoter (SEQ ID NO: 36) for IL-6 gene
was produced. The reporter vector was transfected together with a
RBMS2 expression vector or an empty vector into a MDA-MB-231 cell.
48 hours after the transfection, a luciferase activity was
measured. The results are shown in FIG. 1F.
[0309] As shown in FIG. 1F, the expression of RBMS2 did not affect
the luciferase activity induced by an IL-6 promoter.
Results of Example 1
[0310] From the above-mentioned results, it was strongly suggested
that RBMS2 regulated the expression of IL-6 positively through the
post-transcriptional regulation of IL-6 mRNA rather than the
transcriptional regulation of IL-6 mRNA.
Example 2: Stabilization of IL-6 mRNA by RBMS2 Via ARE
Example 2A
[0311] A RBMS2 expression vector or an empty vector was transfected
into a THP-1 cell. 48 hours after the transfection, LPS was added
to a culture medium (final concentration: 1 .mu.g/ml) to induce the
expression of IL-6 mRNA, and then actinomycin D was added to the
culture medium (final concentration: 5 .mu.g/ml) 5 hours after the
addition to terminate the synthesis of mRNA. The cells were
collected 48 hours after the transfection of the expression vector,
5 hours after the addition of LPS and 3 hours after the addition of
actinomycin D, and the amount/level of IL-6 mRNA was measured by
quantitative PCR. The results are shown in FIG. 2A.
[0312] As shown in FIG. 2A, when an empty vector was transfected,
although the expression of IL-6 mRNA was induced by LPS, IL-6 mRNA
was decomposed rapidly and the amount/level of IL-6 mRNA was
decreased when the transcription was inhibited by the treatment
with actinomycin D. In contrast, when RBMS2 was forcibly expressed,
IL-6 mRNA remained in a high amount/level
[0313] after the treatment with actinomycin D. These results
suggested that the half-life of IL-6 mRNA was prolonged by
RBMS2.
Example 2B
[0314] A tetracycline response expression vector in which a
luciferase ORF and a IL-6 3'UTR were arranged downstream from a
tetracycline response factor (TRE) and a promoter in this order as
observed from the 5'-side was produced. The tetracycline response
expression vector was transfected together with a RBMS2 expression
vector or an empty vector into a HEK293T cell having a Tet-off
regulation plasmid integrated therein. 24 hours after the
transfection, doxycycline was added to the culture medium (final
concentration: 10 .mu.g/ml). 24 hours after the transfection of the
vector and 2 hours after the addition of doxycycline, the cell was
collected, and the amount/level of luciferase mRNA was measured by
quantitative PCR (PCR primer: Forward: SEQ ID NO: 16, Reverse: SEQ
ID NO: 17). The results are shown in FIG. 2B.
[0315] As shown in FIG. 2B, when an empty vector was introduced,
the amount/level of luciferase mRNA containing IL-6 3'UTR was
decreased by the addition of doxycycline (the termination of
synthesis of mRNA). In contrast, when RBMS2 was forcibly expressed,
it was confirmed that a high amount/level of luciferase mRNA
containing IL-6 3'UTR remained after the addition of doxycycline.
From these results, it was suggested that the half-life of
luciferase mRNA containing IL-6 3'UTR was prolonged by RBMS2.
Example 2C
[0316] A mutant 3'UTR reporter vector in which a luciferase ORF and
a mutant 3'UTR of IL-6 (97-267 (nucleotide sequence: SEQ ID NO:
37), 122-193 (nucleotide sequence: SEQ ID NO: 38), .DELTA.ARE'
(nucleotide sequence: SEQ ID NO: 39), .DELTA.ARE2 (nucleotide
sequence: SEQ ID NO: 40) or an ARE mutant (nucleotide sequence: SEQ
ID NO: 41)) were arranged downstream from a promoter in this order
as observed from the 5' region was produced. The mutant 3'UTR
reporter vector was transfected into a HEK293T cell together with a
RBMS2 expression vector or an empty vector. 48 hours after the
transfection, a luciferase activity was measured. The results are
shown in FIG. 2C.
[0317] In a 3'UTR in IL-6 mRNA, there are a stem loop structure
involved in the stabilization of mRNA and an AU-rich element (ARE)
that is rich in AU. The stem loop structure is an element critical
for the recognition by Regnase-1 (ZC3H12A) that is an RNase and the
subsequent degradation. It is reported that ARID5a contributes to
the stabilization of IL-6 mRNA by antagonizing the function of
Regnase-1. However, as shown in FIG. 2C, RBMS2 increased the
activity of a reporter (97-267 and 122-193) in which the stem loop
structure was deleted in a 3'UTR.
[0318] In IL-6 mRNA, there are two adjacent AREs that are close to
each other. It is known that an ARE binding protein, e.g. TTP, is
involved in the degradation of mRNA through this area. As shown in
FIG. 2C, when one of the ARE regions is deleted, the increase in
the reporter activity caused by RBMS2 was not observed (.DELTA.ARE1
and .DELTA.ARE2). When the sequence for ARE was mutated (by
substituting U by G), the increase in the reporter activity by
RBMS2 was not observed, either (ARE mutant). From these results, it
was suggested that RBMS2 was involved in the stabilization of mRNA
through the ARE regions.
Results of Example 2
[0319] From the above-mentioned results, it was strongly suggested
that RBMS2 regulated the expression of IL-6 at a
post-transcriptional amount/level through a mRNA stabilization
mechanism through the ARE regions.
Example 3: Binding of RBMS2 to IL-6 mRNA Through ARE
Example 3A
[0320] The reporter vector produced in Example 1A (wild-type 3'UTR
of IL-6 downstream from a luciferase ORF) or the ARE mutant 3'UTR
reporter vector produced in Example 2C (an ARE mutant) was
transfected into a HEK293T cell together with a RBMS2 expression
vector labeled with FLAG. 48 hours after the introduction, the cell
was collected to prepare a cell lysate. The cell lysate was
immunoprecipitated with an anti-FLAG antibody or a non-specific
IgG, and the amount/level of luciferase mRNA contained in an
immunoprecipitation product was measured by quantitative PCR. The
results are shown in FIG. 3A.
[0321] As shown in FIG. 3A, as the result of the
immunoprecipitation with an anti-FLAG antibody, extreme
concentration of luciferase mRNA containing a wild-type 3'UTR of
IL-6 was observed. In contrast, luciferase mRNA containing ARE
mutant 3'UTR was not concentrated. From these results, it was
suggested that RBMS2 was bonded through an ARE.
Example 3B
[0322] The reporter vector produced in Example 1A (a wild-type
3'UTR of IL-6 downstream from a luciferase ORF) was transfected
into a HEK293T cell together with an expression vector for
FLAG-tagged wild-type RBMS2 or RRM1 domain-deleted RBMS2 (amino
acid sequence: SEQ ID NO: 46). 48 hours after the transfection, the
cell was collected to prepare a cell lysate. The cell lysate was
immunoprecipitated with an anti-FLAG antibody or a non-specific
IgG, and the amount/level of luciferase mRNA contained in the
immunoprecipitation product was measured by quantitative PCR. The
results are shown in FIG. 3B.
[0323] As shown in FIG. 3B, even when a RRM1 domain-deleted mutant
of RBMS2 was overexpressed, the concentration of RNA was not
observed. From this result, it was suggested that a RRM1 domain was
critical for the binding to an ARE.
Example 3C
[0324] The reporter vector produced in Example 1A (a wild-type
3'UTR of IL-6 downstream from a luciferase ORF) was transfected
into a HEK 293 T cell together with an expression vector for
FLAG-tagged wild-type RBMS2 or RRM domain-deleted RBMS2 (RBMS2
.DELTA.RRM1 (amino acid sequence: SEQ ID NO: 46), RBMS2 .DELTA.RRM2
(amino acid sequence: SEQ ID NO: 47), or RBMS2 .DELTA.RRM1/2 (amino
acid sequence: SEQ ID NO: 48)) or an empty vector. 48 hours after
the introduction, a luciferase activity was measured. The results
are shown in FIG. 3C.
[0325] As shown in FIG. 3C, in a RRM1 domain-deleted RBMS2 mutant,
the IL-6 3'UTR-dependent increase in luciferase activity was not
observed.
Results of Example 3
[0326] As mentioned above, it was suggested that RBMS2 enhanced the
expression of IL-6 through the binding to an ARE occurring in a
3'UTR of IL-6 mRNA through a RRM1 domain.
Example 4: Regulation of Expression of ARE-Containing Gene by
RBMS2
Example 4A
[0327] siRNA for RBMS2 (Human RBMS2-1 (Thermo Fisher Scientific
(Ambion), Silencer Select, siRNA ID No. s11867, SEQ ID NO: 8)), or
Human RBMS2-2 (Thermo Fisher Scientific (Ambion), Silencer Select,
siRNA ID No. s11868, SEQ ID NO: 9), or control siRNA (Negative
control (Thermo Fisher Scientific (Ambion), Silencer Select
Negative Control No. 1 siRNA, Product number: 4390843)) was
introduced into a MDA-MB-231 cell. 48 hours after the introduction,
IL-1.beta. was added to the culture medium (final concentration: 20
ng/ml), and then the cell was collected 3 hours after the addition.
cDNA was prepared from the cell, and the expression amount/levels
of RBMS2 mRNA (PCR primer: Forward: SEQ ID NO: 12, Reverse: SEQ ID
NO: 13), IL-6 mRNA (PCR primer: Forward: SEQ ID NO: 14, Reverse:
SEQ ID NO: 15), PTGS2(COX-2) mRNA (PCR primer: Forward: SEQ ID NO:
20, Reverse: SEQ ID NO: 21), IL-8 mRNA (PCR primer: Forward: SEQ ID
NO: 22, Reverse: SEQ ID NO: 23), IL-1.beta. mRNA (PCR primer:
Forward: SEQ ID NO: 18, Reverse: SEQ ID NO: 19), NFKBIA mRNA (PCR
primer: Forward: SEQ ID NO: 24, Reverse: SEQ ID NO: 25) and ZC3H12A
mRNA (PCR primer: Forward: SEQ ID NO: 26, Reverse: SEQ ID NO: 27)
were measured by quantitative PCR. The results are shown in FIG.
4A.
[0328] As shown in FIG. 4A, in a MDA-MB-231 cell, the expression of
IL-1.beta., IL-8 and COX-2 each having an ARE region in a 3'UTR was
suppressed by the inhibition of the expression of RBMS2. In
contrast, no difference in expression was observed between NFKBIA
and ZC3H12A each having no ARE in a 3'-UTR thereof.
Example 4B
[0329] siRNA for RBMS2 (Mouse RBMS2-1 (Thermo Fisher Scientific
(Ambion), Silencer Select, siRNA ID No. s80716, SEQ ID NO: 10), or
Mouse RBMS2-2 (Thermo Fisher Scientific (Ambion), Silencer Select,
siRNA ID No. s80717, SEQ ID NO: 11)), or control siRNA (Negative
control (Thermo Fisher Scientific (Ambion), Silencer Select
Negative Control No. 1 siRNA, Product number: 4390843)) was
introduced into a MEF cell. 48 hours after the introduction, LPS,
Pam3CSK4 or IL-1.beta. was added to a culture medium (final
concentration of LPS: 1 .mu.g/ml, final concentration of Pam3CSK4:
1 .mu.g/ml, or final concentration of IL-1.beta.: 20 ng/ml), and
then the cell was collected 3 hours after the addition. cDNA was
prepared from the cell, and the expression amount/levels of RBMS2
mRNA (PCR primer: Forward: SEQ ID NO: 28, Reverse: SEQ ID NO: 29),
IL-6 mRNA (PCR primer: Forward: SEQ ID NO: 30, Reverse: SEQ ID NO:
31), IL-1.beta. mRNA (PCR primer: Forward: SEQ ID NO: 32, Reverse:
SEQ ID NO: 33) and TNF-.alpha. mRNA (PCR primer: Forward: SEQ ID
NO: 34, Reverse: SEQ ID NO: 35) were measured by quantitative PCR.
The results are shown in FIG. 4B.
[0330] As shown in FIG. 4B, in a mouse embryonic fibroblast (MEF),
the increase in expression of TNF-.alpha., IL-1.beta. and IL-6
caused by the stimulation with LPS, Pam3CSK4 or IL-1.beta. was
suppressed by the inhibition of the expression of RBMS2.
Example 4C
[0331] A reporter vector in which a luciferase ORF, a 3'UTR (a
wild-type 3'UTR of IL-8 (nucleotide sequence: SEQ ID NO: 42), an
ARE mutant 3'UTR of IL-8 (nucleotide sequence: SEQ ID NO: 43), a
wild-type 3'UTR of IL-1.beta. (nucleotide sequence: SEQ ID NO: 44)
and an ARE mutant 3'UTR of IL-1.beta. (nucleotide sequence: SEQ ID
NO: 45)) were arranged downstream from a promoter in this order as
observed from the 5'-side was produced. The reporter vector thus
produced, the reporter vector produced in Example 1A (a wild-type
3'UTR of IL-6 downstream from a luciferase ORF), or the IL-6 ARE
mutant 3'UTR reporter vector (ARE mutant) produced in Example 2C
was transfected into a HEK293T cell together with a RBMS2
expression vector or an empty vector. 48 hours after the
introduction, a luciferase activity was measured. The results are
shown in FIG. 4C.
[0332] As shown in FIG. 4C, when a mutation was caused in an ARE
contained in a 3'UTR of IL-6, IL-8, IL-1.beta. or the like, the
increase in luciferase activity caused by RBMS2 was not
observed.
Results of Example 4
[0333] From the above-mentioned results, it was demonstrated that
RBMS2 was involved in not only the regulation of IL-6 mRNA but also
the regulation of mRNA having an ARE. It was also demonstrated that
the expression amount/level of an inflammation promoting factor
such as IL-6 mRNA was able to be decreased by inhibiting RBMS2.
Example 5: Decrease in Expression of Inflammatory Cytokine in
RBMS2-Deficient Mouse
Example 5A
[0334] A RBMS2-deficient mouse was produced by genome editing using
TALEN. As a result, a mutant mouse from which 110 nucleotides
including the second exon were deleted was produced successfully.
The production scheme is illustrated in FIG. 5A.
Example 5B
[0335] A cell obtained from bone marrow of a RBMS2 homozygously
deficient mouse, a RBMS2 heterozygously deficient mouse or a
wild-type mouse was cultured for 7 days in the presence of M-CSF to
produce a bone marrow-derived macrophage (BMDM). LPS (final
concentration: 100 ng/ml), Pam3CSK4 (final concentration: 300
ng/ml) or poly I:C (final concentration: 10 .mu.g/ml) was added to
a culture medium of the BMDM. The cells were collected before the
addition or 1 hour, 3 hours, 5 hours or 9 hours after the addition,
and the expression amount/level of IL-6 was measured by
quantitative PCR. The results are shown in FIG. 5B.
[0336] As shown in FIG. 5B, the expression of IL-6 induced by the
stimulation with Pam3CSK4 was significantly decreased in a
RBMS2-deficient mouse.
Example 5C
[0337] The same procedure as in Example 5B was carried out, except
that Pam3CSK4 was added to a BMDM culture medium and the expression
amount/levels of IL-1.beta., COX-2 and TNF-.alpha. were
measured.
[0338] As shown in FIG. 5C, the same results as in Example 5B were
obtained with respect to a gene other than IL-6.
Example 6: Resistance of RBMS2-Deficient Mouse to Sepsis
[0339] LPS (15 mg/kg) was administered intraperitoneally to each of
PBMS2 homozygously deficient mice (n=7) or wild-type mice (n=5). 5
hours after the administration, serum was collected, and the
survival time of each mouse was measured. The concentration of IL-6
in the serum was measured by quantitative PCR. The results of the
measurement of the survival time are shown in FIG. 6A, and the
results of the measurement of the concentration of IL-6 are shown
in FIG. 6B.
[0340] As shown in FIGS. 6A and 6B, 60% or more of the wild-type
mice were dead within 81 hours after the intraperitoneal
administration of LPS, while 80% or more of the RBMS2 knockout mice
survived because the expression level of IL-6 that was an
inflammation promoting factor was reduced. From these results, it
was demonstrated that RBMS2 was involved in the worsening of
inflammatory responses in vivo.
Example 7: Correlation Between Expression Amount/Level of RBMS2 and
Expression Amount/Level of IL-6 in Rheumatoid Arthritis Patient
[0341] RNA was reverse-transcribed from a peripheral blood
mononuclear cell from each of rheumatoid arthritis patients (n=12),
and then the expression amount/levels of RBMS2 and IL-6 were
measured by quantitative PCR. The results are shown in FIG. 7.
[0342] As shown in FIG. 7, the expression of RBMS2 and the
expression of IL-6 were positively correlated with each other in
peripheral blood mononuclear cells collected from rheumatoid
arthritis patients.
[0343] With taking the fact that the expression amount/level of
IL-6 in a non-rheumatoid arthritis patient was lower than that in a
rheumatoid arthritis patient and the fact that the expression
amount/level of IL-6 in a rheumatoid arthritis patient correlated
with the degree of progression of the disease into consideration,
these results suggested that it was possible to diagnose rheumatoid
arthritis (with respect to the occurrence of the disease, the
degree of progression of the disease) employing the expression
amount/level of RBMS2 as an index.
Example 8: Search for Factor Capable of Regulating Expression of
RBMS2
[0344] IL-10 protein or TGF.beta. protein was added to a culture
medium of a Jurkat cell that was a human T cell strain (final
concentration: IL-10 protein.fwdarw.20 ng/mL, TGF.beta.
protein.fwdarw.10 ng/mL). cDNA from the cell before the addition
and 8 hours after the addition and 24 hours after the addition was
prepared, and the expression amount/levels of RBMS2 mRNA and HPRT
(control) mRNA were measured by quantitative PCR. The results are
shown in FIG. 8.
[0345] As shown in FIG. 8, the expression amount/level of RBMS2
mRNA was decreased by the addition of IL-10 protein. This result
demonstrated that IL-10 protein had an effect to inhibit the
expression of RBMS2.
Example 9: Comparison of Activity to Regulate Expression of
ARE-Containing Gene
[0346] From the results of Example 4C, it was demonstrated that the
activity of RBMS2 depended on an AU-rich element (ARE) rather than
a stem loop structure in IL-6 3'UTR. On the other hand, it has been
reported that HuR can positively regulate the expression of an
inflammatory cytokine through an ARE, and therefore it has been
believed that the role of HuR is similar to that of RBMS2. Then,
the activities of RBMS2 and HuR were compared with each other by a
luciferase assay.
[0347] Concretely, a reporter vector in which a luciferase ORF and
an IL-6 3'UTR or PTGS2 (COX-2) 3'UTR were arranged downstream from
a promoter in this order as observed from the 5'-region was
produced. The reporter vector was transfected into a HEK293T cell
together with a RBMS2 expression vector, a HuR expression vector or
an empty vector. 48 hours after the transfection, a luciferase
activity was measured. The results are shown in FIG. 9.
[0348] As a result, it was demonstrated that, when an IL-6 3'UTR
was used, the luciferase activity was enhanced by RBMS2, while that
types of enhancement was not observed when HuR was used. When a
PTGS2 (COX-2) 3'UTR, on which it has been reported that HuR can
act, was used, although the enhancement of the luciferase activity
was observed by HuR, it was found that RBMS2 enhanced the activity
more strongly compared with HuR. These results strongly suggested
that RBMS2 targeted different mRNA from mRNA targeted by HuR and
had stronger activity against the same target mRNA than HuR.
Example 10: Binding of RBMS2 to ARE Through RRM1 Domain Example
10A
[0349] Heretofore, it has been reported that a .beta.-sheet in a
RRM domain is essential for a RNA binding activity and an aromatic
amino acid is necessary for the formation of the .beta.-sheet.
Then, a protein .beta.-sheet in a RRM1 domain was estimated using a
secondary structure estimation program, and an expression vector
for a mutant of the .beta.-sheet in which an amino acid was
mutated. The expression vector or an empty vector was transfected
into a MDA-MB-231 cell together with the reporter vector produced
in Example 1A (a wild-type 3'UTR of IL-6 downstream from a
luciferase ORF). 48 hours after the transfection, a luciferase
activity was measured. The results are shown in FIG. 10A.
[0350] As a result, it was demonstrated that two phenylalanine
residues (F101 and F104) in a RRM1 domain were required for the
enhancement of a reporter activity by RBMS2.
Example 10B
[0351] A lentivirus capable of expression a F101/F104A mutant was
transfected into a MDA-MB-231 cell, cDNA was prepared from the cell
after 5 days, and the expression amount/levels of IL-6 mRNA and
IL-8 mRNA were measured by quantitative PCR. The results are shown
in FIG. 10B.
[0352] As shown in FIG. 10B, even when the F101/F104A mutant was
overexpressed, the expression amount/levels of endogenous IL-6 and
IL-8 could not be improved.
Example 10C
[0353] The reporter vector produced in Example 1A (a wild-type
3'UTR of IL-6 downstream from a luciferase ORF) was transfected
into a HEK 293 T cell together with an expression vector for a
FLAG-tagged F101/F104A mutant. 48 hours after the transfection, the
cell was collected to prepare a cell lysate. The cell lysate was
immunoprecipitated with an anti-FLAG antibody or a non-specific
IgG, and the amount/level of luciferase mRNA contained in the
immunoprecipitation product was measured by quantitative PCR. The
results are shown in FIG. 10C.
[0354] As shown in FIG. 10C, the binding to an IL-6 3'UTR was
significantly impaired by double mutation in F101A and F104A.
Example 11
[0355] A 2,4-dinitrofluorobenzene solution (wherein a solvent was a
mixed solution of acetone and olive oil (acetone 4: olive oil 1))
was applied onto an abdominal part of each of RBMS2 homozygously
deficient mice (n=15). After 1 week, a 2,4-dinitrofluorobenzene
solution (wherein a solvent was a mixed solution of acetone and
olive oil (acetone 4: olive oil 1)) or only the mixed solvent was
applied onto an ear of each of the mice. After 6 hours, the
thickness of the auricle was measured, and a value obtained by
subtracting the thickness of the auricle before the test from the
measured value was employed as an index for the swelling of the
auricle. The results are shown in FIG. 11.
[0356] As shown in FIG. 11, the degree of swelling of the auricle
in a RBMS2 homozygously deficient mouse was smaller. This result
suggested that RBMS2 was involved in an allergic disease such as
contact dermatitis.
Example 12
[0357] A MDA-MB-231 cell was transfected with siRNA for a negative
control or RBMS2 (n=3 for each). After 48 hours, RNA was prepared,
an RNA sequencing library was prepared in accordance with a
protocol (Illumina, Inc.), and RNA sequencing was carried out using
Next-seq. Genes each of which the expression amount/level increased
two-fold or more or decreased two-fold or more by the knockdown of
RBMS2 are shown in FIG. 12.
[0358] As shown in FIG. 12, it was demonstrated that the expression
amount/level of mRNA having an ARE in a 3' UTR thereof, such as
IL-6, IL-8, MMP1, c-myc and IL-24 mRNA, was decreased by the
knockdown of RBMS2.
[0359] As demonstrated in Examples 1 to 12 above, RBMS2 regulated
the expression of IL-6 as well as the expression of other
inflammation promoting factors (genes each having an ARE in a 3'
UTR thereof, such as IL-1.beta., IL-8, COX-2, TNF-.alpha., MMP1,
IL-24 and c-Myc). Therefore, it was suggested that it became
possible to diagnose the occurrence and the degree of progression
of a disease of which the development and progression was
associated with any one of these inflammation promoting factors
(e.g., an immune disease, an inflammatory disease, a painful
disease) by employing the expression amount/level of RBMS2 as an
index.
Sequence CWU 1
1
501446DNAHomo sapiens 1catgggcacc tcagattgtt gttgttaatg ggcattcctt
cttctggtca gaaacctgtc 60cactgggcac agaacttatg ttgttctcta tggagaacta
aaagtatgag cgttaggaca 120ctattttaat tatttttaat ttattaatat
ttaaatatgt gaagctgagt taatttatgt 180aagtcatatt tatattttta
agaagtacca cttgaaacat tttatgtatt agttttgaaa 240taataatgga
aagtggctat gcagtttgaa tatcctttgt ttcagagcca gatcatttct
300tggaaagtgt aggcttacct caaataaatg gctaacttat acatattttt
aaagaaatat 360ttatattgta tttatataat gtataaatgg tttttatacc
aataaatggc attttaaaaa 420attcagcaaa aaaaaaaaaa aaaaaa
4462407PRTHomo sapiens 2Met Leu Leu Ser Val Thr Ser Arg Pro Gly Ile
Ser Thr Phe Gly Tyr1 5 10 15Asn Arg Asn Asn Lys Lys Pro Tyr Val Ser
Leu Ala Gln Gln Met Ala 20 25 30Pro Pro Ser Pro Ser Asn Ser Thr Pro
Asn Ser Ser Ser Gly Ser Asn 35 40 45Gly Asn Asp Gln Leu Ser Lys Thr
Asn Leu Tyr Ile Arg Gly Leu Gln 50 55 60Pro Gly Thr Thr Asp Gln Asp
Leu Val Lys Leu Cys Gln Pro Tyr Gly65 70 75 80Lys Ile Val Ser Thr
Lys Ala Ile Leu Asp Lys Thr Thr Asn Lys Cys 85 90 95Lys Gly Tyr Gly
Phe Val Asp Phe Asp Ser Pro Ser Ala Ala Gln Lys 100 105 110Ala Val
Thr Ala Leu Lys Ala Ser Gly Val Gln Ala Gln Met Ala Lys 115 120
125Gln Gln Glu Gln Asp Pro Thr Asn Leu Tyr Ile Ser Asn Leu Pro Leu
130 135 140Ser Met Asp Glu Gln Glu Leu Glu Gly Met Leu Lys Pro Phe
Gly Gln145 150 155 160Val Ile Ser Thr Arg Ile Leu Arg Asp Thr Ser
Gly Thr Ser Arg Gly 165 170 175Val Gly Phe Ala Arg Met Glu Ser Thr
Glu Lys Cys Glu Ala Ile Ile 180 185 190Thr His Phe Asn Gly Lys Tyr
Ile Lys Thr Pro Pro Gly Val Pro Ala 195 200 205Pro Ser Asp Pro Leu
Leu Cys Lys Phe Ala Asp Gly Gly Pro Lys Lys 210 215 220Arg Gln Asn
Gln Gly Lys Phe Val Gln Asn Gly Arg Ala Trp Pro Arg225 230 235
240Asn Ala Asp Met Gly Val Met Ala Leu Thr Tyr Asp Pro Thr Thr Ala
245 250 255Leu Gln Asn Gly Phe Tyr Pro Ala Pro Tyr Asn Ile Thr Pro
Asn Arg 260 265 270Met Leu Ala Gln Ser Ala Leu Ser Pro Tyr Leu Ser
Ser Pro Val Ser 275 280 285Ser Tyr Gln Arg Val Thr Gln Thr Ser Pro
Leu Gln Val Pro Asn Pro 290 295 300Ser Trp Met His His His Ser Tyr
Leu Met Gln Pro Ser Gly Ser Val305 310 315 320Leu Thr Pro Gly Met
Asp His Pro Ile Ser Leu Gln Pro Ala Ser Met 325 330 335Met Gly Pro
Leu Thr Gln Gln Leu Gly His Leu Ser Leu Ser Ser Thr 340 345 350Gly
Thr Tyr Met Pro Thr Ala Ala Ala Met Gln Gly Ala Tyr Ile Ser 355 360
365Gln Tyr Thr Pro Val Pro Ser Ser Ser Val Ser Val Glu Glu Ser Ser
370 375 380Gly Gln Gln Asn Gln Val Ala Val Asp Ala Pro Ser Glu His
Gly Val385 390 395 400Tyr Ser Phe Gln Phe Asn Lys 40538504DNAHomo
sapiens 3gagctcattc tctgcccgca gccccccttc atctctctcc tcctgctcct
tttctcctcc 60tttctcctcc ccctcccttt tttcctccct ccctctcccc ctttccctcg
ctcccctcct 120ccctccctcc ccgtctttct taccccctcc ctttctctct
ctctctctct ctcgctcgtt 180ccctaacatt aaagagaaaa tgctgctatc
cgtgacttcc aggcccggga tttcgacttt 240tggctacaat agaaacaaca
agaagccata tgtgtcattg gctcagcaga tggcaccacc 300tagcccaagc
aacagtacac ctaacagcag tagtggaagc aatggaaatg accagctgag
360caaaaccaac ctatacatcc gaggattgca accaggcact actgaccaag
atcttgtcaa 420gctgtgtcag ccatatggca agattgtttc cactaaggcc
atactggaca agaccacaaa 480caaatgtaaa ggctatggct ttgtagattt
tgacagccct tcagcagcac agaaagctgt 540aacagcactg aaggccagcg
gtgtacaggc acagatggca aagcaacagg aacaggaccc 600cacaaattta
tacatctcaa acctcccact gtcaatggat gagcaggaac tggaggggat
660gctgaagccc tttggccagg ttatctccac ccgtatcctt cgagatacca
gtgggaccag 720cagaggtgtt ggctttgcaa ggatggagtc cacagagaag
tgtgaagcca tcatcaccca 780ctttaatgga aaatatatta agacaccccc
tggagtacca gccccatccg atcccttgct 840ttgcaaattt gctgatggcg
ggccaaagaa acgacagaac caaggaaaat ttgtgcaaaa 900tggacgggct
tggccaagga atgcagacat gggcgtcatg gccttgacct atgaccccac
960cacagctctt cagaatgggt tttacccagc cccctataac atcaccccca
acaggatgct 1020tgctcagtct gcactctccc catacctttc ctctcctgtg
tcttcgtatc agagagtgac 1080tcagacatct cctctacaag tacctaaccc
atcctggatg caccaccatt catacctcat 1140gcagccttca ggttcagttc
tgacaccagg gatggaccat cccatttctc tccagcctgc 1200ctccatgatg
ggacccctta cccagcaact gggccatctc tccctcagca gcacaggcac
1260gtatatgccg acggctgcag ctatgcaagg agcttacatc tcccagtaca
cccctgtgcc 1320ttcttccagt gtttcagtcg aggagagcag cggccaacag
aaccaagtgg cagtggacgc 1380accctcagag catggggtct attctttcca
gttcaacaag taacagtggg attcccctcc 1440ccatctttac tgaatagaaa
tgaattcttg gagatactca tgctcccaga ttccagaggg 1500ttaaccagga
atggagacca tccgtcggcc ctgctaagga ctaacactta gccatcgttt
1560ttcacaggcc tgggcctgga aaaagaaatc tctacgttcc tgccctttac
tattgctgat 1620ggagcctggg ggaaccatca ctttttttgt gtgctacatt
caaggagatc aaaaaaactt 1680ttcttctttt gcaaagaaag ctttttgttt
ttaactgcaa cgtacttttc ccctaccttg 1740aagagacatg gtggtcgcag
cttctcatct atatgaaaaa gttttcgatg tattggaatt 1800atttgggaat
gcttttaaaa caatttgtaa ttatttcttt acaaaccaaa acagaacaga
1860aaggtgtggt gctggaacat cgatgaagga gccctactta ctgagcttag
ttatggactt 1920ttttgatgca tgtgtgtatg tgttttaaaa agtatgcagg
ctctaaaaat gttattttgt 1980aaagctctca gctcatgcac cccatctcct
cttcacccat attatgcctt ctttctcttg 2040tccagattct tctttttctc
ttttctaaac agctgagcct gcctactttg cccttttaca 2100gcttttaatt
ttatggattt ttaaaaatga aatttcatgt ggaatttggg gttggggggc
2160aggctgggca aggaacaagg cagaacacta agtaggccat ggaagtggct
gttctttccc 2220ccaccctgcc acaccctggg agaaaaaact agactttggc
ttcagaaagc acagatgtga 2280cccaggctta ctaaagagac aactccacag
ccctgggaac acacccttga gccaaacttg 2340gttgaagact aggtcttccc
tggcaagttc cggaagaatg gacttactga cttttatcaa 2400ctcttctcac
tgccaaggcc aacagcatct gaggtatagc tttttgggag tacctgcttt
2460cttgcctcct ggaggatatt ttctgtcctg ggcttcatgg cccctctctt
ccctgttaca 2520cattgctgtg ctcagagcct ttgcagctgc gacctagttg
aatccacata ggctccttcc 2580acacggtgga agatctgctg cttcactcac
agaccaggag ttctcaatca gaggtggttt 2640tgtccctcag gcctttggca
acatctagag acagttttga ttgccacgcc tggaggtggg 2700atgtgtgtgc
tactggcatc tagtggctgc taaacatcct acactgcata ggatagtccc
2760cactaccccc agccaagaat tatctgactc caggggtcaa tattgccaag
actgggaaac 2820actgatatag acagtgttgt ccctgcctcc tgggttaggg
taaattttca ccccaaccct 2880ggacaaacag tgccttttga cactcatgca
actgttgggg aaggactgga ctgggatcct 2940tgaattctcc ccagactttg
acttttgata actctgcata tagcaagagt gattcttgaa 3000agagctgtgg
ctccatgctg aatgcacaca tgtactcaga gggattcagg tgggcattgt
3060tctggtgtgt gctgtgcaat ggtgggaaag gacaaagctc tctaaactgt
ccagagcaac 3120ctgccctgcc ccggtgtgct tggacccttt gcccagggaa
ccaggacatc agaaataatg 3180ttccttctgt ggaaggacaa cggggagcta
gggtagcaaa gagcaataaa ttccaactct 3240ttatgaggtc aggagtcctt
taaaaaagcc tatgtcttgg tctttaactg tccccttctg 3300caagatgtga
ctctatacac atggagacag attgaagaaa gacttcaccc atcttctagg
3360aaataaaatc ctgctcttgt cttctcccag tcactccctt atgcactgaa
aggagaattt 3420gactcccttg cacttcaagg ccaggtgcct ctccactaga
cagtcagaca aaggcaaata 3480atggaggata gagagacagg tgttcattag
gctggccata gggagaggaa gggggtgaca 3540gcaggggaaa agaaactgta
gtatcagttg tcacaagtag agctcggccc agctttccct 3600cccttcccag
ccaccctaga attgagagtc ccagtgcccc tagtgtcgac tttctatgta
3660catcctaggg gtgagggagt gcggggcaat gggtaggata taaagcgtaa
aggacaaatt 3720gccgcagtct tctaattgat gcttccaaac tcaggaaggg
cttatccagt gtaaaatagc 3780cccaagccca gcaaccttct agagggctct
ggctgggcag atagcttttg tttttgagtc 3840tccagccctc agtcaaggaa
aatataccat agctgctttc ttgtcagtgg cctttgagag 3900aaagagaaaa
caaatctaga attctgttca tctgatagac cgggggagct ttgctatggt
3960aggaggttta ggaagggcct ttcacgcata gaaaccattg ttgatattgc
cactcccctc 4020tcctctgcct cacagaactc ctgattctgt cttctttctc
tctctatata tatattttaa 4080aatgtttaaa tggctctaat tttttgtttt
gaattttgaa tttacctttt ggagttcttc 4140tcatgtgaat ctacttgtta
gattgtatta atgagtgttt ctggtttggg catgggaagt 4200gatgggggcc
tgaagttatt ttgcagtggc taatggcact gaggaatttc ttttttggtg
4260catttggttt acactcattt cctctcctaa ttgttagttc atttgtaaca
gtgggtgagt 4320gtttggagga aaggagggag aagaaaggag ggatactgtt
tctcccatga aatagtctaa 4380ttggttgggt tgatggcaga aggaaacata
ggggagcctt ccagctcaca gccaagggtt 4440gggctcttaa acactatgcc
tagtgttttc tgaatgctgt cttcatggag cccagctctt 4500actctctttg
tactttacat ctcaccccca ctcattacag atgctcataa cattcttaaa
4560atattttagt acttggcatt tttctgtttt cagtcagcta gaacacacta
gagtcctttc 4620ctcagatggc ataatccttt ataggctctg agcctgccta
gccatctcct atcggtgtta 4680ttactcctca tctcaggctc tgagatgata
ctcagaccct aaactgattg gactttttgg 4740aggagggtgc cagtagagag
gtcaggaaga tgtggagatg atgatggaga gagatgtttt 4800tattttattt
tattttttca gacagagtct tgctctgtcg cccaggctgg agtgcaatgg
4860cacgatctcg gctcactgca acctccgcct cccaggttca agcaattctc
ctgcctcagc 4920ctcccatgta gctgggatta caggcaccca ccaccatgcc
cggctaattt ttttattttt 4980agtagagatg gggtttcacc atgttggcca
ggctggtctc gaactcctga cctcaggtga 5040tctgcccgcc ttagcctccc
aaagtgctgg gattacaggc gtgagccacc gcgcccggcc 5100tttattttat
tttttagaga tggtcttgct gtgtcaccca ggctggagtg cagtgatgca
5160gtcgtagtct actacagtct tgaattcctg ggcccaaggg ttcttcccac
ctaagcctcc 5220caagtagctg gaagtacagg cacatactac caagcccagt
tcgttatttt aagtttttgt 5280agagacaggg gtcttactat gttgcccaga
ctggtcttga actcctgtca agtgagcctc 5340ccacctcagc ctcccaaagt
gctatgatta caggtgtgag cctccatgct tggatgagat 5400gttgttttta
atgtttttgg ttttttggtt ttgttgtttt gtttttttga gacagggtct
5460cactctgtgg cccaggctgg agtgcagtgg caccataatg gctcactgca
gcctcgacct 5520cccaaggctc aggtgatcct cttgcctcag ccaccccttc
cccggccacc aagtagctgg 5580gactatgggc ttgtgccacc atgcctggct
aattttttta tttttagtag agatggggtt 5640ttaccacgtt ggccaggcta
gtgtccaact cctgacctcc aaacagctaa tttttgtatt 5700tttaatagag
acaggttttc gccatgttgc ccagcctcgt ctcgaactcc tgggctcaaa
5760tgatccaccc acctcggcct cccaaagtgc tgggattata ggcatgagcc
accacacctg 5820gccttttcag tttttttatt tttatttttt ctttgagacg
gagtctcgct ctgtcgccca 5880ggctggagtg cagtggcgtg atcttggctc
actgaaacct ccatctcctg ggttcaagca 5940gttctcctgc ctcagcctcc
tgagtagctg ggattacagg tgcctgccac catgcctggc 6000taatttttgt
atttttagta gagacagggt tttgccacat tgcccaggct ggtgttgaac
6060tcctgggctc aaatgatcca cctgccttgg cctcccaaag tgctgggatt
acaggcgtga 6120gccactgcac ccggcccttt gtagtgtttt taactaaaga
atttgtagag ttgcccaggc 6180caggaagcct ggtggctcta aagggtaata
gaccttgtca gtaacagata aggagtggta 6240agaggacatt actcatattg
aagatgaaga ccagactttg ctgcttcaca ggccatgcgc 6300tgggttgggc
cacttcagct ccactccatt cgttttcctt tcctaacttg acaatcagct
6360cactcaccct cccttagtgc ctccagtgcc tactcctgtc actccaatgt
caacccattg 6420ggagttgagg cctgtcactc caatgtcaac ccgtgggctg
ttactttgcg tcatatgatg 6480ctgtgagagg ccttgctgga atgtcctagg
aatccctagt agcagtggct attagtcttc 6540tagaaaagaa ctattgctgc
tgccttgtgc acatgcccca ccttctgggc aagtggcagc 6600attgcgctca
tgaggggctt tgcattctta gccaagggca ataaactggg tgggtgatct
6660ggcccaaact tgcccctagg ctctgctagc cctgaatcag caggcttcag
agacgagggt 6720gggtgttata aaagccagtc tgtaaagggt aaattccaaa
tcttgtgcct tgttatacca 6780atccttcctg attcccgttt aaaccaacta
ctctatttct gtgctgccta cattttcaat 6840cctcccacgc attagcaaat
tcctgaaatt tcctcatttg gtaggccttc cataggagtc 6900agctatggac
ttccatagga gttggcagct aaaaccagac tgtgagcttc tgtctccgtt
6960ctgatttttg ctgcacctcc caggggacag tccccacatg attacaaaag
ccaggtgccc 7020tcatcacccg ttacccctga cctgtccact tgttttgaat
aaaccttcat tctccaagca 7080gatccccaaa cttccttgtc cttgttacac
gtctacctca caacctcaca attcaacaac 7140aggtaaatac ctggattcac
tgatttcttt actgtccttc tctgagggtt gggaaggtgg 7200gggctagaaa
aggctcataa tttttaaact cttgggaatt aaacttggga atttctattc
7260ctacagtgtt ttctctggga tttttagctc actgatcgcc ctagaatagt
gtgaactctc 7320cagataggtc ctgctgtgat aggccagggg agtaggctgt
gcagtgacgg cttagggtaa 7380taagtaatgg ttggaggaca ccagcatagg
agcagagttt agaacttaag aggacaagaa 7440agctgctcag tggctggcgt
atagctgtga aggtaaccaa acaccaaaga gggagtctgt 7500catttttata
aggctggaag cgaatgttcc ttttctacct taacatacag tttagggggt
7560tgcaccaaga caaagtttcc aggctggagg gtaatacata tccagcgcga
gtgaagtccc 7620cactccaaca tatacccttt ttgggtgggt aaacacctct
ttgcatgtgg acagaaaatg 7680tttatactta aacagacata ttttgcatat
ttttatctgg agacttcttc tagttttata 7740ctcttcccac atctatgcca
atgccaccat tctaaaactt acactccttt tctacccctg 7800gtcttttcct
tgtccttccc tacaacttgg tagaggtcca ttttgtctta cttcacactt
7860tttttttttt aaataaaaca caaaagtcta cgtcttggtg tcttatccgt
gagtgggaag 7920tggtaagctg gtgatggtcc catatttgct atgacaggaa
cacagaggtg gcagcttagg 7980aagctggggc cacatctcac aaggcaggac
ctggatgcac tgaatccccc tttgctccag 8040caactcagcc agacatttag
gcaggagcac taatgaccct ttccatccac acagagggta 8100tggaacagga
gcccctgttg ttcccttgcc tgtggtctct taagccagtc atacctatcc
8160caacgccgtc tccccctcag gtgtgtagaa gggaagatga atacacagag
tcttttgaat 8220ctctatcaaa tgtggttttt tttattcaac aactgacaag
cacttttcta cagctgcact 8280tgtggaacat cacatggcaa aaacaggagt
tttttcgcta gacttttttt ttctttttaa 8340ccttattaaa aatgagattg
gtcctaaaaa tatagaaaga aataaattta aattcacaaa 8400aaactgtaca
ttatcaaaaa gtcactaaaa caccacattt ggttatataa aaagtccctt
8460ttgctgtaaa aggaacacag aaacttaaaa aaaaaaaaaa aaaa
850445207DNAMus musculus 4ctttctcctc cccctccctt ctcccttccc
tccctctttc cctcccttcc tctctccctc 60tcctttcccc ctcccctcct ccctccctct
ttctccctcc ctttccttca ctctcttctt 120tctctctctc agctccgtaa
cagtaaagaa aaaatgctgc tatcagtgac ttccaggcct 180gggatttcga
cttttggcta taacaagaac aacaagaagc tatatgtggc tcagcaaatg
240gcacctccaa gtccaaggaa tagcaccccc aacagcagcg gcggaggcgg
cggcgggagt 300ggtgggaacg accagctgag caaaaccaac ctgtatatcc
gaggactgca gccaggcacc 360actgaccagg accttgtcaa gctctgtcag
ccgtatggca agattgtctc cactaaggcc 420atcctggaca agaccacaaa
caagtgcaaa ggctatggct ttgtggactt cgacagtccg 480tcatcagcac
agaaagctgt aactgcactg aaagccagtg gtgtgcaggc acaaatggca
540aagcaacagg agcaagaccc tacaaactta tacatctcaa acctccctct
gtcaatggac 600gagcaggaac tggaggggat gctgaagccc tttggccagg
tcatctctac tcggatcctt 660cgagacacta gtggggccag cagaggggtc
ggctttgcaa ggatggagtc aacagagaag 720tgtgaagcca tcatcaccca
ctttaatgga aagtatataa agacaccccc tggagtagca 780gcaccttctg
accctttgct ttgcaaattt gctgacggtg ggccaaagaa acgacagagc
840caaggaagat acgtgcaaaa tggacgggcc tggccaagga acggagacat
gggcggtatg 900gccttgacct atgaccccac tgctgccctt cagaatgggt
tctacgcagc tccttacagt 960atcgcccaca gcaggatgct tgctcaatct
gcgctagccc cgtatcttcc atctcctgtg 1020tcttcctatc agggctcagt
tctgacacca gggatggacc accccctctc tctccagcct 1080gcctccatga
tgggacctct tacccagcaa ctgggtcacc tgtcactcaa cagtctgggc
1140acgttcatgc cggcagctgc tgctatgcat ggggcttaca tctcccagta
cccagctgtg 1200ccttcttcca gtgtttctgc tgaggagagc aatggccagc
agaatcaact ggcagtggag 1260cccccctcag accacggggt ctatcctttc
cagttcagca aataatgagg tccagacctg 1320gggagagaag cctatgcagt
cctccttggg ctccttcctg tggctgatgg aacctgagga 1380atcacgttgg
tttttttttt tgaatgtgct acattcaaga tcaaaaagac ttttttttct
1440tttgcaaaag aagtttcttg ctttgtattt aattgcagcg aacctttctc
ctaccccaaa 1500gacaccagtt gaggtgcttc tcatcttcga gaggtttggg
tatgttgggc ttttaaaatg 1560gtttaagaac aatcgcagtg ctgtgaagtc
agtgaagggg cgctgcttgc caggttcagt 1620tagggactat tttcaatgtg
tgttttagat atgcaggctt aaaacttttg ttttgcaaag 1680ctctcaacac
acactttgca cacacccacc cacctccctc ccacgcactg tttaaggcag
1740ggtctcacag tgcagtcctg gctggctgac aatgtagacc aggctggctt
tgaactcagc 1800gatccttccc actctgtctg agtgcaccgc catgcctagc
tagttgtttt tttttttttg 1860gttttttttt tttcaagaca gggtttctct
gtgtagccct ggctgtcctg gcgctcactt 1920tgtagaccaa gctagcctcg
aacttagaaa tccacctgcc tctgcctccc aagtgctggg 1980attaaaggcg
tgcgccacca ccgcccggct agttgttttt taaaacttct ccacaatggt
2040ctttttttct tttccattta gcttagcctg cctactttgc ctttttacaa
tttttaattt 2100tatgaatttg atttaaattt catgtggaat tgggggaagg
gagagcaacg cacaggtaga 2160gaagtgtggc agtagaggtt gctttccctc
tgccctcctg gcctaggagg ggttagactt 2220tgacttcagg aagcagacat
gtgacaacca tgcagccttt ggaaaccacc ttcaaccaac 2280tctggtggca
agtgccactg agaagtggca tccagcttca gcatcctgga tacctgctct
2340gcctcctgga gggtcttggg tcttcatcct gccccaggtc tcacagcccc
tgcccctgct 2400gagcaagttc tgccttccac tttaccagga ggttgttttg
tccccgagag gctgctgtct 2460tgtccatcat catccttgat taccaagcct
ggggagtatc atgtgtgctg gtcctgaaca 2520cctgacagaa cctggcccca
ctcctccacc ccttattccc tggcagttat ttggtgtaga 2580ctaaacccca
gtccagcctt ggacatggag cctgttagtt catgtacttg gccagcagtg
2640acatctgtga ctttgcctcc tggaggatct tttctgcctg ggttctcaca
gcctctcatc 2700ccagctgcac atggctgtac tcaagtggta cttggctgaa
tcacttaatt cagcaggagc 2760catcttattt agcagggctc acccttggtt
gtatgtacat gccaagtgca cacagtttga 2820agggatgaaa gcaagaactg
ttgtgtatac tgtgcatgct tgagaatgga ctgagctttc 2880tctatgcagc
atacccagcc cagccctggt gtttggaccc cttccccagg gaaccagacc
2940accagaaata attttccttc ttgtggaagg acgacggggt gctaggatag
aaaaaggcaa 3000gactcacagc tccctggcca ctcaaaggtc aggagggact
tgtaaacatg cctgccgtta 3060actattccct tctgcaagac tttcagcaca
aaatgtgact tgttacatca catcccttca 3120cgtcataagt acacctccag
tagacaaagc aatggagatg gggaagtgtg tccagaggtc 3180agtgtgaaag
aggaagggca gggcatggtg atggtgagaa actggctcag cagctgtgca
3240cactacagaa ttgggaatcc tggcctgact gtgctgctgt gcgttctaga
ctgaggaagg 3300ggctgaggac aatggacagc ataaagtaca aaatgaccag
ttttctcatt ggtgcttcca 3360acatcaggaa ggtcataccc agttatcgac
ctctagccct ctagcactca gtgacagact 3420cagagctctg tcctgtggaa
accctgctga cagtcttgct gccactgctg ctccttcact 3480gcctattaaa
aattgttcct ttttctgtag
ttctaaagca atttgaataa ctgacagttt 3540taaaatttga aattaccttt
ttaggttttt cttttgagta tactggttag ggttctagtg 3600tttctggttg
aacaggtgtg agcatgggaa atgagggcaa cctgtaattt tgtttttata
3660gtggttaatg gcactcactg tgtaatttct gcttagtgat ttccatccac
tcttttcctc 3720attagctcat tactcagaaa ggtagcgact gaagacagga
agtagtatgc aagccatgag 3780ggatgagttg taagcctcct ctccatcatc
ctgcccaagg ctcctaacag tctcctgagt 3840cctgtttgca tggaggccag
atagatggct ctgcactcta tatctcacct gtactcattt 3900tagtacattg
tggttttgct tccaatcaat cagagtggac tagagctcac acctgacctc
3960gtgtgactct tcactaggct ccaggcctat cccatctcat tgggtcagac
tgtagatgga 4020ctggactttt ggagacaggt cctgctatta cttaggaata
tgtgggaaca atgataggga 4080ccaagcttgt gaagcaaaac ggctttggaa
agggcaagtg ctcaataata aaaaatgggg 4140tggaagtgaa gcgttatttg
ctgagggttt cggaaggact agtttgtttt tgtccctggc 4200ccttccggtc
aagtgggcac ctcccatttt gtcgccaaga gcaatacagt tgagtaggtg
4260gtagacccaa actcacccag ccatttctag acctgaattc agaggataag
gtgagtctag 4320aggtatatac aaccaatgct tgttagacca attcttacca
attcggagca agccggtggc 4380tctacttctg ggtaacctgc ctcttaaaag
tgcctttgat cccagtagta gattcagggt 4440ctacaggagc cttccctcag
cagctaaaac caatttgtct cctctactct ttgctgcacc 4500tcccggaggt
ggtctccatg tggcagtgat gccctcatca cccttgtgac caccgctctg
4560aatgttcaaa ataagcattt cctgtccaag caaatcccta aactccctcg
tcctcattac 4620tcaactacct cagtctgaca actcaaggca cacaagcacc
tacttgcctc ccaccatcct 4680ttactgccct tgtctttgtg gggttggaac
aaatgcaaac tcctcagttc cacaacccaa 4740aactgctctt tctggatcgt
cttctccagt gacgttggct ccctacaaga gcgaactctc 4800catatgggtc
agtccctgta ggacaagcca gggactatgc tgtgcagtca aatgccaggt
4860ttggagcata gcttaagacc gcagagagta aggacttgag tggtggtggt
gtgcagtttg 4920agagagctgg aatggacgtc tctgctcctg ccctctgggc
gggtcgggtg atgacttcta 4980gatgcactcc ttaccctgga gggaagttaa
tttatactta agatgcattt tgtacatttc 5040ccctttaccc agaaacctgt
agtttcatac tcccatgtct atgccaacac tacctatcgc 5100tcactctccg
tggctgactt ctggttccat ttccttcctg tgacttggaa gagctttact
5160tttgctcacg tgaacctttc ttttttaaat aaaacacaaa agcctcc
52075383PRTMus musculus 5Met Leu Leu Ser Val Thr Ser Arg Pro Gly
Ile Ser Thr Phe Gly Tyr1 5 10 15Asn Lys Asn Asn Lys Lys Leu Tyr Val
Ala Gln Gln Met Ala Pro Pro 20 25 30Ser Pro Arg Asn Ser Thr Pro Asn
Ser Ser Gly Gly Gly Gly Gly Gly 35 40 45Ser Gly Gly Asn Asp Gln Leu
Ser Lys Thr Asn Leu Tyr Ile Arg Gly 50 55 60Leu Gln Pro Gly Thr Thr
Asp Gln Asp Leu Val Lys Leu Cys Gln Pro65 70 75 80Tyr Gly Lys Ile
Val Ser Thr Lys Ala Ile Leu Asp Lys Thr Thr Asn 85 90 95Lys Cys Lys
Gly Tyr Gly Phe Val Asp Phe Asp Ser Pro Ser Ser Ala 100 105 110Gln
Lys Ala Val Thr Ala Leu Lys Ala Ser Gly Val Gln Ala Gln Met 115 120
125Ala Lys Gln Gln Glu Gln Asp Pro Thr Asn Leu Tyr Ile Ser Asn Leu
130 135 140Pro Leu Ser Met Asp Glu Gln Glu Leu Glu Gly Met Leu Lys
Pro Phe145 150 155 160Gly Gln Val Ile Ser Thr Arg Ile Leu Arg Asp
Thr Ser Gly Ala Ser 165 170 175Arg Gly Val Gly Phe Ala Arg Met Glu
Ser Thr Glu Lys Cys Glu Ala 180 185 190Ile Ile Thr His Phe Asn Gly
Lys Tyr Ile Lys Thr Pro Pro Gly Val 195 200 205Ala Ala Pro Ser Asp
Pro Leu Leu Cys Lys Phe Ala Asp Gly Gly Pro 210 215 220Lys Lys Arg
Gln Ser Gln Gly Arg Tyr Val Gln Asn Gly Arg Ala Trp225 230 235
240Pro Arg Asn Gly Asp Met Gly Gly Met Ala Leu Thr Tyr Asp Pro Thr
245 250 255Ala Ala Leu Gln Asn Gly Phe Tyr Ala Ala Pro Tyr Ser Ile
Ala His 260 265 270Ser Arg Met Leu Ala Gln Ser Ala Leu Ala Pro Tyr
Leu Pro Ser Pro 275 280 285Val Ser Ser Tyr Gln Gly Ser Val Leu Thr
Pro Gly Met Asp His Pro 290 295 300Leu Ser Leu Gln Pro Ala Ser Met
Met Gly Pro Leu Thr Gln Gln Leu305 310 315 320Gly His Leu Ser Leu
Asn Ser Leu Gly Thr Phe Met Pro Ala Ala Ala 325 330 335Ala Met His
Gly Ala Tyr Ile Ser Gln Tyr Pro Ala Val Pro Ser Ser 340 345 350Ser
Val Ser Ala Glu Glu Ser Asn Gly Gln Gln Asn Gln Leu Ala Val 355 360
365Glu Pro Pro Ser Asp His Gly Val Tyr Pro Phe Gln Phe Ser Lys 370
375 3806355PRTMus musculus 6Met Ala Pro Pro Ser Pro Arg Asn Ser Thr
Pro Asn Ser Ser Gly Gly1 5 10 15Gly Gly Gly Gly Ser Gly Gly Asn Asp
Gln Leu Ser Lys Thr Asn Leu 20 25 30Tyr Ile Arg Gly Leu Gln Pro Gly
Thr Thr Asp Gln Asp Leu Val Lys 35 40 45Leu Cys Gln Pro Tyr Gly Lys
Ile Val Ser Thr Lys Ala Ile Leu Asp 50 55 60Lys Thr Thr Asn Lys Cys
Lys Gly Tyr Gly Phe Val Asp Phe Asp Ser65 70 75 80Pro Ser Ser Ala
Gln Lys Ala Val Thr Ala Leu Lys Ala Ser Gly Val 85 90 95Gln Ala Gln
Met Ala Lys Gln Gln Glu Gln Asp Pro Thr Asn Leu Tyr 100 105 110Ile
Ser Asn Leu Pro Leu Ser Met Asp Glu Gln Glu Leu Glu Gly Met 115 120
125Leu Lys Pro Phe Gly Gln Val Ile Ser Thr Arg Ile Leu Arg Asp Thr
130 135 140Ser Gly Ala Ser Arg Gly Val Gly Phe Ala Arg Met Glu Ser
Thr Glu145 150 155 160Lys Cys Glu Ala Ile Ile Thr His Phe Asn Gly
Lys Tyr Ile Lys Thr 165 170 175Pro Pro Gly Val Ala Ala Pro Ser Asp
Pro Leu Leu Cys Lys Phe Ala 180 185 190Asp Gly Gly Pro Lys Lys Arg
Gln Ser Gln Gly Arg Tyr Val Gln Asn 195 200 205Gly Arg Ala Trp Pro
Arg Asn Gly Asp Met Gly Gly Met Ala Leu Thr 210 215 220Tyr Asp Pro
Thr Ala Ala Leu Gln Asn Gly Phe Tyr Ala Ala Pro Tyr225 230 235
240Ser Ile Ala His Ser Arg Met Leu Ala Gln Ser Ala Leu Ala Pro Tyr
245 250 255Leu Pro Ser Pro Val Ser Ser Tyr Gln Gly Ser Val Leu Thr
Pro Gly 260 265 270Met Asp His Pro Leu Ser Leu Gln Pro Ala Ser Met
Met Gly Pro Leu 275 280 285Thr Gln Gln Leu Gly His Leu Ser Leu Asn
Ser Leu Gly Thr Phe Met 290 295 300Pro Ala Ala Ala Ala Met His Gly
Ala Tyr Ile Ser Gln Tyr Pro Ala305 310 315 320Val Pro Ser Ser Ser
Val Ser Ala Glu Glu Ser Asn Gly Gln Gln Asn 325 330 335Gln Leu Ala
Val Glu Pro Pro Ser Asp His Gly Val Tyr Pro Phe Gln 340 345 350Phe
Ser Lys 35575390DNAMus musculus 7acttcctgtc ctgttggaaa tgaaagttag
gaattggggg tggtagccgt gcacgtattt 60attctgcagc atgccggcac tgtctgtttc
gttatcacag caaagcagcc gcaggaaaaa 120cagagagcag agctttgttt
tagagagagt ctgggcttga ggtctgttgt gacaaagacg 180gagttaattt
acttctggag aatgtgtgtg gcagcagtgg gaggcctgag gaggggctct
240gaaggcattc agtctccctc cctgttctag gtggatggat aggcttcttt
ggtatcactt 300gtaagttttc ttttcctgtt ccaactcctc cttggagggc
atattggagc tcagagagga 360ctctgtgaac gtcatttggc ttggccataa
attccacacc tgctatatgt ggctcagcaa 420atggcacctc caagtccaag
gaatagcacc cccaacagca gcggcggagg cggcggcggg 480agtggtggga
acgaccagct gagcaaaacc aacctgtata tccgaggact gcagccaggc
540accactgacc aggaccttgt caagctctgt cagccgtatg gcaagattgt
ctccactaag 600gccatcctgg acaagaccac aaacaagtgc aaaggctatg
gctttgtgga cttcgacagt 660ccgtcatcag cacagaaagc tgtaactgca
ctgaaagcca gtggtgtgca ggcacaaatg 720gcaaagcaac aggagcaaga
ccctacaaac ttatacatct caaacctccc tctgtcaatg 780gacgagcagg
aactggaggg gatgctgaag ccctttggcc aggtcatctc tactcggatc
840cttcgagaca ctagtggggc cagcagaggg gtcggctttg caaggatgga
gtcaacagag 900aagtgtgaag ccatcatcac ccactttaat ggaaagtata
taaagacacc ccctggagta 960gcagcacctt ctgacccttt gctttgcaaa
tttgctgacg gtgggccaaa gaaacgacag 1020agccaaggaa gatacgtgca
aaatggacgg gcctggccaa ggaacggaga catgggcggt 1080atggccttga
cctatgaccc cactgctgcc cttcagaatg ggttctacgc agctccttac
1140agtatcgccc acagcaggat gcttgctcaa tctgcgctag ccccgtatct
tccatctcct 1200gtgtcttcct atcagggctc agttctgaca ccagggatgg
accaccccct ctctctccag 1260cctgcctcca tgatgggacc tcttacccag
caactgggtc acctgtcact caacagtctg 1320ggcacgttca tgccggcagc
tgctgctatg catggggctt acatctccca gtacccagct 1380gtgccttctt
ccagtgtttc tgctgaggag agcaatggcc agcagaatca actggcagtg
1440gagcccccct cagaccacgg ggtctatcct ttccagttca gcaaataatg
aggtccagac 1500ctggggagag aagcctatgc agtcctcctt gggctccttc
ctgtggctga tggaacctga 1560ggaatcacgt tggttttttt ttttgaatgt
gctacattca agatcaaaaa gacttttttt 1620tcttttgcaa aagaagtttc
ttgctttgta tttaattgca gcgaaccttt ctcctacccc 1680aaagacacca
gttgaggtgc ttctcatctt cgagaggttt gggtatgttg ggcttttaaa
1740atggtttaag aacaatcgca gtgctgtgaa gtcagtgaag gggcgctgct
tgccaggttc 1800agttagggac tattttcaat gtgtgtttta gatatgcagg
cttaaaactt ttgttttgca 1860aagctctcaa cacacacttt gcacacaccc
acccacctcc ctcccacgca ctgtttaagg 1920cagggtctca cagtgcagtc
ctggctggct gacaatgtag accaggctgg ctttgaactc 1980agcgatcctt
cccactctgt ctgagtgcac cgccatgcct agctagttgt tttttttttt
2040ttggtttttt ttttttcaag acagggtttc tctgtgtagc cctggctgtc
ctggcgctca 2100ctttgtagac caagctagcc tcgaacttag aaatccacct
gcctctgcct cccaagtgct 2160gggattaaag gcgtgcgcca ccaccgcccg
gctagttgtt ttttaaaact tctccacaat 2220ggtctttttt tcttttccat
ttagcttagc ctgcctactt tgccttttta caatttttaa 2280ttttatgaat
ttgatttaaa tttcatgtgg aattggggga agggagagca acgcacaggt
2340agagaagtgt ggcagtagag gttgctttcc ctctgccctc ctggcctagg
aggggttaga 2400ctttgacttc aggaagcaga catgtgacaa ccatgcagcc
tttggaaacc accttcaacc 2460aactctggtg gcaagtgcca ctgagaagtg
gcatccagct tcagcatcct ggatacctgc 2520tctgcctcct ggagggtctt
gggtcttcat cctgccccag gtctcacagc ccctgcccct 2580gctgagcaag
ttctgccttc cactttacca ggaggttgtt ttgtccccga gaggctgctg
2640tcttgtccat catcatcctt gattaccaag cctggggagt atcatgtgtg
ctggtcctga 2700acacctgaca gaacctggcc ccactcctcc accccttatt
ccctggcagt tatttggtgt 2760agactaaacc ccagtccagc cttggacatg
gagcctgtta gttcatgtac ttggccagca 2820gtgacatctg tgactttgcc
tcctggagga tcttttctgc ctgggttctc acagcctctc 2880atcccagctg
cacatggctg tactcaagtg gtacttggct gaatcactta attcagcagg
2940agccatctta tttagcaggg ctcacccttg gttgtatgta catgccaagt
gcacacagtt 3000tgaagggatg aaagcaagaa ctgttgtgta tactgtgcat
gcttgagaat ggactgagct 3060ttctctatgc agcataccca gcccagccct
ggtgtttgga ccccttcccc agggaaccag 3120accaccagaa ataattttcc
ttcttgtgga aggacgacgg ggtgctagga tagaaaaagg 3180caagactcac
agctccctgg ccactcaaag gtcaggaggg acttgtaaac atgcctgccg
3240ttaactattc ccttctgcaa gactttcagc acaaaatgtg acttgttaca
tcacatccct 3300tcacgtcata agtacacctc cagtagacaa agcaatggag
atggggaagt gtgtccagag 3360gtcagtgtga aagaggaagg gcagggcatg
gtgatggtga gaaactggct cagcagctgt 3420gcacactaca gaattgggaa
tcctggcctg actgtgctgc tgtgcgttct agactgagga 3480aggggctgag
gacaatggac agcataaagt acaaaatgac cagttttctc attggtgctt
3540ccaacatcag gaaggtcata cccagttatc gacctctagc cctctagcac
tcagtgacag 3600actcagagct ctgtcctgtg gaaaccctgc tgacagtctt
gctgccactg ctgctccttc 3660actgcctatt aaaaattgtt cctttttctg
tagttctaaa gcaatttgaa taactgacag 3720ttttaaaatt tgaaattacc
tttttaggtt tttcttttga gtatactggt tagggttcta 3780gtgtttctgg
ttgaacaggt gtgagcatgg gaaatgaggg caacctgtaa ttttgttttt
3840atagtggtta atggcactca ctgtgtaatt tctgcttagt gatttccatc
cactcttttc 3900ctcattagct cattactcag aaaggtagcg actgaagaca
ggaagtagta tgcaagccat 3960gagggatgag ttgtaagcct cctctccatc
atcctgccca aggctcctaa cagtctcctg 4020agtcctgttt gcatggaggc
cagatagatg gctctgcact ctatatctca cctgtactca 4080ttttagtaca
ttgtggtttt gcttccaatc aatcagagtg gactagagct cacacctgac
4140ctcgtgtgac tcttcactag gctccaggcc tatcccatct cattgggtca
gactgtagat 4200ggactggact tttggagaca ggtcctgcta ttacttagga
atatgtggga acaatgatag 4260ggaccaagct tgtgaagcaa aacggctttg
gaaagggcaa gtgctcaata ataaaaaatg 4320gggtggaagt gaagcgttat
ttgctgaggg tttcggaagg actagtttgt ttttgtccct 4380ggcccttccg
gtcaagtggg cacctcccat tttgtcgcca agagcaatac agttgagtag
4440gtggtagacc caaactcacc cagccatttc tagacctgaa ttcagaggat
aaggtgagtc 4500tagaggtata tacaaccaat gcttgttaga ccaattctta
ccaattcgga gcaagccggt 4560ggctctactt ctgggtaacc tgcctcttaa
aagtgccttt gatcccagta gtagattcag 4620ggtctacagg agccttccct
cagcagctaa aaccaatttg tctcctctac tctttgctgc 4680acctcccgga
ggtggtctcc atgtggcagt gatgccctca tcacccttgt gaccaccgct
4740ctgaatgttc aaaataagca tttcctgtcc aagcaaatcc ctaaactccc
tcgtcctcat 4800tactcaacta cctcagtctg acaactcaag gcacacaagc
acctacttgc ctcccaccat 4860cctttactgc ccttgtcttt gtggggttgg
aacaaatgca aactcctcag ttccacaacc 4920caaaactgct ctttctggat
cgtcttctcc agtgacgttg gctccctaca agagcgaact 4980ctccatatgg
gtcagtccct gtaggacaag ccagggacta tgctgtgcag tcaaatgcca
5040ggtttggagc atagcttaag accgcagaga gtaaggactt gagtggtggt
ggtgtgcagt 5100ttgagagagc tggaatggac gtctctgctc ctgccctctg
ggcgggtcgg gtgatgactt 5160ctagatgcac tccttaccct ggagggaagt
taatttatac ttaagatgca ttttgtacat 5220ttccccttta cccagaaacc
tgtagtttca tactcccatg tctatgccaa cactacctat 5280cgctcactct
ccgtggctga cttctggttc catttccttc ctgtgacttg gaagagcttt
5340acttttgctc acgtgaacct ttctttttta aataaaacac aaaagcctcc
5390821DNAArtificial SequenceHuman RBMS2-1 siRNA 8uuugcacaaa
uuuuccuugg t 21921DNAArtificial SequenceHuman RBMS2-2 siRNA
9ugcucaucca uugacagugg g 211021DNAArtificial SequenceMouse RBMS2-1
siRNA 10uaaggagcug cguagaaccc a 211121DNAArtificial SequenceMouse
RBMS2-2 siRNA 11agauguauaa guuuguaggg t 211220DNAArtificial
SequenceHumanRBMS2 Forward primer 12catctctccc tcagcagcac
201320DNAArtificial SequenceHumanRBMS2 Reverse primer 13gctgctctcc
tcgactgaaa 201420DNAArtificial SequenceHuman IL-6 Forward primer
14ctccaggagc ccagctatga 201520DNAArtificial SequenceHuman IL-6
Reverse primer 15gaggtgagtg gctgtctgtg 201620DNAArtificial
SequenceLuciferase Forward primer 16gaaatgtccg ttcggttggc
201720DNAArtificial SequenceLuciferase Reverse primer 17tccgataaat
aacgcgccca 201820DNAArtificial SequenceHuman IL1B Forward primer
18cgaggcacaa ggcacaacag 201920DNAArtificial SequenceHuman IL1B
Reverse primer 19agaggtccag gtcctggaag 202020DNAArtificial
SequenceHuman PTGS2 Forward primer 20gttccacccg cagtacagaa
202120DNAArtificial SequenceHuman PTGS2 Reverse primer 21agggcttcag
cataaagcgt 202220DNAArtificial SequenceHuman IL8 Forward primer
22accggaagga accatctcac 202321DNAArtificial SequenceHuman IL8
Reverse primer 23ggcaaaactg caccttcaca c 212420DNAArtificial
SequenceHuman NFKBIA Forward primer 24gggccagctg acactagaaa
202520DNAArtificial SequenceHuman NFKBIA Reverse primer
25gtcatcatag ggcagctcgt 202620DNAArtificial SequenceHuman ZC3H12A
Forward primer 26gtctgacggg atcgtggttt 202720DNAArtificial
SequenceHuman ZC3H12A Reverse primer 27cagggggcat aaacttgtca
202821DNAArtificial SequenceMouse RBMS2 Forward primer 28tgctgctatc
agtgacttcc a 212922DNAArtificial SequenceMouse RBMS2 Reverse primer
29tgctgagcca catatagctt ct 223020DNAArtificial SequenceMouse IL6
Forward primer 30aggcataacg cactaggttt 203120DNAArtificial
SequenceMouse IL6 Reverse primer 31agctggagtc acagaaggag
203225DNAArtificial SequenceMouse IL1b Forward primer 32caaccaacaa
gtgatattct ccatg 253321DNAArtificial SequenceMouse IL1b Reverse
primer 33gatccacact ctccagctgc a 213420DNAArtificial SequenceMouse
TNFa Forward primer 34atggcctccc tctcatcagt 203520DNAArtificial
SequenceMouse TNFa Reverse primer 35cttggtggtt tgctacgacg
20361240DNAArtificial SequenceIL-6 promoter 36cctactttca gcctggaatc
attctgaatg ctagctagat atctggagac aggtggacag 60aaaaccagga actagtctga
aaaagaaact aaccaaaggg aagaagtctg tttaagtttg 120acccagccta
gaagacttga
gcattggagg ggttattcag agtgagacgt accaccttca 180gattcaaatc
ctgtcatcca gtagaaggga gcttcaaaca caagctagct aagatacaat
240gaggtccttc ttcgatatct ttatcttcca tataccatga atcaaagaaa
cttcaacaac 300atgaggactg caacagacct tcaagcctcc ttgcatgacc
tggaaatgtt ttggggtgtc 360ctggcagcag tgggatcagc actaacagat
aagggcaact ctcacagaga ctaaaggtct 420taactaagaa gatagccaag
agaccactgg ggagaatgca gagaataggc ttggacttgg 480aagccaagat
tgcttgacaa cagacagaag atatttctgt acttcaccca ctttacccac
540ctggcaactc ctggaaacaa ctgcacaaaa tttggaggtg aacaaaccat
tagaaacaac 600tggtcctgac aagacacagg aaaaacaagc aatatgcaac
attactgtct gttgtccagg 660ttgggtgctg ggggtgggag agggagtgtg
tgtctttgta tgatctgaaa aaactcaggt 720cagaacatct gtagatcctt
acagacatac aaaagaatcc tagcctctta ttcatgtgtg 780tgtgtgtgtg
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tatgtgtgtg tcgtctgtca
840tgcgcgcgtg cctgcgttta aataacatca gctttagctt ctctttctcc
ttataaaaca 900ttgtgaattt cagttttctt tcccatcaag acatgctcaa
gtgctgagtc acttttaaag 960aaaaaaaaga agagtgctca tgcttcttag
ggctagcctc aaggatgact taagcacact 1020ttccccttcc tagttgtgat
tctttcgatg ctaaacgacg tcacattgtg caatcttaat 1080aaggtttcca
atcagcccca cccactctgg ccccaccccc accctccaac aaagattttt
1140atcaaatgtg ggattttccc atgagtctca aaattagaga gttgactcct
aataaatatg 1200agactgggga tgtctgtagc tcattctgct ctggagccca
124037171DNAArtificial SequenceIL-6 3'UTR 97-267 37ctaaaagtat
gagcgttagg acactatttt aattattttt aatttattaa tatttaaata 60tgtgaagctg
agttaattta tgtaagtcat atttatattt ttaagaagta ccacttgaaa
120cattttatgt attagttttg aaataataat ggaaagtggc tatgcagttt g
1713872DNAArtificial SequenceIL-6 3'UTR 122-193 38tattttaatt
atttttaatt tattaatatt taaatatgtg aagctgagtt aatttatgta 60agtcatattt
at 723939DNAArtificial SequenceIL-6 3'UTR delta ARE1 39tattttaatt
atttttaatt tattaatatt taaatatgt 3940101DNAArtificial SequenceIL-6
3'UTR delta ARE2 40agttaattta tgtaagtcat atttatattt ttaagaagta
ccacttgaaa cattttatgt 60attagttttg aaataataat ggaaagtggc tatgcagttt
g 1014171DNAArtificial SequenceIL-6 3'UTR ARE mutant 41tattttaatt
atttttaagg gattaatagg gaaatatgtg aagctgagtt aagggatgta 60agtcataggg
a 7142357DNAArtificial SequenceIL-8 3'UTR wild type 42ggatccacaa
gtccttgttc cactgtgcct tggtttctcc tttatttcta agtggaaaaa 60gtattagcca
ccatcttacc tcacagtgat gttgtgagga catgtggaag cactttaagt
120tttttcatca taacataaat tattttcaag tgtaacttat taacctattt
attatttatg 180tatttattta agcatcaaat atttgtgcaa gaatttggaa
aaatagaaga tgaatcattg 240attgaatagt tataaagatg ttatagtaaa
tttattttat tttagatatt aaatgatgtt 300ttattagata aatttcaatc
agggttttta gattaaacaa acaaacaatt gggtacc 35743357DNAArtificial
SequenceIL-8 3'UTR mutant 43ggatccacaa gtccttgttc cactgtgcct
tggtttctcc tttatttcta agtggaaaaa 60gtattagcca ccatcttacc tcacagtgat
gttgtgagga catgtggaag cactttaagt 120tttttcatca taacataaat
tattttcaag tgtaacttat taacctaggg attagggatg 180tagggaggga
agcatcaaat atttgtgcaa gaatttggaa aaatagaaga tgaatcattg
240attgaatagt tataaagatg ttatagtaaa tttattttat tttagatatt
aaatgatgtt 300ttattagata aatttcaatc agggttttta gattaaacaa
acaaacaatt gggtacc 35744584DNAArtificial SequenceIL1B 3'UTR wild
type 44agagagctgt acccagagag tcctgtgctg aatgtggact caatccctag
ggctggcaga 60aagggaacag aaaggttttt gagtacggct atagcctgga ctttcctgtt
gtctacacca 120atgcccaact gcctgcctta gggtagtgct aagaggatct
cctgtccatc agccaggaca 180gtcagctctc tcctttcagg gccaatcccc
agcccttttg ttgagccagg cctctctcac 240ctctcctact cacttaaagc
ccgcctgaca gaaaccacgg ccacatttgg ttctaagaaa 300ccctctgtca
ttcgctccca cattctgatg agcaaccgct tccctattta tttatttatt
360tgtttgtttg ttttattcat tggtctaatt tattcaaagg gggcaagaag
tagcagtgtc 420tgtaaaagag cctagttttt aatagctatg gaatcaattc
aatttggact ggtgtgctct 480ctttaaatca agtcctttaa ttaagactga
aaatatataa gctcagatta tttaaatggg 540aatatttata aatgagcaaa
tatcatactg ttcaatggtt ctga 58445584DNAArtificial SequenceIL1B 3'UTR
mutant 45agagagctgt acccagagag tcctgtgctg aatgtggact caatccctag
ggctggcaga 60aagggaacag aaaggttttt gagtacggct atagcctgga ctttcctgtt
gtctacacca 120atgcccaact gcctgcctta gggtagtgct aagaggatct
cctgtccatc agccaggaca 180gtcagctctc tcctttcagg gccaatcccc
agcccttttg ttgagccagg cctctctcac 240ctctcctact cacttaaagc
ccgcctgaca gaaaccacgg ccacatttgg ttctaagaaa 300ccctctgtca
ttcgctccca cattctgatg agcaaccgct tccctattta tttatttatt
360tgtttgtttg ttttattcat tggtctaatt tattcaaagg gggcaagaag
tagcagtgtc 420tgtaaaagag cctagttttt aatagctatg gaatcaattc
aatttggact ggtgtgctct 480ctttaaatca agtcctttaa ttaagactga
aaatatataa gctcagatta tttaaatggg 540aatatttata aatgagcaaa
tatcatactg ttcaatggtt ctga 58446304PRTArtificial Sequence3xFLAG-
RBMS2 delta RRM1 domain 46Met Asp Tyr Lys Asp His Asp Gly Asp Tyr
Lys Asp His Asp Ile Asp1 5 10 15Tyr Lys Asp Asp Asp Asp Lys Lys Leu
Leu Glu Glu Gln Asp Pro Thr 20 25 30Asn Leu Tyr Ile Ser Asn Leu Pro
Leu Ser Met Asp Glu Gln Glu Leu 35 40 45Glu Gly Met Leu Lys Pro Phe
Gly Gln Val Ile Ser Thr Arg Ile Leu 50 55 60Arg Asp Thr Ser Gly Thr
Ser Arg Gly Val Gly Phe Ala Arg Met Glu65 70 75 80Ser Thr Glu Lys
Cys Glu Ala Ile Ile Thr His Phe Asn Gly Lys Tyr 85 90 95Ile Lys Thr
Pro Pro Gly Val Pro Ala Pro Ser Asp Pro Leu Leu Cys 100 105 110Lys
Phe Ala Asp Gly Gly Pro Lys Lys Arg Gln Asn Gln Gly Lys Phe 115 120
125Val Gln Asn Gly Arg Ala Trp Pro Arg Asn Ala Asp Met Gly Val Met
130 135 140Ala Leu Thr Tyr Asp Pro Thr Thr Ala Leu Gln Asn Gly Phe
Tyr Pro145 150 155 160Ala Pro Tyr Asn Ile Thr Pro Asn Arg Met Leu
Ala Gln Ser Ala Leu 165 170 175Ser Pro Tyr Leu Ser Ser Pro Val Ser
Ser Tyr Gln Arg Val Thr Gln 180 185 190Thr Ser Pro Leu Gln Val Pro
Asn Pro Ser Trp Met His His His Ser 195 200 205Tyr Leu Met Gln Pro
Ser Gly Ser Val Leu Thr Pro Gly Met Asp His 210 215 220Pro Ile Ser
Leu Gln Pro Ala Ser Met Met Gly Pro Leu Thr Gln Gln225 230 235
240Leu Gly His Leu Ser Leu Ser Ser Thr Gly Thr Tyr Met Pro Thr Ala
245 250 255Ala Ala Met Gln Gly Ala Tyr Ile Ser Gln Tyr Thr Pro Val
Pro Ser 260 265 270Ser Ser Val Ser Val Glu Glu Ser Ser Gly Gln Gln
Asn Gln Val Ala 275 280 285Val Asp Ala Pro Ser Glu His Gly Val Tyr
Ser Phe Gln Phe Asn Lys 290 295 30047321PRTArtificial
Sequence3xFLAG- RBMS2 delta RRM2 domain 47Met Asp Tyr Lys Asp His
Asp Gly Asp Tyr Lys Asp His Asp Ile Asp1 5 10 15Tyr Lys Asp Asp Asp
Asp Lys Lys Leu Ala Pro Pro Ser Pro Ser Asn 20 25 30Ser Thr Pro Asn
Ser Ser Ser Gly Ser Asn Gly Asn Asp Gln Leu Ser 35 40 45Lys Thr Asn
Leu Tyr Ile Arg Gly Leu Gln Pro Gly Thr Thr Asp Gln 50 55 60Asp Leu
Val Lys Leu Cys Gln Pro Tyr Gly Lys Ile Val Ser Thr Lys65 70 75
80Ala Ile Leu Asp Lys Thr Thr Asn Lys Cys Lys Gly Tyr Gly Phe Val
85 90 95Asp Phe Asp Ser Pro Ser Ala Ala Gln Lys Ala Val Thr Ala Leu
Lys 100 105 110Ala Ser Gly Val Gln Ala Gln Met Ala Lys Gln Gln Glu
Gln Asp Pro 115 120 125Cys Lys Phe Ala Asp Gly Gly Pro Lys Lys Arg
Gln Asn Gln Gly Lys 130 135 140Phe Val Gln Asn Gly Arg Ala Trp Pro
Arg Asn Ala Asp Met Gly Val145 150 155 160Met Ala Leu Thr Tyr Asp
Pro Thr Thr Ala Leu Gln Asn Gly Phe Tyr 165 170 175Pro Ala Pro Tyr
Asn Ile Thr Pro Asn Arg Met Leu Ala Gln Ser Ala 180 185 190Leu Ser
Pro Tyr Leu Ser Ser Pro Val Ser Ser Tyr Gln Arg Val Thr 195 200
205Gln Thr Ser Pro Leu Gln Val Pro Asn Pro Ser Trp Met His His His
210 215 220Ser Tyr Leu Met Gln Pro Ser Gly Ser Val Leu Thr Pro Gly
Met Asp225 230 235 240His Pro Ile Ser Leu Gln Pro Ala Ser Met Met
Gly Pro Leu Thr Gln 245 250 255Gln Leu Gly His Leu Ser Leu Ser Ser
Thr Gly Thr Tyr Met Pro Thr 260 265 270Ala Ala Ala Met Gln Gly Ala
Tyr Ile Ser Gln Tyr Thr Pro Val Pro 275 280 285Ser Ser Ser Val Ser
Val Glu Glu Ser Ser Gly Gln Gln Asn Gln Val 290 295 300Ala Val Asp
Ala Pro Ser Glu His Gly Val Tyr Ser Phe Gln Phe Asn305 310 315
320Lys48216PRTArtificial Sequence3xFLAG- RBMS2 delta RRM1/2 domain
48Met Asp Tyr Lys Asp His Asp Gly Asp Tyr Lys Asp His Asp Ile Asp1
5 10 15Tyr Lys Asp Asp Asp Asp Lys Lys Leu Leu Glu Asp Gly Gly Pro
Lys 20 25 30Lys Arg Gln Asn Gln Gly Lys Phe Val Gln Asn Gly Arg Ala
Trp Pro 35 40 45Arg Asn Ala Asp Met Gly Val Met Ala Leu Thr Tyr Asp
Pro Thr Thr 50 55 60Ala Leu Gln Asn Gly Phe Tyr Pro Ala Pro Tyr Asn
Ile Thr Pro Asn65 70 75 80Arg Met Leu Ala Gln Ser Ala Leu Ser Pro
Tyr Leu Ser Ser Pro Val 85 90 95Ser Ser Tyr Gln Arg Val Thr Gln Thr
Ser Pro Leu Gln Val Pro Asn 100 105 110Pro Ser Trp Met His His His
Ser Tyr Leu Met Gln Pro Ser Gly Ser 115 120 125Val Leu Thr Pro Gly
Met Asp His Pro Ile Ser Leu Gln Pro Ala Ser 130 135 140Met Met Gly
Pro Leu Thr Gln Gln Leu Gly His Leu Ser Leu Ser Ser145 150 155
160Thr Gly Thr Tyr Met Pro Thr Ala Ala Ala Met Gln Gly Ala Tyr Ile
165 170 175Ser Gln Tyr Thr Pro Val Pro Ser Ser Ser Val Ser Val Glu
Glu Ser 180 185 190Ser Gly Gln Gln Asn Gln Val Ala Val Asp Ala Pro
Ser Glu His Gly 195 200 205Val Tyr Ser Phe Gln Phe Asn Lys 210
2154934PRTArtificial SequenceDeleted RBMS2 sequence 49Leu Thr Pro
Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys1 5 10 15Gln Ala
Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp 20 25 30His
Gly5013DNAArtificial SequenceTALEN sequence 50atcgagagcc aat 13
* * * * *