U.S. patent application number 12/990330 was filed with the patent office on 2011-06-16 for cancer marker, method for evaluation of cancer by using the cancer marker, and evaluation reagent.
This patent application is currently assigned to NEC CORPORATION. Invention is credited to Seiko Iizuka, Yoshinori Kosugi, Masahiko Kuroda, Takayuki Mizutani, Kazuma Ohyashiki, Kosuke Oikawa, Masakatsu Takanashi, Masami Tanaka, Akihiko Tsuchida.
Application Number | 20110143360 12/990330 |
Document ID | / |
Family ID | 41255131 |
Filed Date | 2011-06-16 |
United States Patent
Application |
20110143360 |
Kind Code |
A1 |
Kuroda; Masahiko ; et
al. |
June 16, 2011 |
CANCER MARKER, METHOD FOR EVALUATION OF CANCER BY USING THE CANCER
MARKER, AND EVALUATION REAGENT
Abstract
The present invention provides a novel cancer marker for
evaluating the onset, the preclinical stage, the clinical stage, or
the prognosis of a cancer in a subject, and an evaluation method
using the same. A cancer marker containing at least one miRNA
selected from hsa-miR-92 and hsa-miR-494 is used as a marker for
cancers excluding breast cancer. A method for evaluating the
possibility of cancers excluding breast cancer includes the step of
detecting the expression level of a cancer marker in a biological
sample collected from a subject. In this method, the cancer marker
contains at least one miRNA selected from hsa-miR-92 and
hsa-miR-494.
Inventors: |
Kuroda; Masahiko; (Tokyo,
JP) ; Tanaka; Masami; (Fukuoka, JP) ; Oikawa;
Kosuke; (Tokyo, JP) ; Mizutani; Takayuki;
(Tokyo, JP) ; Takanashi; Masakatsu; (Tokyo,
JP) ; Iizuka; Seiko; (Tokyo, JP) ; Kosugi;
Yoshinori; (Tokyo, JP) ; Ohyashiki; Kazuma;
(Tokyo, JP) ; Tsuchida; Akihiko; (Tokyo,
JP) |
Assignee: |
NEC CORPORATION
Tokyo
JP
TOKYO MEDICAL UNIVERSITY
Tokyo
JP
NEC CORPORATION
Tokyo
JP
TOKYO MEDICAL UNIVERSITY TSUCHIDA
Tokyo
JP
|
Family ID: |
41255131 |
Appl. No.: |
12/990330 |
Filed: |
April 28, 2009 |
PCT Filed: |
April 28, 2009 |
PCT NO: |
PCT/JP2009/058421 |
371 Date: |
January 5, 2011 |
Current U.S.
Class: |
435/6.14 ;
536/23.1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/158 20130101; C12Q 2600/178 20130101 |
Class at
Publication: |
435/6.14 ;
536/23.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/02 20060101 C07H021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 2008 |
JP |
2008-119280 |
Claims
1. A cancer marker applicable to a cancer other than breast cancer,
comprising at least one miRNA selected from hsa-miR-92 and
hsa-miR-494.
2. The cancer marker according to claim 1, wherein the hsa-miR-92
is hsa-miR-92a.
3. The cancer marker according to claim 1, wherein the miRNA is at
least one of hsa-miR-92a and hsa-miR-494.
4. The cancer marker according to claim 1, wherein the miRNA is
miRNA contained in plasma or serum.
5. The cancer marker according to claim 1, wherein the cancer is at
least one cancer selected from the group consisting of colon
cancer, gallbladder cancer, stomach cancer, lung cancer, leukemia,
pancreas cancer, prostate cancer, bladder cancer, kidney cancer,
uterine body cancer, cervical cancer, hepatocyte cancer, biliary
tract cancer, brain tumor, laryngeal cancer, tongue cancer, rectal
cancer, and osteosarcoma.
6. The cancer marker according to claim 1, used for judging an
onset, a preclinical stage, a clinical stage, or prognosis of the
cancer.
7. An evaluation method for evaluating a possibility of a cancer
other than breast cancer, the evaluation method comprising the step
of: detecting an expression level of a cancer marker in a
biological sample, wherein the cancer marker is the cancer marker
according to claim 1.
8. The evaluation method according to claim 7, wherein the
hsa-miR-92 is hsa-miR-92a.
9. The evaluation method according to claim 7, wherein the miRNA is
at least one of hsa-miR-92a and hsa-miR-494.
10. The evaluation method according to claim 7, wherein the miRNA
is miRNA contained in plasma or serum.
11. The evaluation method according to claim 7, wherein the
biological sample is a sample containing plasma or serum.
12. The evaluation method according to claim 7, wherein the cancer
is at least one cancer selected from the group consisting of colon
cancer, gallbladder cancer, stomach cancer, lung cancer, leukemia,
pancreas cancer, prostate cancer, bladder cancer, kidney cancer,
uterine body cancer, cervical cancer, hepatocyte cancer, biliary
tract cancer, brain tumor, laryngeal cancer, tongue cancer, rectal
cancer, and osteosarcoma.
13. The evaluation method according to claim 7, further comprising
the step of: determining the possibility of the cancer based on the
expression level of the cancer marker in the biological sample
detected in the cancer marker-detecting step by at least one method
selected from the group consisting of methods (1), (2), and (3):
(1) the expression level of the cancer marker in the biological
sample of a subject is compared with an expression level of the
cancer marker in a biological sample of a normal subject, and when
the expression level in the subject is lower than the expression
level in the normal subject, it is determined that the subject has
a high possibility of the cancer; (2) the expression level of the
cancer marker in the biological sample of a subject is compared
with an expression level of the cancer marker in a biological
sample of a normal subject, and as the expression level in the
subject becomes relatively lower than the expression level in the
normal subject, it is determined that the cancer in the subject is
relatively advanced; and (3) the expression level of the cancer
marker in the biological sample of a subject is compared with an
expression level of the cancer marker in a biological sample of
each of cancer patients at different progression stages, and it is
determined that the cancer in the subject is in a same progression
stage as the cancer in the patient exhibiting a same or similar
expression level.
14. The evaluation method according to claim 7, wherein the
expression level of the cancer marker is represented as an amount
of the cancer marker expressed in the biological sample.
15. The evaluation method according to claim 7, further comprising
the step of: detecting an expression level of a correction marker
in the biological sample, wherein the expression level of the
cancer marker is represented as a ratio between an amount of the
cancer marker expressed in the biological sample and an amount of
the correction marker expressed in the biological sample.
16. The evaluation method according to claim 15, wherein the
correction marker is hsa-miR-638.
17. An evaluation reagent for evaluating a possibility of a cancer
other than breast cancer, the evaluation reagent comprising: a
miRNA detection reagent for detecting at least one miRNA selected
from hsa-miR-92 and hsa-miR-494, wherein the evaluation reagent is
used in the evaluation method according to claim 7.
18. The evaluation reagent according to claim 17, wherein the
hsa-miR-92 is hsa-miR-92a.
19. The evaluation reagent according to claim 17, wherein the miRNA
is at least one of hsa-miR-92a and hsa-miR-494.
20. The evaluation reagent according to claim 17, further
comprising: a correction marker detection reagent for detecting a
correction marker.
21. The evaluation reagent according to claim 20, wherein the
correction marker is hsa-miR-638.
22. A correction marker comprising: hsa-miR-638, wherein the
correction marker is used to correct an expression level of the
cancer marker according to claim 1 in a biological sample in the
evaluation method according to claim 15.
23. The correction marker according to claim 22, wherein the miRNA
is miRNA contained in plasma or serum.
24. A correction method for correcting an expression level of a
cancer marker in a biological sample, the correction method
comprising, in order to correct an expression level of the cancer
marker according to claim 1 in a biological sample in the
evaluation method according to claim 7, the steps of: measuring an
expression level of the cancer marker in a biological sample;
measuring an amount of a correction marker expressed in the
biological sample; and correcting the expression level of the
cancer marker by setting a ratio between an amount of the cancer
marker expressed and the amount of the correction marker expressed
to a corrected expression level of the cancer marker in the
biological sample.
25. The correction method according to claim 24, wherein the
correction marker is the correction marker according to claim
22.
26. The correction method according to claim 24, wherein the
biological sample is a sample containing plasma or serum.
27. A correction reagent comprising: a miRNA detection reagent for
detecting hsa-miR-638, wherein the correction reagent is used to
correct an expression level of the cancer marker according to claim
1 in a biological sample in the evaluation method according to
claim 15.
Description
TECHNICAL FIELD
[0001] The present invention relates to a cancer marker, a method
for evaluating the possibility of cancers using the same, and an
evaluation reagent. The present invention also relates to a
correction marker, correction method, and correction reagent for
correcting the cancer marker in the evaluation method.
BACKGROUND ART
[0002] In the field of clinical medical practice, it is required to
easily judge the presence or absence of a disease, the degree of
progression of the disease, the effect obtained after a treatment,
etc. Under these circumstances, as a method for judging them
indirectly, detecting a marker whose expression amount changes
specifically accompanying the onset or progression of each disease
has been proposed, and attempts actually are made to put this into
practical use.
[0003] Among various diseases, detecting malignant tumors, which
are so-called cancers, early and selecting and changing a treatment
strategy therefor appropriately are particularly important. Thus,
in order to realize indirect judgment by the detection of a marker
as described above, various cancer markers (tumor markers) have
been reported. Specific examples of the cancer marker include PSA
(Prostate-Specific Antigen), CEA (Carcinoembryonic Antigen), CA
19-9 (Carcinoembryonic Antigen 19-9), and CA 72-4 (Carcinoembryonic
Antigen 72-4). Furthermore, it is described in Non-Patent Documents
1 and 2 that the expression of miRNAs such as has-mir-15,
has-mir-16, miR-143, and miR-145 is downregulated in lymphocytic
leukemia, colon cancer, and the like (Non-Patent Documents 1,
2).
PRIOR ART DOCUMENTS
Non-Patent Documents
[0004] [Non-Patent Document 1] Calin G A, Dumitru C D, Shimizu M et
al., Frequent deletions and down-regulation of micro-RNA genes
miR15 and miR16 at 13q14 in chronic lymphocytic leukemia, Proc Natl
Acad Sci USA, 2002, vol. 99, pp. 15524-9 [0005] [Non-Patent
Document 2] Michael M Z, SM O C, van Holst Pellekaan N G, Young G
P, James R J, Reduced accumulation of specific microRNAs in
colorectal neoplasia, Mol cancer Res, 2003, vol. 1, pp. 882-91
BRIEF SUMMARY OF THE INVENTION
Problem to be Solved by the Invention
[0006] However, in the field of clinical medical practice, since
cancer markers with which the onset of cancers and their
progression can be judged with excellent reliability are necessary,
there still is a demand for the provision of a novel cancer marker.
Thus, with the foregoing mind, it is an object of the present
invention to provide a novel cancer marker for evaluating cancers,
an evaluation method using the cancer marker, and an evaluation
reagent to be used in the evaluation method. Furthermore, it is
another object of the present invention to provide a novel
correction marker for correcting the expression level of the cancer
marker, a correction method for correcting the expression level
using the correction marker, and a correction reagent to be used in
the correction method.
Means for Solving Problem
[0007] A cancer marker of the present invention is a cancer marker
applicable to a cancer other than breast cancer, containing at
least one miRNA selected from hsa-miR-92 and hsa-miR-494.
[0008] An evaluation method of the present invention is an
evaluation method for evaluating a possibility of a cancer other
than breast cancer. The evaluation method includes the step of
detecting an expression level of a cancer marker in a biological
sample. In the evaluation method, the cancer marker is the cancer
marker of the present invention.
[0009] An evaluation reagent of the present invention is an
evaluation reagent for evaluating a possibility of a cancer other
than breast cancer. The evaluation reagent contains a miRNA
detection reagent for detecting at least one miRNA selected from
hsa-miR-92 and hsa-miR-494. The evaluation reagent is used in the
evaluation method of the present invention.
[0010] A correction marker of the present invention contains
hsa-miR-638. The correction marker is used to correct an expression
level of the cancer marker of the present invention in a biological
sample in the evaluation method of the present invention.
[0011] A correction method of the present invention includes, in
order to correct an expression level of the cancer marker of the
present invention in a biological sample in the evaluation method
of the present invention, the steps of:
[0012] measuring an expression level of the cancer marker in a
biological sample;
[0013] measuring an amount of a correction marker expressed in the
biological sample; and
[0014] correcting the expression level of the cancer marker by
setting a ratio between an amount of the cancer marker expressed
and the amount of the correction marker expressed to a corrected
expression level of the cancer marker in the biological sample.
[0015] A correction reagent of the present invention contains a
miRNA detection reagent for detecting hsa-miR-638. The correction
reagent is used to correct an expression level of the cancer marker
of the present invention in a biological sample in the evaluation
method of the present invention.
Effects of the Invention
[0016] The inventors of the present invention conducted a diligent
study, and as a result, they found that the expression levels of
hsa-miR-92 and hsa-miR-494 in a biological sample decrease
accompanying the development of cancers, thereby achieving the
present invention. According to the cancer marker of the present
invention, by detecting the expression level thereof in a
biological sample, it is possible to judge the presence or absence
of cancer development or the cancer progression, for example.
Furthermore, the cancer marker of the present invention provides a
significant difference between negative and positive regarding the
canceration, for example. Thus, according to the cancer marker of
the present invention, it becomes possible to detect cancers at an
initial stage easily whereas such detection is difficult by general
palpation and the like. Still further, according to the correction
marker of the present invention, the accuracy of detection using
the cancer marker further can be improved.
BRIEF DESCRIPTION OF DRAWINGS
[0017] FIGS. 1A and 1B are graphs showing the expression profiles
of various miRNAs in samples derived from normal subjects in
Example 2 of the present invention. FIG. 1A is a graph showing the
signal intensity values of various miRNAs contained in each of the
samples, and FIG. 1B is a graph showing a logarithmic value
(log.sub.10[individual rank_Normal/Average rank_Normal]) of a value
obtained by dividing a signal value rank of each miRNA in each of
the samples derived from the seven normal subjects by the signal
value average rank thereof.
[0018] FIG. 2 is a graph showing the ratio (Average
rank_AML/Average rank_Normal) between the signal value average rank
of each miRNA between the samples derived from the AML patients and
the signal value average rank of each miRNA among the samples
derived from the normal subjects in Example 2 of the present
invention.
[0019] FIG. 3 is a graph showing the ratio
(hsa-miR-92a/hsa-miR-638) between the fluorescence intensity of
hsa-miR-92a as a cancer marker and the fluorescence intensity of
hsa-miR-638 a correction marker in samples derived from normal
subjects and in samples derived from acute leukemia patients in
Example 3 of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
Cancer Marker
[0020] The cancer marker of the present invention is, as described
above, a cancer marker applicable to a cancer other than breast
cancer, containing at least one miRNA selected from hsa-miR-92 and
hsa-miR-494.
[0021] As described above, by the inventors of the present
invention, it has been revealed that the expression levels of these
miRNAs change specifically accompanying the canceration of cells of
various tissues. More specifically, it has been revealed that the
expression levels of the miRNAs in, for example, plasma or serum
decreases accompanying the canceration. From this fact, it is
interpreted that, for example, the expression levels of the miRNAs
decrease significantly: after the onset of a cancer as compared
with before the onset of the cancer; in the preclinical stage as
compared with before the preclinical stage; in the clinical stage
as compared with before the clinical stage, in the initial stage as
compared with before the initial stage; after the initial stage as
compared with in the initial stage. Therefore, by detecting the
expression levels of these miRNAs, it becomes possible to carry out
evaluation or the like of the possibility that a subject may
develop a cancer, whether or not canceration has occurred, the
stage of cancer progression such as a preclinical stage (an initial
stage) or a clinical stage (an advanced stage or a disease stage),
or prognosis, for example.
[0022] The meaning of the terms used in the present invention is as
follows. The term "cancer" generally means malignant tumor. The
term "canceration" generally means the onset of a cancer and also
encompasses "malignant transformation". Regarding the term "onset",
for example, the time point at which one is diagnosed as having a
specific disease through synthetic judgment based on
disease-specific clinical symptoms, test data, etc., is referred to
as the onset of the disease. The term "preclinical stage" generally
refers to a condition before the onset of a disease where
disease-specific clinical symptoms have not appeared yet but in an
early stage of the disease in which a trace amount of malignant
tumor cells are present already. In cervical cancer, the
preclinical stage generally indicates precancerous lesions such as
CIN1, CIN2, and CIN3. In colon cancer, the preclinical stage
indicates a condition of having adenoma. The term "clinical stage"
generally refers to cancers found through image findings and
cancers that can be identified by the use of existing tumor
markers. The term "initial stage" generally indicates a condition
of having an early cancer. The term "prognosis" means, for example,
a postoperative course of a disease. Since the cancer marker of the
present invention can provide useful information for, for example,
predicting prognosis, foreseeing the course of a disease, and
selecting an appropriate treatment method, it also can be referred
to as a "prognostic factor". The "stage of cancer progression" can
be judged as appropriate based on, for example, the kind of
cancerous tissues or the like. In general, Stage 0 and Stage I can
be classified as an initial cancer, Stage II can be classified as
an early cancer, and Stage III and Stage IV can be classified as an
advanced cancer.
[0023] Cancers to which the cancer marker of the present invention
is applicable are not particularly limited, and examples thereof
include colon cancer, gallbladder cancer, stomach cancer, lung
cancer, leukemia, pancreas cancer, prostate cancer, bladder cancer,
kidney cancer, uterine body cancer, cervical cancer, hepatocyte
cancer, biliary tract cancer, brain tumor, laryngeal cancer, tongue
cancer, rectal cancer, and osteosarcoma.
[0024] In the present invention, each miRNA may be, for example, a
single strand (monomer) or a double strand (dimer). Furthermore, in
the present invention, each miRNA preferably is the miRNA in its
mature form cleaved by ribonuclease such as Dicer.
[0025] In the present invention, the hsa-miR-92 may be, for
example, hsa-miR-92a, which preferably is the miRNA in its mature
form as described above.
[0026] The sequence of the mature hsa-miR-92a is registered under
Accession No. MIMAT0000092, for example. This sequence is shown as
SEQ ID NO: 1 below.
TABLE-US-00001 hsa-miR-92a (SEQ ID NO: 1)
5'-uauugcacuugucccggccugu-3'
[0027] The hsa-miR-494 may be, for example, the miRNA in its mature
form as described above. The sequence of the mature miRNA is
registered under Accession No. MIMAT0002816. The sequence of the
mature hsa-miR-494 is shown as SEQ ID NO: 2.
TABLE-US-00002 hsa-miR-494 (SEQ ID NO: 2)
5'-ugaaacauacacgggaaaccuc-3'
[0028] As disclosed in the documents listed below, the 5' end and
the 3' end of each of the miRNAs respectively have some variations,
for example. Therefore, each of the miRNAs of the present invention
also encompasses variants having a sequence different from the
sequence thereof in its mature form by a few bases. [0029] Wu H. et
al., 2007, PLoS ONE 2 (10): e1020 miRNA profiling of naive,
effector and memory CD8 T cells. [0030] Pablo Landgraf et al.,
2007, Cell, vol. 129, pp. 1401-1414 A Mammalian microRNA Expression
Atlas Based on Small RNA Library Sequencing. [0031] Neilson et al.,
2007, Genes Dev, vol. 21, pp. 578-589 Dynamic regulation of miRNA
expression in order to stage of cellular development. [0032] Ruby
et al., 2006, Cell, vol. 127, pp. 1193-1207 Large-scale sequencing
reveals 21U-RNAs and additional microRNAs and endogeneous siRNAs in
C. elegans. [0033] Obernoster et al., RNA 2006 12: pp. 1161-1167
Post-transcriptional regulation of microRNA expression. [0034]
Lagos-Quintana et al., 2002, Curr Biol, vol. 12, pp. 735-739
Identification of tissue-specific microRNAs from mouse.
[0035] The miRNAs in the present invention encompass, for example,
polynucleotides having a base sequence with a homology to the base
sequences of the respective SEQ ID NOs: 1 and 2, and
polynucleotides having a base sequence complementary thereto. The
"homology" refers to the degree of identity between sequences to be
compared with each other when they are aligned appropriately, and
represents the occurrence ratio (%) of perfect match of amino acids
between these sequences. When it is described that the base
sequence of a polynucleotide "has a homology" to the base sequences
of the miRNAs of the present invention, it means that the
polynucleotide is similar enough to the miRNAs to be able to
maintain the function as the miRNAs of the present invention. The
alignment can be achieved by using an arbitrary algorithm such as
BLAST, for example. Even when the base sequences differ from each
other by, for example, point mutation, deletion, or addition, it
can be said that they are homologous as long as such a difference
does not affect the function of the miRNAs. The number of bases
different between the base sequences is, for example, 1 to 20, 1 to
15, 1 to 10, 1 to 5, 1 to 3, 1 to 2, or 1. Furthermore, when base
sequences of two polynucleotides to be compared with each other
have an identity of, for example, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or about 99%, it can be said that they are
homologous. Furthermore, for example, when one of the two
polynucleotides hybridizes to a polynucleotide having a base
sequence complementary to the other polynucleotide under stringent
conditions, it can be said that the two polynucleotides are
homologous. The stringent conditions are not particularly limited,
and may be such that, for example, the two polynucleotides are kept
at a temperature of "Tm-25.degree. C." overnight in a solution
containing 6.times.SSC, 0.5% SDS, 5.times.Denhardt's solution, and
0.01% denatured salmon sperm nucleic acid.
[0036] The cancer marker of the present invention is, for example,
a miRNA present in a biological sample. The biological sample in
the present invention is a liquid fraction collected from a living
organism (e.g., a subject), and examples thereof include blood,
saliva, urine, and diluted solutions thereof. Among them, a blood
sample is preferable because change in expression levels of the
various miRNAs accompanying the canceration is particularly
noticeable. The blood sample is not particularly limited. It may
be, for example, a sample containing a liquid fraction of blood,
such as a so-called plasma-containing sample or serum-containing
sample. Specific examples of the sample include whole blood, a
liquid fraction, plasma fraction, and serum fraction of whole
blood, samples containing them, and samples obtained by diluting
them.
[0037] <Evaluation Method>
[0038] The evaluation method of the present invention is, as
described above, an evaluation method for evaluating a possibility
of a cancer other than breast cancer. The evaluation method
includes the step of detecting an expression level of a cancer
marker in a biological sample. In the evaluation method, the cancer
marker is the cancer marker of the present invention.
[0039] The present invention is characterized in that the cancer
marker of the present invention is detected as a cancer marker, and
for example, a method for detecting the expression level of each
miRNA is by no means limited. The cancer marker of the present
invention is as described above. In the present invention, the
cancer marker to be detected may be, for example, either one of
hsa-miR-92 and hsa-miR-494 or both of them. When the cancer marker
to be detected is hsa-miR-92, it is preferable that the hsa-miR-92
is in its mature form, for example. When the cancer marker to be
detected is hsa-miR-92a, it is preferable that the hsa-miR-92a is
in its mature form.
[0040] In the present invention, cancers to be evaluated are not
particularly limited, and as described above, examples thereof
include colon cancer, gallbladder cancer, stomach cancer, lung
cancer, leukemia, pancreas cancer, prostate cancer, bladder cancer,
kidney cancer, uterine body cancer, cervical cancer, hepatocyte
cancer, biliary tract cancer, brain tumor, laryngeal cancer, tongue
cancer, rectal cancer, and osteosarcoma.
[0041] In the present invention, the biological sample is not
particularly limited, and examples thereof include, as described
above, blood, saliva, and urine. Among them, the biological sample
preferably is the above-described blood sample, more preferably a
sample containing plasma, a sample containing serum, or the like,
because change in expression levels of the various miRNAs
accompanying the canceration is particularly noticeable. Such a
blood sample is particularly preferable also because, for example,
a test can be carried out merely by collecting blood and the pain
and burden placed on a subject can be reduced considerably.
[0042] The evaluation method of the present invention further may
include the step of determining the possibility of the cancer. More
specifically, the evaluation method of the present invention may
include the step of determining the possibility of the cancer based
on the expression level of the cancer marker in the biological
sample detected in the cancer marker-detecting step by at least one
method selected from the group consisting of methods (1), (2), and
(3):
(1) the expression level of the cancer marker in the biological
sample of a subject is compared with an expression level of the
cancer marker in a biological sample of a normal subject, and when
the expression level in the subject is lower than the expression
level in the normal subject, it is determined that the subject has
a high possibility of the cancer; (2) the expression level of the
cancer marker in the biological sample of a subject is compared
with an expression level of the cancer marker in a biological
sample of a normal subject, and as the expression level in the
subject becomes relatively lower than the expression level in the
normal subject, it is determined that the cancer in the subject is
relatively advanced; and (3) the expression level of the cancer
marker in the biological sample of a subject is compared with an
expression level of the cancer marker in a biological sample of
each of cancer patients at different progression stages, and it is
determined that the cancer in the subject is in the same
progression stage as the cancer in the patient exhibiting the same
or similar expression level.
[0043] In the present invention, the possibility of a cancer
encompasses, for example, the possibility that the subject may
develop a cancer, whether or not canceration has occurred, the
stage of cancer progression such as a preclinical stage or a
clinical stage, prognosis, or the like. In the present invention,
the term "normal subject" means, for example, a subject who is not
judged as having developed a cancer to be evaluated or having a
possibility that he has developed the cancer.
[0044] The expression level of the cancer marker in the normal
subject in the methods (1) and (2) can be determined previously
using a biological sample collected from the normal subject, for
example. Furthermore, the expression levels of the cancer marker in
the cancer patients in the method (3) also can be determined by,
for example, previously classifying the patients according to the
progression stage and using biological samples collected from the
patients at the respective progression stages. In the methods (1)
to (3), the kind of the biological samples of the normal subject
and the patients preferably are the same as the kind of the
biological sample of the subject, for example, and the biological
sample preferably is a blood sample, particularly preferably a
sample containing plasma or a sample containing serum, for example.
Furthermore, the biological samples of the normal subject and the
patients preferably are prepared in the same manner and under the
same conditions as the biological sample of the subject, for
example.
[0045] In the present invention, the expression level of the cancer
marker may be, for example, the amount of the cancer marker
expressed in the biological sample. The amount of the cancer marker
expressed may be, for example, the actual amount of miRNA or a
value correlated with the actual amount of miRNA. Examples of the
latter include a signal value obtained when detecting the miRNA.
This signal value can be determined as appropriate depending on,
for example, the method for detecting the miRNA and the kind of a
detector for detecting the signal value. When the detection method
is, for example, a PCR method such as a real-time RT-PCR (real-time
reverse transcription-polymerase chain reaction) method, the signal
value can be expressed with a unit of copies/.mu.l or the like, for
example.
[0046] It is preferable that the expression level of the cancer
marker is corrected with the expression level of a correction
marker, for example. The correction marker is not particularly
limited, and may be, for example, a substance exhibiting a
constitutive expression level in a biological sample. In
particular, the correction marker preferably is a substance
exhibiting a constitutive expression level in a biological sample
regardless of the presence or absence of canceration. By detecting
the expression level of such a correction marker as an internal
standard in a biological sample collected to detect the cancer
marker, the expression levels of the cancer marker between
different subjects and the expression levels in the same subject
over time can be compared with still higher reliability, for
example. For the reasons stated above, the evaluation method of the
present invention further may include the step of detecting the
expression level of the correction marker in the biological sample
collected from the subject.
[0047] The above-described corrected expression level of the cancer
marker can be represented as the ratio between the amount of the
cancer marker expressed and the amount of the correction marker
expressed in the biological sample, for example. Specifically, it
can be represented as, for example, "the amount of the cancer
marker expressed/the amount of the correction marker expressed" or
"the amount of the correction marker expressed/the amount of the
cancer marker expressed". The expression of the cancer marker of
the present invention decreases accompanying the canceration. Thus,
in the case where it is represented as "the amount of the cancer
marker expressed/the amount of the correction marker expressed" as
the former, for example, it can be judged that the possibility of a
cancer becomes higher as the value becomes relatively smaller. On
the other hand, in the case where it is represented as "the amount
of the correction marker expressed/the amount of the cancer marker
expressed" as the latter, it can be judged that the possibility of
a cancer becomes higher as the value becomes greater, for
example.
[0048] The correction marker is not particularly limited, and a
known marker can be used as the correction marker. In the present
invention, it is preferable to use, in particular, hsa-miR-638 as
the correction marker. The correction marker containing this miRNA
corresponds to the correction marker of the present invention to be
described later, and it exhibits constitutive expression in a
biological sample regardless of the presence or absence of
canceration, for example. Since the correction marker of the
present invention exhibits constitutive expression regardless of
the presence or absence of canceration as described above, it can
serve as an internal standard in a biological sample.
[0049] The correction marker of the present invention, i.e., the
hsa-miR-638, preferably is the miRNA in its mature form cleaved by
a ribonuclease such as Dicer, for example.
[0050] The hsa-miR-638 may be, for example, the miRNA in its mature
form as described above. The sequence of the mature miRNA is
registered under Accession No. MIMAT00033088. The sequence of the
mature hsa-miR-638 is shown as SEQ ID NO: 3. hsa-miR-638 (SEQ ID
NO: 3)
[0051] 5'-agggaucgcgggcggguggcggccu-3'
[0052] In the present invention, the subject is not particularly
limited, and examples thereof include: mammals including humans,
dogs, and cats; primates; and rodents.
[0053] In the following, the evaluation method of the present
invention will be described with reference to an example where
plasma or serum is used as a biological sample. It is to be noted,
however, the present invention is not limited thereto.
[0054] First, total RNA is extracted from plasma or serum of a
subject. The plasma or serum can be recovered by a known method,
and for example, it can be prepared by collecting whole blood from
the subject and removing a blood cell fraction through
centrifugation. Furthermore, the method for extracting the total
RNA from the plasma or serum is not particularly limited, and any
of known methods can be employed. Examples of the extraction method
include a guanidine-CsCl ultracentrifugation method and an AGPC
(Acid Guanidinium-Phenol-Chloroform) method.
[0055] Next, the cancer marker of the present invention, i.e., at
least one of hsa-miR-92 and hsa-miR-494, in the extracted total RNA
is detected. The method for detecting the cancer marker of the
present invention is by no means limited, as long as it can detect
the expression of the miRNA as described above. Specific examples
of the detection method include, for example, Northern blot
analysis, a real-time RT-PCR detection method, and a microarray
analysis method. The present invention is characterized in that the
miRNA to be detected as a cancer marker is at least one of
hsa-miR-92 and hsa-miR-494. A method for detecting it is by no
means limited, and those skilled in the art can carry out such a
method based on common general technical knowledge.
[0056] Northern blot analysis can be carried out, for example,
using a probe as described below. The probe is not particularly
limited, and probes that can detect the above-described miRNAs can
be used. For example, the probe may be the one that can hybridize
to any of the above-described miRNAs. A commercially available
product may be used as the probe, or the probe may be prepared on
your own, for example. The sequence of the probe can be designed as
appropriate based on the base sequences of the miRNAs and common
general technical knowledge, for example. Specific examples of the
probe include the one having a sequence complementary to the miRNA
to be detected. It is preferable that the sequence of the probe is,
for example, at least about 70% complementary to the miRNA to be
detected, more preferably at least 90% complementary to the same,
and particularly preferably 100% complementary to the same. The
constitutional unit of the probe is not particularly limited, and,
for example, a known constitutional unit can be employed. Specific
examples of the constitutional unit include nucleotides such as
deoxyribonucleotide and ribonucleotide, PNA (Peptide Nucleic Acid),
and LNA (Locked Nucleic Acid). Examples of the LNA include BNA
(Bridged Nucleic Acid) such as 2',4'-bridged nucleic acid. Bases in
the nucleotides are not particularly limited. They may be, for
example, natural bases such as adenine, guanine, cytosine, thymine,
and uracil, or may be unnatural bases (artificial bases). The
length of the probe is not particularly limited, and is, for
example, 10 to 100 mer, preferably 10 to 40 mer, more preferably 10
to 25 mer, and still more preferably 15 to 20 mer.
[0057] As a specific example, first, the extracted total RNA is
fractionated depending on the strand length by electrophoresis, and
the fractionated total RNA is transcribed onto a membrane from the
gel used in the electrophoresis. Examples of the membrane include a
nitrocellulose membrane and a nylon membrane. Subsequently, the
membrane on which the fractionated RNAs have been transcribed was
incubated in a predetermined buffer in the presence of the
above-described probe labeled with a radioactive material such as
.sup.32P. Then, by detecting the label of the probe, miRNA that has
hybridized with the probe can be detected. When the probe labeled
with a radioactive material is used as described above, the miRNA
that has hybridized with the probe can be quantified by, for
example, autoradiography, based on the band intensity. Conditions
of the Northern blot analysis are not particularly limited, and it
is preferable that, for example, prehybridization, hybridization,
and washing are carried out under stringent conditions. For
example, in the case where DNA labeled with .sup.32P is used as a
probe, the stringent conditions are at 37.degree. C. in a
hybridization buffer. The hybridization buffer may be, for example,
a buffer containing 0.25 mol/l sodium phosphate (pH 7.2), 7% SDS,
and 0.5% sodium pyrophosphate. Furthermore, in the washing step,
the stringent conditions are at 37.degree. C. in a washing buffer
containing 2.times.SSC and 1% SDS and further at room temperature
in a washing buffer containing 0.1.times.SSC. It should be note
that the stringent conditions are not limited thereto, and
depending on the selected detection method, conditions standard
therefor can be employed.
[0058] The real-time RT-PCR detection method can be carried out
using a fluorescent reagent as described below, for example. First,
for example, a linker is ligated to each of 5' end and 3' end of
the extracted total RNA. Then, with the total RNA having the linker
ligated thereto as a template, cDNA is amplified. Further, with the
thus-obtained cDNA as a template, PCR is carried out using a primer
that can amplify the miRNA to be detected. The primer is by no
means limited, and examples thereof include primers that can
hybridize to the above-described miRNA or a peripheral region of
the miRNA. The primer can be designed as appropriate based on the
base sequence of the above-described miRNA and common general
technical knowledge, for example. Specific examples of the primer
include a probe having a sequence complementary to the miRNA to be
detected or a peripheral region of the miRNA. It is preferable that
the sequence of the primer is, for example, at least about 70%
complementary to, for example, the miRNA to be detected or a
peripheral region of the miRNA, preferably at least 80%
complementary to the same, more preferably at least 90%
complementary to the same, still more preferably at least 95%
complementary to the same, and particularly preferably 100%
complementary to the same. The constitutional unit of the primer is
not particularly limited, and is, for example, the same as that of
the above-described probe. The length of the primer is not
particularly limited, and may be a general length.
[0059] At the time of carrying out the PCR, it is preferable to
cause a fluorescent reagent to be present in a PCR reaction
solution, for example. Examples of the fluorescent reagent include
a fluorescent dye that specifically binds to a double-stranded
nucleic acid and a fluorescent dye that intercalates into a
double-stranded nucleic acid. When such a fluorescent reagent forms
a double-stranded nucleic acid by nucleic acid amplification, the
fluorescent dye binds to or intercalates into the double-stranded
nucleic acid. Then, by measuring the fluorescence intensity of the
fluorescent dye that has bound to or intercalated into the
double-stranded nucleic acid, it is possible to quantify the miRNA
to be detected. Examples of the fluorescent dye include SYBR
(trademark) Green. Such a real-time RT-PCR detection method can be
carried out by a known method, for example. Also, it can be carried
out, for example, using a commercially available reagent such as
SYBR (trademark) Green PCR Master Mix (trade name, Perkin-Elmer
Applied Biosystems) and a commercially available detector such as
ABI Prism 7900 Sequence Detection System (trade name, Perkin-Elmer
Applied Biosystems) in accordance with their manuals. Furthermore,
the above-described primer also may serve as the fluorescent
reagent. Such a primer may be, for example, a labeled primer
labeled with a fluorescent dye, whose fluorescence is quenched when
a fluorescent double-stranded nucleic acid has not yet been formed
and the quenching is released when the double-stranded nucleic acid
is formed. Such a labeled primer can be designed based on common
general technical knowledge, for example.
[0060] When the expression amount is measured by such a detection
method, it is preferable that an internal standard such as a
correction marker is measured, and with regard to the miRNA to be
detected, the expression amount corrected with the internal
standard is calculated as will be described later, for example.
[0061] On the other hand, the cancer marker of the present
invention in plasma or serum collected from a normal subject also
is detected in the same manner. At this time, it is preferable that
the kind of the cancer marker used for the subject is the same as
that used for the normal subject. It is preferable that the
expression level of the cancer marker of the present invention in
the normal subject is determined previously, for example, and it is
not necessary to determine the expression level in the normal
subject every time evaluation is made. That is, it is preferable
that the previously detected expression level of the cancer marker
of the present invention in the normal subject is set to a standard
value of the normal subject. The expression level in the normal
subject may be, for example, a value obtained from a single normal
subject or may be a value calculated from the expression levels in
a plurality of normal subjects by a statistical method.
[0062] Then, from the expression level in the subject and the
expression level in the normal subject, the possibility of a cancer
in the subject can be evaluated by, for example, the following
method (1) or (2).
[0063] In the method (1), for example, the expression level of the
cancer marker in the subject is compared with the expression level
of the cancer marker in the biological sample of the normal
subject, and when the expression level in the subject is
significantly lower than the expression level in the normal
subject, it is determined that the possibility of the cancer is
high. As described above, the expression level of the cancer marker
of the present invention in blood decreases accompanying the
canceration, for example. Therefore, when the expression level in
the subject is equal to or higher than that in the normal subject,
it can be judged that the subject has a low possibility of
canceration. On the other hand, when the expression level in the
subject is lower than that in the normal subject, it can be judged
that the subject has a high possibility of canceration.
[0064] In the method (2), for example, the expression level of the
cancer marker in the subject is compared with the expression level
of the cancer marker in the biological sample of a normal subject,
and when the expression level in the subject becomes relatively
lower than the expression level in the normal subject, it is
determined that the cancer in the subject is relatively advanced.
As described above, the expression level of the cancer marker of
the present invention in blood decreases accompanying the
progression of canceration, for example. Therefore, as the
expression level in the subject is lower than the expression level
in the normal subject and the difference in expression level
between the subject and the normal subject is relatively small, it
can be judged that the cancer progression is less severe, and as
the above-described difference is relatively large, it can be
judged that the cancer progression is severe.
[0065] In the methods (1) and (2), for example, when the expression
level in the subject is at least 50% lower than the expression
level in the normal subject, for example, it can be judged that the
subject has a high possibility of the cancer, and as the expression
level in the subject decreases further to be at least 85%, at least
90%, and at least 95% lower than the expression level in the normal
subject, it can be judged that the cancer is advanced further. The
same applies to the case where the expression level is corrected
with a correction marker as will be described later.
[0066] Furthermore, instead of or in addition to the detection of
the cancer marker with regard to the normal subject, the cancer
marker of the present invention may be detected in the same manner
with regard to serum or plasma collected from cancer patients at
different progression stages. At this time, it is preferable that
the kind of the cancer marker used for the subject is the same as
that used for the cancer patients. The expression levels of the
cancer marker of the present invention in the patients preferably
are determined previously, and it is not necessary to determine the
expression levels in the patients every time evaluation is made.
That is, it is preferable that the previously detected expression
level of the cancer marker of the present invention in each cancer
patient is set to a standard value for each progression stage. Note
here that the expression level in the cancer patient may be a value
obtained from, for example, a single cancer patient, or may be a
value calculated from the expression levels in a plurality of
cancer patients by a statistical method.
[0067] Then, from the expression level in the subject and the
expression level in each cancer patient, the possibility of the
cancer in the subject can be evaluated by, for example, the
following method (3).
[0068] In the method (3), for example, the expression level of the
cancer marker in the subject is compared with the expression level
of the cancer marker in the biological sample of each of the cancer
patients at different progression stages, and it is determined that
the cancer in the subject is in the same progression stage as the
cancer in the patient exhibiting the same or similar expression
level. As described above, the expression level of the cancer
marker of the present invention in blood decreases with the
progression of canceration. Therefore, by determining the
expression level in the cancer patients at different progression
stages, not only the possibility of canceration of the subject but
also the progression stage of the cancer can be evaluated through
comparison with the thus-determined expression levels.
[0069] As described in the methods (1) to (3), when comparing the
expression level in the subject with that of the normal subject or
each of the cancer patients, the significant difference
therebetween can be judged by a statistical method such as a
t-test, an F-test, or a chi-square test, for example.
[0070] Furthermore, in order to improve the reliability of the
evaluation method of the present invention, the evaluation may be
carried out using the expression level of the cancer marker of the
present invention corrected as described above.
[0071] As described above, the corrected expression level can be
represented as the ratio between the amount of the cancer marker
expressed and the amount of the correction marker expressed in a
biological sample, for example. As a specific example, it can be
represented as "the amount of the cancer marker expressed/the
amount of the correction marker expressed". In the method (1) or
(2), the ratio calculated with regard to the subject may be
compared with the ratio calculated with regard to the normal
subject, for example. Furthermore, in order to make the comparison
of these ratios easier, it is preferable that, assuming the ratio
calculated with regard to the normal subject is "1", a relative
value of the ratio calculated with regard to the subject compared
to this is determined, for example. This allows the judgment to be
made more easily based on whether the calculated value with regard
to the subject is smaller or greater than "1". Furthermore, also in
the method (3), for example, the ratio calculated with regard to
the subject may be compared with the ratio calculated with regard
to each of the cancer patients at the different progression
stages.
[0072] According to such an evaluation method, for example, with
regard to a subject having a cancer at a preclinical stage, it is
possible to judge that the subject has a high possibility of the
cancer with high reliability whereas such judgment has been
difficult conventionally. Furthermore, for example, the stage of
cancer progression also can be judged with high reliability. Thus,
in prevention or treatments of cancers, information important in
determining strategies for medication, operation, etc. for example,
can be obtained with high reliability.
[0073] <Evaluation Reagent>
[0074] The evaluation reagent of the present invention is, as
described above, an evaluation reagent to be used in the evaluation
method of the present invention and characterized in that it
contains a reagent for detecting the cancer marker of the present
invention, i.e., a miRNA detection reagent for detecting at least
one miRNA selected from hsa-miR-92 and hsa-miR-494. According to
such an evaluation reagent, it is possible to carry out the
evaluation method of the present invention conveniently.
[0075] The present invention is characterized in that, as described
above, at least one of hsa-miR-92 and hsa-miR-494 is detected as a
cancer marker, and a method for detecting these miRNAs is by no
means limited. Therefore, it is only necessary that the miRNA
detection reagent contained in the evaluation reagent of the
present invention can detect either of these miRNAs, and for
example, the kind, composition, etc. of the reagent are by no means
limited. Furthermore, those skilled in the art can set detection
reagents for these miRNAs based on common general technical
knowledge.
[0076] The evaluation reagent of the present invention further may
contain any of various enzymes, buffer solutions, washing
solutions, dissolving solutions, dispersions, diluents and the
like, for example, depending on various detection methods.
Furthermore, the form of the evaluation reagent of the present
invention is not particularly limited. For example, it may be a
wet-type reagent in the liquid form or a dry-type reagent in the
dry form.
[0077] The miRNA detection reagent is not particularly limited, and
examples thereof include reagents to be used in the Northern blot
analysis and real-time RT-PCR detection method described above.
Specific examples of the miRNA detection reagent to be used in the
Northern blot analysis include labeled probes that can hybridize to
either of these miRNAs, such as those described above. Furthermore,
specific examples of the reagent to be used in the real-time RT-PCR
detection method include primers for amplifying cDNA from total
RNA, primers for amplifying either of these miRNAs, fluorescent
reagents that specifically bind to or intercalate into
double-stranded nucleic acids, and various reagents that can be
used in nucleic acid amplification, such as those described above.
Examples of the various reagents include nucleotide triphosphate
(dNTP) and enzymes such as DNA polymerase.
[0078] <Evaluation Kit>
[0079] The evaluation kit of the present invention is, as described
above, an evaluation kit to be used in the evaluation method of the
present invention and characterized in that it includes a miRNA
detection reagent for detecting at least one miRNA selected from
hsa-miR-92 and hsa-miR-494 (e.g., the evaluation reagent of the
present invention). According to such an evaluation kit, the
evaluation method of the present invention can be carried out
conveniently. In the evaluation kit of the present invention, the
evaluation reagent of the present invention is as described above,
for example.
[0080] It is preferable that the evaluation kit of the present
invention further includes a correction marker detection reagent
for detecting a correction marker. The correction marker detection
reagent is not particularly limited, and can be determined as
appropriate depending on the kind of the correction marker, the
method for detecting the correction marker, etc. described above.
As described above, the correction marker used in the evaluation
method of the present invention preferably is the correction marker
of the present invention. Thus, the correction marker detection
reagent preferably is a reagent for detecting the correction marker
of the present invention, i.e., a miRNA detection reagent for
detecting hsa-miR-638, for example. This miRNA detection reagent is
not particularly limited. As in the case of the reagent for
detecting the cancer marker of the present invention, examples of
the miRNA detection reagent include reagents to be used in the
Northern blot analysis and the real-time RT-PCR detection
method.
[0081] The form of the evaluation kit of the present invention is
not particularly limited. It may be a wet-type kit in the liquid
form or a dry-type kit in the dry form. Various reagents in the
evaluation kit of the present invention may be provided separately
and used together when the kit is used, or may be mixed together
before the kit is used, for example.
[0082] The evaluation kit of the present invention may be, for
example, a test tool in which the above-described evaluation
reagent of the present invention and other various reagents are
arranged. The form of the test tool is not particularly limited,
and examples of the test tool include reactors such as a
microreactor, chips such as a microchip, plates such as a
microtiter plate, and arrays such as a microarray. According to
such a test tool, for example, by detecting a necessary signal with
various kinds of existing detectors such as a real-time PCR device,
the evaluation method of the present invention can be carried out
easily. Furthermore, the test tool may include, for example, a
system for carrying out the following operations by computer
processing: numerical conversion of a detected signal; correction
of a measured signal value by the expression level of the
correction marker as described above; creation of a data file for
each subject; storage of the data file in a predetermined
directory; statistical analysis of measurement results obtained as
to subjects, normal subjects, and patients; and the like. Those
skilled in the art can design such a data processing system from
existing techniques, methods, and procedures based on common
general technical knowledge.
[0083] The evaluation kit of the present invention further may
include, for example, any of various appliances and the like that
can be used to carry out the evaluation method of the present
invention. Furthermore, it is preferable that the evaluation kit of
the present invention further include instructions for use, for
example.
[0084] <Correction Marker>
[0085] As described above, the correction marker of the present
invention contains hsa-miR-638 and is characterized in that it is
used to correct the expression level of the cancer marker of the
present invention in a biological sample in the evaluation method
of the present invention.
[0086] The inventors of the present invention conducted a diligent
study in order to obtain a novel correction marker that serves as
an internal standard in a biological sample. As a result, they
found out that the above-described hsa-miR-638 exhibits
constitutive expression in a biological sample, in particular, in a
blood sample, thus achieving the present invention. miRNA that
serves as a correction marker has not yet been reported, and the
miRNAs found by the inventors of the present invention are the
first such miRNA reported. According to such a correction marker,
for example, the comparison as to the cancer marker of the present
invention to be detected between subjects or in the same subject
over time can be carried out more reliably. The miRNA in the
correction marker of the present invention is as described
above.
[0087] The correction marker of the present invention is a miRNA
present in a biological sample, and the biological sample is not
particularly limited. The biological sample is not particularly
limited, and examples thereof include, as described above, blood,
saliva, and urine. Among them, the blood samples as described above
are preferable because the expression of the correction marker
therein remains constitutive regardless of whether the subject has
a cancer or not. Among the blood samples, for example, a sample
containing plasma, a sample containing serum, a sample containing
plasma and serum, and the like are more preferable.
[0088] The correction marker of the present invention can be used
to correct the expression level of a marker to be detected in a
biological sample. Specifically, the correction marker of the
present invention preferably is used to correct the expression
level of the cancer marker of the present invention. The correction
marker of the present invention is present constitutively
regardless of whether the subject has a cancer or not, for example.
Accordingly, the kind of the marker to be detected is not limited
to the cancer marker of the present invention. For example, it may
be a cancer marker whose expression level changes accompanying the
onset of a cancer. Note here that the marker to be detected may be,
for example, a marker whose expression level in a biological sample
varies depending on a disease.
[0089] <Correction Method by Correction Marker>
[0090] As described above, in order to correct the expression level
of the cancer marker of the present invention in a biological
sample in the evaluation method of the present invention, the
correction method of the present invention includes the steps
of:
[0091] measuring the expression level of the cancer marker of the
present invention in a biological sample;
[0092] measuring the amount of the correction marker expressed in
the biological sample; and
[0093] correcting the expression level of the cancer marker by
setting the ratio between the amount of the cancer marker expressed
and the amount of the correction marker expressed to a corrected
expression level of the cancer marker in the biological sample.
[0094] According to the correction method of the present invention,
since the correction marker is present constitutively in a
biological sample, by correcting the expression level of the cancer
marker of the present invention in a biological sample with this
correction marker, it becomes possible to compare, for example, the
expression levels of the cancer marker between different subjects,
the expression levels in the same subject over time, etc. with
still higher reliability.
[0095] It is to be noted that, as described above, the correction
method using the correction marker of the present invention can be
used not only for the cancer marker of the present invention but
also for other markers to be detected in a biological sample, for
example. Since the correction marker is present constitutively
regardless of whether the subject has a cancer or not, for example,
the marker to be detected preferably is a cancer marker whose
expression level changes accompanying the onset of a cancer.
Specific examples such a cancer marker include, in addition to the
cancer marker of the present invention, for example, miR-16-1-15a,
miR-145, let-7 family, miR-155, miR-17-92 cluster, miR-21, miR-221,
miR10-b, miR-128, miR-181a, miR-181b, miR-125b, miR-145, miR-143,
miR-133b, miR-31, miR-135b, miR-96, miR-183, miR-18, miR-224,
miR-199a, miR-195, miR-200a, miR-125a, miR-122, miR-126, miR-21,
miR-205, miR-15a, miR-16-1, miR-150, miR-222, miR-103, miR-107,
miR-204, miR-372, miR-373, miR-146b, miR-197, and miR-346.
[0096] The present invention is characterized in that the amount of
the cancer marker of the present invention expressed is corrected
by the correction marker of the present invention. Thus, methods
for measuring the amount of the cancer marker of the present
invention expressed and the amount of the correction marker of the
present invention expressed are by no means limited. For the
correction marker of the present invention, for example, the method
may be any method that can detect each miRNA as described above, as
in the case of the cancer marker of the present invention.
[0097] <Correction Reagent>
[0098] As described above, the correction reagent of the present
invention includes a miRNA detection reagent for detecting
hsa-miR-638 and is characterized in that it is used to correct the
expression level of the cancer marker of the present invention in a
biological sample in the evaluation method of the present
invention. According to such a correction reagent, the correction
method of the present invention can be carried out
conveniently.
[0099] The present invention is characterized in that, as described
above, hsa-miR-638 is detected as a correction marker, and a method
for detecting the miRNA is by no means limited. Therefore, it is
only necessary that the miRNA detection reagent contained in the
correction reagent of the present invention can detect this miRNA
as described above, and for example, the kind, composition, etc. of
the reagent are by no means limited. Furthermore, those skilled in
the art can set a miRNA detection reagent for detecting this miRNA
as appropriate based on common general technical knowledge.
[0100] The correction reagent of the present invention may contain
any of various enzymes, buffer solutions, washing solutions,
dissolving solutions, dispersions, diluents, and the like, for
example, depending on various detection methods for detecting the
miRNA. Furthermore, the form of the correction reagent of the
present invention is not particularly limited. For example, it may
be a wet-type reagent in the liquid form or a dry-type reagent in
the dry form.
[0101] The miRNA detection reagent is not particularly limited, and
examples thereof include reagents to be used in the Northern blot
analysis and real-time RT-PCR detection method described above.
Specific examples of the miRNA detection reagent to be used in the
Northern blot analysis include labeled probes that can hybridize to
either of these miRNAs, such as those described above. Furthermore,
specific examples of the reagent to be used in the real-time RT-PCR
detection method include primers for amplifying cDNA from total
RNA, primers for amplifying either of these miRNAs, fluorescent
reagents that specifically bind to or intercalate into
double-stranded nucleic acids, and various reagents that can be
used in nucleic acid amplification, such as those described above.
Examples of the various reagents include nucleotide triphosphate
(dNTP) and enzymes such as DNA polymerase.
EXAMPLES
[0102] Next, the present invention will be described with reference
to examples. It is to be noted, however, the present invention is
by no means limited by the following examples.
Example 1
[0103] Bloods were collected from a normal subject and cancer
patients shown in the following tables, and a fraction containing
serum and plasma (hereinafter referred to as a sample) was
separated from each blood. From the thus-obtained sample, total RNA
was extracted using Isogen-LS (trade name) (NIPPON GENE CO., LTD.),
and the concentration of the total RNA was adjusted so as to be 100
ng/.mu.l. Then, the total RNA was dephosphorylated using alkaline
phosphatase derived from calf small intestine (trade name "Alkaline
Phosphatase (Calf intestine) (CIAP)", TAKARA BIO INC.). Thereafter,
the total RNA was labeled with a cyanine dye using ligase (trade
name "T4 RNA Ligase (Cloned)", Ambion). Note here that these
operations were carried out using a kit (trade name "miRNA Labeling
Reagent and Hybridization Kit", catalog No. 5190-0408, Agilent
Technologies, Inc.) in accordance with a protocol attached thereto.
Furthermore, using a microarray slide (trade name "Human miRNA
Microarray kit 8.times.15K V2", catalog No. G4470B, Agilent
Technologies, Inc.), hybridization of the cyanine labeled total RNA
was caused, and signals were scanned using a scanner (trade name
"DNA Microarray Scanner", Agilent Technologies, Inc.). For the
signal detection, software programs "Feature Extraction 9.5.3
Software and Agilent Scan Control Software (ver.7.0)" accompanying
the scanner were used.
[0104] In the above-described manner, signal values indicating the
expression levels of hsa-miR-92a and hsa-miR-494 as the cancer
markers and hsa-miR-638 as the correction marker in the sample of
each of the subjects were obtained. Still further, with regard to
each subject, the following (A) and (B) were calculated.
(A) the ratio between the expression level of the cancer marker and
the expression level of the correction marker, represented by the
following formulae:
[0105] hsa-miR-92a/hsa-miR-638 (hereinafter, "92a/638")
[0106] hsa-miR-494/hsa-miR-638 (hereinafter, "494/638")
(B) a relative value obtained by dividing the ratios "92a/638" and
"494/638" of each of the subjects respectively by ratios "92a/638"
and "494/638" of the normal subject (BS63), represented by the
following formulae:
[0107] [subject 92a/638]/[normal subject 92a/638] (hereinafter,
"92a/638/BS63")
[0108] [subject 494/638]/[normal subject 494/638] (hereinafter,
[494/638/BS63])
[0109] The results thereof are shown in Table 1 and Table 2 below,
respectively. Table 1 shows the result obtained when the cancer
marker was hsa-miR-92a and the correction marker was hsa-miR-638.
Table 2 shows the result obtained when the cancer marker was
hsa-miR-494 and the correction marker was hsa-miR-638.
TABLE-US-00003 TABLE 1 Subject Normal Cancer patient subject No.
BS63 BS51 BS53 BS55 BS58 BS62 Sex woman woman man woman man woman
Disease normal colon gallbladder stomach colon stomach Ca cancer
cancer cancer cancer (sm2) (mac) hsa-miR-92a 318.97 359.199 294.319
25.146 928.489 258.925 hsa-miR-638 2317.28 59098 3215.28 8209.97
16700.4 25753.2 92a/638 0.138 0.006 0.092 0.003 0.056 0.01
92a/638/BS63 1.000 0.044 0.665 0.022 0.404 0.073
TABLE-US-00004 TABLE 2 Subject Normal Cancer patient subject No.
BS63 BS51 BS53 BS55 BS58 BS62 Sex woman woman man woman man woman
Disease normal colon gallbladder stomach colon stomach Ca cancer
cancer cancer cancer (sm2) (mac) hsa-miR-494 23.145 39.2726 2.74749
2.48419 13.6667 14.2209 hsa-miR-638 2317.28 59098 3215.28 8209.97
16700.4 25753.2 494/638 0.010 0.001 0.001 0 0.001 0.001
494/638/BS63 1.000 0.067 0.086 0.03 0.082 0.055
[0110] As can be seen from Table 1, it was found that, when
hsa-miR-92a was detected as the cancer marker and corrected with
hsa-miR-638 as the correction marker, the expression levels
92a/638/BS63 in the cancer patients were from 0.022 to 0.665, which
were much lower than "1.0" as the expression level 92a/638/BS63 in
the normal subject. Also, as can be seen from Table 2, it was found
that, when hsa-miR-494 was detected as the cancer marker and
corrected with hsa-miR-638 as the correction marker, the expression
levels 494/638/BS63 in the cancer patients were from 0.03 to 0.086,
which were much lower than "1.0" as the expression level
494/638/BS63 in the normal subject. These results demonstrate that,
by detecting the cancer marker of the present invention, the
presence or absence of canceration can be judged with high
reliability.
Example 2
[0111] Bloods were collected from acute myelogenous leukemia (AML)
patients (n=2) and normal subjects (n=7), and a fraction containing
serum and plasma (hereinafter referred to as a "sample") was
recovered by centrifuging each of the bloods at 15,680 m/s.sup.2
for 15 minutes. Except that the thus-obtained samples were used,
the expression of miRNAs in each sample was analyzed by microarray
analysis in the same manner as in Example 1. Then, with regard to
each of the samples derived from the normal subjects, the signal
value ranks (Individual rank_Normal) of the various miRNAs
expressed were determined, and also the signal value average ranks
(Average rank_Normal) of the various miRNAs among the samples
derived from the normal subjects were determined. Furthermore, in
order to examine the variation in expression of each miRNA among
the samples, the signal value rank of each of the various miRNAs in
each of the samples derived from the normal subjects was divided by
the signal value average rank of the corresponding miRNA among the
samples derived from the normal subjects, and the logarithm thereof
(log.sub.10(individual rank_Normal/Average rank_Normal]) was
determined (The base was 10).
[0112] Using these results, first, with regard to the samples
prepared from seven specimens derived from the normal subjects,
expression profiles of various miRNAs were compared with each
other. The result thereof is shown in FIGS. 1A and 1B. FIG. 1A
shows the signal intensity values of the various miRNAs contained
in each of the samples prepared from the seven specimens. In FIG.
1A, 1 to 7 on the horizontal axis indicate the respective
specimens, and the vertical axis indicates the signal intensity. In
FIG. 1A, "hsa-miR" is represented by "miR" (hereinafter the same).
FIG. 1B shows a logarithmic value (log.sub.10[individual
rank_Normal/Average rank_Normal]) of a value obtained by dividing
the rank of each of the various miRNAs in the seven normal subjects
by the average rank thereof. In FIG. 1B, the horizontal axis
indicates the respective miRNAs, and the vertical axis indicates
the logarithmic value.
[0113] As a result, as can be seen from FIG. 1A, in any of the
samples prepared from the seven specimens derived from the normal
subjects, expression of hsa-miR-638 and hsa-miR-92a was observed,
and further, it was found that hsa-miR-638 exhibited the highest
expression level. Furthermore, as can be seen from FIG. 1B, with
regard to hsa-miR-638, the above-described logarithmic value
regarding the signal value rank was 0. The absolute value of this
logarithmic value varies greatly about 0 as the difference in
behavior of the expression profile of the miRNA among the
respective samples of the normal subjects becomes noticeable, for
example. However, since this logarithmic value was "0" with regard
to hsa-miR-638, it was found that hsa-miR-638 exhibits similar
expression profile in any of the samples of the normal subjects.
These results demonstrate that hsa-miR-638 serves as a correction
marker in quantification of miRNA in a sample.
[0114] Next, with regard to the samples derived from the AML
patients, the signal value average ranks (Average rank_AML) of the
various miRNAs between the samples were determined. Then, the
signal value average rank (Average rank_AML) of each of the various
miRNAs between the samples derived from the two AML patients was
divided by the signal value average rank (Average rank_Normal) of
each of the various miRNAs among the samples derived from the seven
normal subjects to compare the expression profiles of the various
miRNAs in the samples derived from the AML patients. The result
thereof is shown in FIG. 2. FIG. 2 is a graph showing the ratio
(Average rank_AML/Average rank_Normal) between the signal value
average rank of each miRNA between the samples derived from the AML
patients and the signal value average rank of each miRNA among the
samples derived from the normal subjects. As the value of this
ratio becomes greater, it means that the difference in expression
profile is noticeable between the patients and the normal subjects.
In FIG. 2, the vertical axis indicates Average rank_AML/Average
rank_Normal, and the horizontal axis indicates the respective
miRNAs.
[0115] As can be seen from FIG. 2, the signal value average rank of
hsa-miR-92a in the AML patients was about 20 times greater than
that of hsa-miR-92a in the normal subjects. This indicates that the
expression of hsa-miR-92a decreased markedly in the AML patients.
This result demonstrates that hsa-miR-92a can serve as a cancer
marker for AML.
Example 3
[0116] From each of 77 subjects consisting of 39 men and 38 women,
a fraction containing plasma and serum (hereinafter referred to as
a "sample") was recovered in the same manner as in Example 2. Among
these subjects, 16 subjects were normal subjects and 61 subjects
were acute leukemia patients. The FAB classification of the acute
leukemia patients is as follows:
TABLE-US-00005 AML M0 2 (3.2%) AML M1 11 (18.0%) AML M2 19 (31.1%)
AML M3 10 (16.3%) AML M4 3 (4.9%) AML M4E 1 (1.6%) AML M5 1 (1.6%)
AML M5b 1 (1.6%) AML M6 1 (1.6%) AML M7 2 (3.2%) AML (with
multilineage dysplasia) 3 (4.9%) ALL L2 2 (3.2%) ALL ph+ 1 (1.6%)
ALL preB 4 (6.5%)
[0117] The expression levels of hsa-miR-92a and hsa-miR-638 in each
of the samples were analyzed by quantitative RT-PCR using a
commercially available TaqMan (trademark) MicroRNA Assay (Applied
Biosystems). Unless otherwise stated, the analysis was conducted in
accordance with the instructions for use accompanying the
commercially available reagent used.
[0118] First, the total RNA was isolated from the sample derived
from each of the subjects in the same manner as in Example 2. With
20 ng of the total RNA as a template, a reverse transcription
reaction was carried out using "TaqMan (trademark) MicroRNA RT Kit"
(trade name) (Applied Biosystems). Specifically, 20 ng of the total
RNA (input RNA) were added to 15 .mu.l of a reverse transcription
reaction solution having the following composition, and the
resultant mixture was incubated at 16.degree. C. for 30 minutes, at
42.degree. C. for 30 minutes, and at 85.degree. C. for 5 minutes,
thereby causing a reverse transcription reaction. Note here that,
in the following reaction solution, the following primer for
hsa-miR-92a and primer for hsa-miR-638 were both miRNA-specific
stem-loop primers (Looped RT primers). Specifically, as the
following primer for hsa-miR-92a, Assay Name hsa-miR-92, Product
number 4373013 of TaqMan (trademark) MicroRNA Assay (Applied
Biosystems) was used, and as the primer for hsa-miR-638, Assay Name
hsa-miR-638, Product number 4380986 of TaqMan (trademark) MicroRNA
Assay (Applied Biosystems) was used.
TABLE-US-00006 TABLE 3 (Reverse Transcription Reaction Solution) 10
.times. RT buffer*.sup.1 1.5 .mu.l 100 mmol/l dNTPs containing dTTP
0.15 .mu.l 20 units/.mu.l RNase inhibitor*.sup.1 0.188 .mu.l 50
units/.mu.l reverse transcriptase*.sup.2 1 .mu.l Primer for
hsa-miR-92a 1 .mu.l Primer for hsa-miR-638 1 .mu.l Input RNA 10.16
.mu.l Total 15 .mu.l *.sup.1trade name "TaqMan (trademark) MicroRNA
RT Kit" (Applied Biosystems) *.sup.2trade name "MultiScribe
(trademark) Reverse Transcriptase (Applied Biosystems)
[0119] Subsequently, with a transcription product obtained through
the reverse transcription reaction as a template, quantitative
real-time PCR was carried out using Mx3005P (trademark) QPCR system
(STRATAGENE). As the reverse transcription product, 1 .mu.l of
diluted solution of the reverse transcription product obtained by
mixing the reaction solution after the above-described reverse
transcription reaction and nuclease-free water at a ratio of 1:2
(volume ratio) was used. Specifically, 20 .mu.l of a PCR reaction
solution having the following composition to which 1 .mu.l of the
diluted solution had been added was treated at 95.degree. C. for 2
minutes and then was subjected to 50 cycles of incubation with a
treatment at 95.degree. C. for 15 seconds and 60.degree. C. for 1
minute as one cycle. In the following PCR reaction solution, the
following buffer contained a primer set included in the
above-described TaqMan (trademark) MicroRNA Assay (Assay Name
hsa-miR-92: Product number 4373013: Applied Biosystems) for
hsa-miR-92a detection and TaqMan (trademark) Probe. Alternatively,
the buffer contained a primer set included in the above-described
TaqMan (trademark) MicroRNA Assay (Assay Name hsa-miR-638: Product
number 4380986: Applied Biosystems) for hsa-miR-638 detection and
TaqMan (trademark) Probe. In the probe for detecting hsa-miR-92a
and the probe for detecting hsa-miR-638, the 5' end was labeled
with FAM and the 3' end was labeled with TAMRA.
TABLE-US-00007 TABLE 4 (PCR Reaction Solution) 20 .times. PCR
buffer*.sup.1 1 .mu.l diluted solution of reverse transcription
product 1 .mu.l TaqMan (trademark) reagent*.sup.2 10 .mu.l
nuclease-free water 8 .mu.l Total 20 .mu.l *.sup.1trade name
"TaqMan (trademark) MicroRNA Assays" (Applied Biosystems) *.sup.22
.times. TaqMan (trademark) Universal Master (Applied Biosystems)
MixAmpErase (trademark) UNG (Applied Biosystems) was not added
[0120] Data obtained through the quantitative RT-PCR was analyzed
with MxPro-Mx3005P (trademark) version 3.00 (STRATAGENE), and the
automatic Ct setting for baseline adaptation and threshold for Ct
determination also were conducted. Then, with regard to the sample
derived from each subject, the ratio (hsa-miR-92a/hsa-miR-638)
between the fluorescence intensity indicating hsa-miR-92a as the
cancer marker and the fluorescence intensity indicating hsa-miR-638
as the correction marker was determined. Furthermore, the
Mann-Whitney U test was conducted to examine the statistical
significance of the difference between the ratios determined from
the samples derived from the normal subjects and the ratios
determined from the samples derived from the acute leukemia
patients.
[0121] The result thereof is shown in FIG. 3. FIG. 3 is a graph
showing the fluorescence intensity ratio (hsa-miR-92a/hsa-miR-638)
with regard to the samples derived from the normal subjects and the
samples derived from the acute leukemia patients. As can be seen
from FIG. 3, with regard to the samples derived from the normal
subjects, the fluorescence intensity ratios were as follows:
median=0.4672; 25%=0.0051; 75%=0.0138; maximum value=2.3388;
minimum value=0.0189; and average value=0.74587. On the other hand,
with regard to the samples derived from the acute leukemia
patients, the fluorescence intensity ratios were as follows:
median=0.0129; 25%=0.0058; 75%=0.0274; maximum value=0.1085;
minimum value=0.0004; and average value=0.020585. From this result,
it was found that hsa-miR-92a expression decreased markedly in the
samples derived from the patients. Furthermore, P value, which
indicates the statistical significance of the difference between
the normal subjects and the acute leukemia patients, was
2.0.times.10.sup.-8, from which it was found that the difference
was significant enough. The result described above demonstrates
that hsa-miR-92a in serum or plasma is a particularly highly
sensitive marker for acute leukemia.
Example 4
[0122] With regard to two pancreas cancer patients, hsa-miR-92a and
hsa-miR-638 were measured in the same manner as in Example 3 before
and after operation, and the fluorescence intensity ratio
(hsa-miR-92a/hsa-miR-638) was calculated. As a result, relative
values of the fluorescence intensity ratio after operation in the
two patients assuming that the fluorescence intensity ratio before
operation was 1 were 2.67 and 1.40, respectively. It can be seen
from this result that the amount of hsa-miR-92a expressed decreases
when a subject has pancreas cancer, and the amount of hsa-miR-92a
expressed increases after the pancreas cancer is treated. That is,
it can be said that it is possible to judge whether or not a
subject has pancreas cancer based on the amount of hsa-miR-92a
expressed.
[0123] Similarly, also with regard to four liver cancer patients,
hsa-miR-92a and hsa-miR-638 were measured in the same manner as in
Example 3 before and after operation, and the fluorescence
intensity ratio (hsa-miR-92a/hsa-miR-638) was calculated. As a
result, relative values of the fluorescence intensity ratio after
operation in the four patients assuming that the fluorescence
intensity ratio before operation was 1 were 6.13, 12.6, 156.52, and
12.12, respectively. It can be seen from this result that the
amount of hsa-miR-92a expressed decreases when a subject has liver
cancer, and the amount of hsa-miR-92a expressed increases after the
liver cancer is treated. That is, it can be said that it is
possible to judge whether or not a subject has liver cancer based
on the amount of hsa-miR-92a expressed. It is to be noted that,
with regard to other cancers, the similar results were
obtained.
INDUSTRIAL APPLICABILITY
[0124] According to the present invention, by detecting the
expression level of the cancer marker of the present invention in a
biological sample, it becomes possible to judge the presence or
absence of the development or progression of cancers with high
reliability, for example. Furthermore, the cancer marker of the
present invention provides a marked difference between negative and
positive regarding the canceration, for example. Thus, according to
the cancer marker of the present invention, it is possible to
detect cancers at an initial stage whereas such detection is
difficult by general palpation and the like
SEQUENCE LISTING
[0125] TF09011-0.5T25.txt
Sequence CWU 1
1
3122RNAHomo sapiens 1uauugcacuu gucccggccu gu 22222RNAHomo sapiens
2ugaaacauac acgggaaacc uc 22325RNAHomo sapiens 3agggaucgcg
ggcggguggc ggccu 25
* * * * *