U.S. patent application number 14/099685 was filed with the patent office on 2014-04-10 for method for early diagnosis of liver cancer.
This patent application is currently assigned to National Tsing Hua University. The applicant listed for this patent is National Tsing Hua University. Invention is credited to Yu-Ting Chou, Hsiao-Chen Tu, Horng-Dar Wang, Chiou-Hwa Yuh.
Application Number | 20140099647 14/099685 |
Document ID | / |
Family ID | 47597498 |
Filed Date | 2014-04-10 |
United States Patent
Application |
20140099647 |
Kind Code |
A1 |
Wang; Horng-Dar ; et
al. |
April 10, 2014 |
METHOD FOR EARLY DIAGNOSIS OF LIVER CANCER
Abstract
Disclosed is a method for early diagnosis of liver cancer. The
method comprises the steps of:(A) providing a sample obtained from
a subject; (B) assessing the expression level of four subtypes of
.alpha.-mannosidase genes consisting of MAN1C1 in the sample; (C)
comparing the expression level of .alpha.-mannosidase genes in the
sample with a normal control; and (D) determining whether the
subject having a risk of suffering liver cancer in accordance with
the result of step (C); wherein while the MAN1C1 expression level
of the sample is lower than that in the normal control, the subject
is determined to have a risk of suffering liver cancer.
Additionally, while MAN1A1, MAN1A2 and MAN1B1 expression levels in
the sample are higher than those in control group, the subject is
determined to suffer from liver cancer and has a risk of
metastasis. In the future, MAN1C1 can be applied to early diagnosis
of liver cancer and metastasis, suppression of liver metastasis,
and screening agents for treating liver cancer.
Inventors: |
Wang; Horng-Dar; (Hsinchu,
TW) ; Yuh; Chiou-Hwa; (Hsinchu, TW) ; Tu;
Hsiao-Chen; (Hsinchu, TW) ; Chou; Yu-Ting;
(Hsinchu, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
National Tsing Hua University |
Hsinchu |
|
TW |
|
|
Assignee: |
National Tsing Hua
University
Hsinchu
TW
|
Family ID: |
47597498 |
Appl. No.: |
14/099685 |
Filed: |
December 6, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
13220055 |
Aug 29, 2011 |
8628920 |
|
|
14099685 |
|
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/455 |
Current CPC
Class: |
G01N 33/5011 20130101;
G01N 33/57438 20130101 |
Class at
Publication: |
435/6.12 ;
435/455 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 27, 2011 |
TW |
100126558 |
Claims
1. A method for early diagnosis of liver cancer, comprising the
steps of: (A) providing a sample obtained from a subject; (B)
assessing MAN1C1 expression levels in the sample by detecting
MAN1C1 RNA or protein expression levels in the sample; (C)
comparing the MAN1C1 expression levels in the sample with MAN1C1
expression levels in a normal control; and (D) determining whether
the subject having a risk of suffering liver cancer in accordance
with the result of step (C); wherein while the MAN1C1 expression
levels of the sample is lower than that in the normal control, the
subject is determined to have a risk of suffering liver cancer;
wherein the sample and the normal control are liver biopsies.
2. The method as claimed in claim 1, wherein the MAN1C1 expression
levels in the sample are at least two folds lower than the MAN1C1
expression levels in the normal control.
3. The method as claimed in claim 1, wherein the subject is
hepatitis B virus carrier.
4. A method of inhibiting metastasis of liver cancer, comprising a
step of overexpressing MAN1C1 by increasing .alpha.-mannosidase IC
expression levels in a liver cancer cell.
5. The method as claimed in claim 4, wherein overexpressing MAN1C1
can inhibit the MMP9 expression levels.
6. A method of screening a drug for liver cancer, comprising the
steps of: (A) providing a liver cancer cell treated with a drug;
(B) assessing MAN1C1 expression levels in the liver cancer cell;
(C) determining whether the drug has a therapeutical effect
according to the MAN1C1 expression levels.
7. The method as claimed in claim 6, further comprising assessing
MMP9 expression levels in the liver cancer cell, and determining
whether the drug has a therapeutical effect according to the MMP9
expression levels.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/220,055, filed Aug. 29, 2011, which claims priority
under 35 U.S.C. .sctn.119(a) on Patent Application No(s). 100126558
filed in Taiwan, Republic of China, on Jul. 27, 2011, the entire
contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to a method for diagnosis of
liver cancer and metastasis, particularly relates to a method using
MAN1C1 for early diagnosis of liver cancer, inhibition of
metastasis, and screening drugs for treating liver cancer.
BACKGROUND OF THE INVENTION
[0003] Liver cancer is one of the most common malignant tumors in
Taiwan, more than 7,000 people died as a result of liver cancer
each year. The symptom was not obvious in early stage; the patients
feel nothing after having liver cancer for long time. Until the
progression of the disease to some degree, it will gradually
produce some symptoms such as liver pain, loss of appetite,
fatigue, weakness, losing weight etc. At the later stage, patients
develop jaundice, ascites, vomiting, coma and other symptoms.
Patients with liver cancer often palpable huge tumor on abdominal,
however this has come in late, and even metastasis to the lungs and
other organs. The overall duration of liver cancer is about two and
half years, of which first two years are the early stage without
symptoms. Once the symptoms appear, the survival time is only six
months. Liver cancer is very difficult to diagnosis. For most of
the patients, liver transplantation is their only hope. The method
for early diagnosis could save countless lives. Some of the current
method of detection of tumor growth is often based on the existence
of the blood concentrations of specific markers. For the detection
of liver cancer, commonly use .alpha.-fetoprotein (AFP) in
diagnosing liver cancer. AFP is a normal fetal serum protein
synthesized by the liver, yolk sac, and gastrointestinal tract that
shares sequence homology with albumin. It is a major component of
fetal plasma, reaching a peak concentration of 3 mg/ml at 12 weeks
of gestation. AFP can be found in 95% primary liver cancer
patients' blood, it is also used as a marker for screening liver
cirrhosis and hepatitis. Due to AFP's low specificity, fake
positive results are frequently occurs. It is estimated that 6
billion NTD commercial potential exist in Taiwan's market regarding
liver caner early diagnosis, and a more gigantic potential exists
in foreign market.
[0004] The process of N-glycosylation consists of a covalent
linkage of a specific oligosaccharide (Blc3Man9GlcNAc2) on a
nascent protein. Once the oligosaccharide is transferred, several
subsequent steps of maturation will occur along the secretory
pathway. N-glycosylation is ubiquitous in eukaryotes. First steps
of N-glycosylation are conserved through eukaryotes from yeast to
human, which take place in the endoplasmic reticulum. The following
and last steps of maturation leading to polymannosylated
glycoprotein, which occur in the Golgi apparatus, and are species
specific. The function of .alpha.-mannosidase is to trim the
mammose of the glycoprotein in the process of N-glycosylation.
There are many types of .alpha.-mannosidase in human. Previous
studies revealed that some specific types of mannosidase are
related to the formation of cancer, supported with high expression
level of mannosidase in particular cancer. Swainsonine (SW),
.alpha.-mannosidase II inhibitor can efficiently decrease the tumor
size in nude mice injected with leukemia cell (MDAY-D2) (Goss,
1995). Deoxymannojirimyci (DMJ), .alpha.-mannosidase I inhibitor
decreased migration ability of bladder cancer cells (T24)
(Przybylo, 2005). DMJ also can induce liver cancer cell (7721)
toward apoptosis (Przybylo, 2005). Based on these literatures, the
present invention further discover the expression level of four
.alpha.-mannosidase genes in different stages of liver cancer and
their correlation to migration ability. Furthermore, early
diagnosis of cancer using .alpha.-mannosidase has not been reported
previously, and we identified one type of
.alpha.-mannosidase-MAN1C1 can predict the early stage.
SUMMARY OF THE INVENTION
[0005] Though high expression level of .alpha.-mannosidase has been
known to be associated with specific cancers, and suppressing the
activity of .alpha.-mannosidase may inhibit growth, induce
apoptosis even decrease migration ability of cancer cells. However,
early diagnosis of liver cancer using MAN1C1 has not been reported
before. Furthermore, expression levels of four .alpha.-mannosidase
subtypes have never been identified in different liver cancer
stages.
[0006] One object of the present invention is to provide a method
for early diagnosis of liver cancer by low expression of
MAN1C1.
[0007] Another object of the present invention is to provide a
method for determining liver cancer and metastasis by high
expression of MAN1A1, MAN1A2 and MAN1B1.
[0008] Yet another object of the present invention is to provide a
method for inhibiting metastasis by overexpressing MAN1C1 in liver
cancer cells.
[0009] Yet another object of the present invention is to provide a
marker for screening target drug for treating liver cancer.
[0010] In one embodiment, the method for early diagnosis of liver
cancer comprises the steps of: (A) providing a sample obtained from
a subject; (B) assessing the expression level of four subtypes of
.alpha.-mannosidase genes consisting of MAN1A1, MAN1A2, MAN1B1 and
MAN1C1 in the sample; (C) comparing the expression level of
.alpha.-mannosidase genes in the sample with a normal control; and
(D) determining whether the subject having a risk of suffering
liver cancer in accordance with the result of step (C); wherein
while the MAN1C1 expression level of the sample is lower than that
in the normal control, the subject is determined to have a risk of
suffering liver cancer. Additionally, while MAN1A1, MAN1A2 and
MAN1B1 expression levels in the sample are higher than those in
control group, the subject is determined to suffer from liver
cancer and has a risk of metastasis.
[0011] Preferably, the expression levels of MAN1A1, MAN1A2, MAN1B1
and MAN1C1 in the sample are at least two folds higher or lower
than those in the normal control; wherein step (D) further
comprises comparing MMP9 expression level in the sample with a
normal control, while MAN1A1, MAN1A2 and MAN1B1 expression levels
in the sample are higher than those in control group, and the MMP9
expression level in the sample is higher than in the normal
control, the subject is determined to have a risk of liver
metastasis. The expression level of .alpha.-mannosidase (MAN1A1,
MAN1A2, MAN1B1 and MAN1C1) and MMP9 mentioned above can be either
RNA or protein, and the subject is hepatitis B virus carrier, and
the sample is a liver tissue obtained from the subject.
[0012] In another embodiment, the method of inhibiting metastasis
in liver cancer cell comprises a step of overexpressing MAN1C1 in a
liver cancer cell so as to inhibit liver metastasis. Preferably,
overexpressing MAN1C1 can inhibit the MMP9 expression level in the
liver cancer cell.
[0013] In yet another embodiment, the method of screening a drug
for liver cancer, comprises the steps of: (A) providing a liver
cancer cell treated with a drug; (B) assessing MAN1C1 expression
level of the liver cancer cell; (C) determining whether the drug
has a therapeutical effect according to the MAN1C1 expression
level.
[0014] In the future, MAN1C1 can be applied to early diagnosis of
liver cancer and metastasis, suppression of liver metastasis, and
screening agents for treating liver cancer.
[0015] The embodiments of the present invention are further
described through below detailed examples and the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 demonstrates the Golgi N-linked carbohydrate
processing pathway.
[0017] FIG. 2A-2B demonstrate a flowchart of experiments in the
present invention.
[0018] FIG. 3A-3E demonstrates MAN1A1, MAN1A2, MAN1B1, MAN1C and
MAN1C1 expression levels using the samples of human liver cancer
patients carry hepatitis B virus.
[0019] FIG. 4 demonstrates the Q-PCR result of endogenous MAN1A1,
MAN1A2, MAN1B1, and MAN1C1 expression levels in different cell
lines.
[0020] FIG. 5A-5E demonstrate the cell migration assay results
after overexpression of MAN1A1 and MAN1C1.
[0021] FIG. 6A-6E demonstrate the cell migration assay results
after shRNA knockdown of MAN1A1, MAN1A2 and MAN1B1.
[0022] FIG. 7A-7C demonstrate the cell migration assay results
after stable overexpression of MAN1C1 in Hep3B (MAN1C1/Hep3B).
[0023] FIG. 8 demonstrate the in vivo cell migration assay results
of 293T, PLC5 and Hep3B cells by xenotransplantation into
zebrafish.
[0024] FIG. 9 demonstrate the cell migration assay results of
MAN1C1-overexpressed Hep3B cells (MAN1C1/Hep3B) by in vivo
xenotransplantation into zebrafish.
[0025] FIG. 10A-10B demonstrate the MTT assay result.
[0026] FIG. 11A-11B demonstrate MMP9 expression level in
MAN1C1-overexpressed cells.
DETAILED DESCRIPTION
[0027] A method for early diagnosis of liver cancer and prediction
of metastasis is described with reference to the preferred
embodiments below, it is apparent to those skilled in the art that
a variety of modifications and changes may be made without
departing from the scope of the present invention which is intended
to be defined by the appended claims.
[0028] FIG. 1 demonstrates the Golgi N-linked carbohydrate
processing pathway. As shown in FIG. 1, .alpha.-1, 2 mannosidase I
plays a role for trimming carbohydrate branches in carbohydrate
processing. The inhibitors such as DMJ and SW can inhibit
oligosaccharide chain trimming. Oligosaccharide chain synthesis and
processing includes a series of glycoside hydrolases, such as
glucosidase (i.e. glucosidase II) and mannosidase (i.e. mannosidase
I and mannosidase II). Mannosidase I and mannosidase II mainly
present in Golgi apparatus, these enzymes can be inhibited and
terminate the Golgi N-linked carbohydrate processing, so as to
produce high-mannose and complex-type N-linked glycans. .alpha.-1,
2 mannosidase I inhibitor DMJ and .alpha.-mannosidase II inhibitor
SW can influence glycan epitope expression and further inhibit
tumor cell migration, invasion and growth in vitro, which shows a
potential ability of inhibiting cancer metastasis.
[0029] FIG. 2A-2B demonstrate a flowchart of experiments in the
present invention. As shown in FIG. 2A, the quantitative PCR was
performed to assess endogenous .alpha.-1, 2 mannosidase mRNA in
mice and human. As shown in FIG. 2B, MAN1A1, MAN1A2, MAN1B1 and
MAN1C1 cloning was performed. Overexpression and shRNA knockdown of
.alpha.-1, 2 mannosidase were performed in the in vitro cell
migration assay. Also a MAN1C1 stable transfected cell line was
established and injected into zebrafish embryo, and an in vivo cell
migration assay was then performed. Simultaneously, Q-PCR
experiment was performed to assess if MMP9 mRNA level has changed
due to .alpha.-1, 2 mannosidase overexpression.
[0030] FIG. 3 demonstrates MAN1A1, MAN1A2, MAN1B1, MAN1C and MAN1C1
expression levels in liver tissue obtained from hepatitis B virus
positive liver cancer patients. As shown in FIG. 3A-3E, MANA1,
MANA2, MANB1, MAN1C and MAN1C1 mRNA level of liver cancer patients
were assessed by Q-PCR, wherein MANIC was part of MAN1C1. Those
patients were separated into three stages: stages I, II and III,
the latter stage represents the more aggressive cancerous
condition. The cancerous liver tissue was used as experiment group,
and the non-cancerous liver tissue obtained from the same patient
was used as a control group. In cancerous tissue, two-fold
expression higher than control group was defined as overexpression,
and two-fold expression lower than control group was defined as
decreased expression. As shown in FIG. 3A-3E, MAN1A1, MAN1A2 and
MAN1B1 were elevated aggressive cancerous condition, particularly
two folds than normal liver tissue. However, MAN1C1 expression in
the early stage liver cancer patient was lower than that in normal
liver tissue with 2 folds.
[0031] FIG. 4 demonstrates the Q-PCR result including: (A)
endogenous MAN1A1, MAN1A2 and MAN1B1 RNA levels; performing in
vitro cell migration assay in different cell lines (B) PLC5 and (C)
Hep3B, to determined which cell line for further overexpression and
knockdown experiments (D) endogenous MAN1C1 RNA levels in different
cell lines. A serial dilution (10.sup.-3.about.10.sup.-9) was
performed by using green fluorescent protein (GFP) DNA with known
molecule numbers to be a standard curve. By using the standard
curve, Ct value was calculated to molecule numbers by
interpolation, such that endogenous MAN1A1, MAN1A2, MAN1B1 and
MAN1C1 mRNA numbers in PLC5, Hep3B and HepG2 cells were obtained.
10.sup.5 cells were added in 3000 of serum free DMEM and incubated
on a transwell culture dish which had a membrane with 6.4 mm
diameter and 8.0 .mu.m pore size. 500 .mu.l medium (10% FBS in
DMEM) was added into lower chamber, and the dish was incubated at
37.degree. C. for 48 hours, and the cells in the lower chamber was
stained with 1.times. DAPI and calculated. The result was shown in
figure, endogenous .alpha.-1, 2 mannosidase mRNA levels were varied
in different cells, and the migration ability of Hep3B was greater
than PLC5. According to endogenous mRNA level and cell migration
ability (E), PLC5 cell line was determined to be used in MAN1A1
overexpression experiment, Hep3B was determined to be used in
MAN1A1, MAN1A2 and MAN1B1 knockdown experiments, and MAN1C1
overexpression was performed in Hep3B.
[0032] FIG. 5A-5E demonstrate the cell migration assay results
after overexpression of MAN1A1 and MAN1C1 for two days. As shown in
FIG. 5A, Q-PCR was performed to quantify MAN1A1 and MAN1C1 mRNA
expression level, and untransfected cells (mock) were used as
control. Cell migration assay results were shown in FIGS. 5B, 5C,
5D and 5E, and the method used was similar to FIG. 4B and 4C.
Results shown in FIG. 5A demonstrated that MAN1A1 and MAN1C1 mRNA
were successfully elevated to 11.73 and 160278 folds as compared
with Hep3B. Furthermore, MAN1A1 overexpression advanced the ability
of cell migration to 2.41 folds, and MAN1C1 overexpression reduced
the ability of cell migration to 0.48 folds. FIG. 5B and 5C are
photos which demonstrate cell migration assay results of PLC5 and
MAN1A1 overexpression in PLC5. FIG. 5D and 5E are photos which
demonstrate cell migration assay results of Hep3B and MAN1C1
overexpression in Hep3B.
[0033] FIG. 6A-6E demonstrate the cell migration assay results
after shRNA knockdown of MAN1A1, MAN1A2 and MAN1B1 for two days. As
shown in FIG. 5A, Q-PCR was performed to quantify MAN1A1, MAN1A2
and MAN1B1 mRNA expression level, and untransfected cells (mock)
were used as control. Cell migration assay results were shown in
FIGS. 6B, 6C, 6D and 6E, and the method used was similar to FIG. 4B
and 4C. Results shown in FIG. 6A demonstrated that MAN1A1, MAN1A2
and MAN1B1 mRNA after knockdown with shRNA were successfully
reduced to 55%, 62% and 64% as compared with untransfected Hep3B
cells. Furthermore, MAN1A1, MAN1A2 and MAN1B1 knockdown reduced the
ability of cell migration to 31%, 38% and 45% as compared with
untransfected Hep3B cells. FIG. 6B-6E are photos which demonstrate
cell migration assay results of Hep3B before (B) and after shRNA
knockdown of MAN1A1(C), MAN1A2(D) and MAN1B1(E).
[0034] FIG. 7A-7C demonstrate the cell migration assay results
after stable overexpression of MANIC1 in Hep3B (MAN1C1/Hep3B).
Stable overexpressed Hep3B was used as experiment group, and
original Hep3B was used as control group. Methods used in FIG. 7A:
Q-PCR was performed to quantify MAN1C1 mRNA expression level, and
untransfected cells (mock) were used as control. The cell migration
assay performed in FIGS. 7B and 7C were similar to FIGS. 4B and 4C,
except the analyzing time was after 24 hr. As shown in figures,
cell migration ability of cells with stable overexpression of
MAN1C1 was reduced to 16% as compared with original Hep3B. FIGS. 7B
and 7C are photos which demonstrate cell migration assay results of
Hep3B and stable overexpression of MAN1C1 in Hep3B.
[0035] FIG. 8 demonstrate in vivo cell migration assay results of
293T, PLC5 and Hep3B cells by xenotransplantation to zebrafish
embryos. 293T is a transformed human kidney cell line. PCL5 and
Hep3B are human hepatoma cell lines. Establishment of the
xenotransplantation zebrafish animal model in FIG. 8: using DiI to
label the suspension of cells and incubated in PBS before
injection, and the labeled cells were microinjected into 2 days old
zebrafish embryos. Each injection amount was 4.6 nl and contained
400 cells, and the injection position was the rear of yolk. The
injected cells were monitored for 3 days after injection. Previous
in vitro experiment results found that migration ability of Hep3B
was greater than PLC5, and the identical conclusion was once
identified by in vivo experiment, and the 293T were non-cancerous
control. According to statistic analysis results, cell migration
was observed in 3.33% of zebrafish after injection of 293T cells.
Three days after injection, cell migration was observed in 62% of
zebrafish after injection of Hep3B cells. These results were
consistent with the previous in vitro experiments. After 3 days of
microinjection, 293T and PLC5 cells were still retained in yolk
cavity. The position of yolk cavity where Hep3B cells injected. The
cells were found to migrate to tail after 1 day of injection.
[0036] FIG. 9 demonstrate the cell migration assay results of
MAN1C1-overexpressed Hep3B cells (MAN1C1/Hep3B) by in vivo
xenotransplantation. Stable overexpression of MAN1C1 in Hep3B was
used as experiment group, and Hep3B was used as control. The cells
of experiment group and control were microinjected into zebrafish
yolk. Each injection contains 400 labeled cells, and these cells
were observed and recorded at 1.sup.St and 3.sup.rd day. The method
used was similar to FIG. 8, however experiment group was stable
overexpressed MAN1C1 Hep3B, and the control contained large amount
of expression vectors. According to statistic analysis results,
cell migration ability was 18.6% in experiment group, and cell
migration ability was 36.2% in control group. According to the
results, three days after injection, cell migration ability of
stable overexpressed MAN1C1 in Hep3B was two folds than original
Hep3B. The cells were found to migrate to tail in control group
after 1 day of injection, this result was also found after 3 days
of injection. 80% of MAN1C1/Hep3B cells were observed that Hep3B
cells were retained in yolk cavity after 3 days of injection.
[0037] FIG. 10 demonstrates MTT assay result. X-axis represents
time (hour), and Y-axis represents proliferation fold. Experiment
method: 1. 6,000 cells were seeded on a 96-well culture dish. 2.
200 .mu.l of mixed medium (MTT:DMEM=1:9) was added into the dish
every 24 hr, and cells were incubated for 3 hr at 37.degree. C. 3.
100 .mu.l of DMSO was used to break the cells, and absorbance value
(OD.sub.570nm) of each sample was measured. Experiment group:
overexpression of MAN1A1 in Hep3B (A) or stable overexpression of
MAN1C1 in Hep3B (B). Control group: nontransfected Hep3B cells. As
shown in MTT assay results of FIG. 10A, overexpression of MAN1A1
promotes Hep3B cells growing faster than nontransfected cells. As
shown in FIG. 10B, the growth curves of experiment group
(overexpression of MAN1C1) and control has no difference.
[0038] FIG. 11A-11B demonstrate MMP9 expression level in MAN1A1 or
MAN1C1-overexpressed cells. As shown in FIG. 11A, Q-PCR was used to
assess MMP9 expression level, untransfected cells were used as
control and expression more than 2 folds was defined as
overexpression. The results suggest overexpression of MAN1A1
increased MMP9 expression to 2.3 folds than control, and
overexpression of MAN1C1 suppressed MMP9 expression to 0.33 folds.
As shown in FIG. 11B, RT-PCR was performed to amplify MAN1C1 and
MMP9 cDNA, and 18s RNA was used as control. MMP9 expression in
stable overexpression MAN1C1 Hep3B cellsd was reduced in comparison
with control group.
[0039] As results disclosed in the present invention, three genes:
MAN1A1, MAN1A2 and MAN1B1 were overpressed in liver cancer when
compare to the normal counterpart. However, the expression of
MAN1C1 was down-regulated in HCC patients when compare to normal
liver tissues. Moreover, 94% of HBV carrier HCC patients exhibit
over two-fold decreased MAN1C1 expression as early as stage I. This
result indicated that the decreasing expression of MAN1C1 might be
a potential biomarker for early diagnosis for HCC. Those expression
patterns implied MAN1A1, MAN1A2 and MAN1B1 probably are potential
oncogenes, and the MAN1C1 might functions as tumor suppressor. In
order to study the role of four .alpha.-mannosidase genes during
hepatocarcinogenesis, we first cloned the genes for MAN1A1, MAN1A2,
MAN1B1 and MAN1C1, and used cell line to investigate the
proliferation, migration and other genes' expression after
over-expressed or knockdown those genes. It was found that
overexpression of MAN1A1 into PLC5 cells can enhance the migration
ability, and knockdown of MAN1A1, MAN1A2 and MAN1B1 can decrease
the migration ability in Hep3B cells. On the other hand,
overexpression MAN1C1 in Hep3B cell decreased migration ability by
in vitro transwell assay. To further determine how .alpha.-1, 2
mannosidase I influenced migration ability, hepatic cell lines with
stable overexpression of .alpha.-1, 2 mannosidase I were thus
established. Zebrafish embryo was used to perform in vivo
xenotransplantation to observe hepatic cancerous cells migration in
vivo, and found that migration ability of MAN1C1/Hep3B stable cell
line was reduced in zebrafish embryo. To further study the
relationship between .alpha.-mannosidase genes and cell migration,
we focus on matrix metalloproteinases (MMPs) which are proteases to
promoted cancer cells growth, migration, invasion and metastasis
(Egeblad and Werb, 2002). According to Q-PCR results, it was
suggested that overexpression of MAN1A1 increased MMP9 mRNA
expression level, and overexpression of MAN1C1 decreased MMP9 mRNA
expression level. Due to MMPs are capable of degrading all kinds of
extracellular matrix proteins, decreased MMP9 expression means that
cell migration and invasion ability is inhibited. According to
disclosure of the present invention, it is demonstrated that early
reduction of MAN1C1 overexpression in liver cancer patients has
potential to be a molecular marker for screening early liver
cancer. As proved in cell migration assay, no matter in vivo or in
vitro experiment results suggest that MAN1C1 is capable of
inhibiting cell migration ability of hepatic cancerous cells.
[0040] In conclusion, MAN1C1 has potential to be a tumor suppressor
gene and apply to early diagnosis for liver cancer. In one
embodiment, the method for early diagnosis of liver cancer
comprises the steps of:(A) providing a sample obtained from a
subject; (B) assessing the expression level of four subtypes of
.alpha.-mannosidase genes consisting of MAN1A1, MAN1A2, MAN1B1 and
MAN1C1 in the sample; (C) comparing the expression level of
.alpha.-mannosidase genes in the sample with a normal control; and
(D) determining whether the subject having a risk of suffering
liver cancer in accordance with the result of step (C); wherein
while the MAN1C1 expression level of the sample is lower than that
in the normal control, the subject is determined to have a risk of
suffering liver cancer. Additionally, while MAN1A1, MAN1A2 and
MAN1B1 expression levels in the sample are higher than those in
control group, the subject is determined to suffer from liver
cancer and has a risk of metastasis.
[0041] Preferably, the expression levels of MAN1A1, MAN1A2, MAN1B1
and MAN1C1 in the sample are at least two folds higher or lower
than those in the normal control; wherein step (D) further
comprises comparing MMP9 expression level in the sample with a
normal control, while MAN1A1, MAN1A2 and MAN1B1 expression levels
in the sample are higher than those in control group, and the MMP9
expression level in the sample is higher than in the normal
control, the subject is determined to have a risk of liver
metastasis. The expression level of .alpha.-mannosidase (MAN1A1,
MAN1A2, MAN1B1 and MAN1C1) and MMP9 mentioned above can be either
RNA or protein, and the subject is hepatitis B carrier, and the
sample is a liver tissue obtained from the subject.
[0042] In another embodiment, the method of inhibiting metastasis
in liver cancer cell comprises a step of overexpressing MAN1C1 in a
liver cancer cell so as to inhibit liver metastasis. Preferably,
overexpressing MAN1C1 can inhibit the MMP9 expression level in the
liver cancer cell.
[0043] In yet another embodiment, the method of screening a drug
for liver cancer, comprises the steps of: (A) providing a liver
cancer cell treated with a drug; (B) assessing MAN1C1 expression
level of the liver cancer cell; (C) determining whether the drug
has a therapeutical effect according to the MAN1C1 expression
level.
[0044] Although the present invention is described with reference
to the preferred embodiments thereof, it is apparent to those
skilled in the art that a variety of modifications and changes may
be made without departing from the scope of the present invention
which is intended to be defined by the appended claims.
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