The Progression of Liver Fibrosis Is Related with Overexpression of the miR-199 and 200 Families
Chronic hepatitis C (CH) can develop into liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Liver fibrosis and HCC development are strongly correlated, but there is no effective treatment against fibrosis because the critical mechanism of progression of liver fibrosis is not fully understood. microRNAs (miRNAs) are now essential to the molecular mechanisms of several biological processes. In order to clarify how the aberrant expression of miRNAs participates in development of the liver fibrosis, we analyzed the liver fibrosis in mouse liver fibrosis model and human clinical samples. In a CCL(4)-induced mouse liver fibrosis model, we compared the miRNA expression profile from CCL(4) and olive oil administrated liver specimens on 4, 6, and 8 weeks. We also measured expression profiles of human miRNAs in the liver biopsy specimens from 105 CH type C patients without a history of anti-viral therapy. Eleven mouse miRNAs were significantly elevated in progressed liver fibrosis relative to control. By using a large amount of human material in CH analysis, we determined the miRNA expression pattern according to the grade of liver fibrosis. We detected several human miRNAs whose expression levels were correlated with the degree of progression of liver fibrosis. In both the mouse and human studies, the expression levels of miR-199a, 199a*, 200a, and 200b were positively and significantly correlated to the progressed liver fibrosis. The expression level of fibrosis related genes in hepatic stellate cells (HSC), were significantly increased by overexpression of these miRNAs. Four miRNAs are tightly related to the grade of liver fibrosis in both human and mouse was shown. This information may uncover the critical mechanism of progression of liver fibrosis. miRNA expression profiling has potential for diagnostic and therapeutic applications.
The Progression of Liver Fibrosis Is Related with
Overexpression of the miR-199 and 200 Families
*, Hidenori Toyoda
, Masami Tanaka
, Masahiko Kuroda
, Yoshinori Harada
, Atsushi Tajima
, Nobuyoshi Kosaka
, Takahiro Ochiya
, Kunitada Shimotohno
1 Center for Genomic Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan, 2 Department of Gastroenterology, Ogaki Municipal Hospital, Ogaki, Japan,
3 Department of Molecular Pathology, Tokyo Medical University, Tokyo, Japan, 4 Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine,
Kyoto, Japan, 5 Department of Molecular Life Science, Tokai Unive rsity School of Medicine, Isehara, Japan, 6 Division of Molecular and Cellular Medicine, National Cancer
Center Research Institute, Tokyo, Japan, 7 Research Institute, Chiba Institute of Technology, Narashino, Japan
Chronic hepatitis C (CH) can develop into liver cirrhosis (LC) and hepatocellular carcinoma (HCC). Liver fibrosis
and HCC development are strongly correlated, but there is no effective treatment against fibrosis because the critical
mechanism of progression of liver fibrosis is not fully understood. microRNAs (miRNAs) are now essential to the molecular
mechanisms of several biological processes. In order to clarify how the aberrant expression of miRNAs participates in
development of the liver fibrosis, we analyzed the liver fibrosis in mouse liver fibrosis model and human clinical samples.
In a CCL
-induced mouse liver fibrosis model, we compared the miRNA expression profile from CCL
olive oil administrated liver specimens on 4, 6, and 8 weeks. We also measured expression profiles of human miRNAs in the
liver biopsy specimens from 105 CH type C patients without a history of anti-viral therapy.
Eleven mouse miRNAs were significantly elevated in progressed liver fibrosis relative to control. By using
a large amount of human material in CH analysis, we determined the miRNA expression pattern according to the grade of
liver fibrosis. We detected several human miRNAs whose expression levels were correlated with the degree of progression
of liver fibrosis. In both the mouse and human studies, the expression levels of miR-199a, 199a*, 200a, and 200b were
positively and significantly correlated to the progressed liver fibrosis. The expression level of fibrosis related genes in
hepatic stellate cells (HSC), were significantly increased by overexpression of these miRNAs.
Four miRNAs are tightly related to the grade of liver fibrosis in both human and mouse was shown. This
information may uncover the critical mechanism of progression of liver fibrosis. miRNA expression profiling has potential for
diagnostic and therapeutic applications.
Citation: Murakami Y, Toyoda H, Tanaka M, Kuroda M, Harada Y, et al. (2011) The Progression of Liver Fibrosis Is Related with Overexpression of the miR-199 and
200 Families. PLoS ONE 6(1): e16081. doi:10.1371/journal.pone.0016081
Editor: Chad Creighton, Baylor College of Medicine, United States of America
Received September 15, 2010; Accepted December 6, 2010; Published January 24, 2011
Copyright: ß 2011 Murakami et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Japanese Ministry of Health, Labour and Welfare (Y.M, and K.S). This work was also supported by the ‘Strategic
Research-Based Support’ Project for private universities; with matching funds from the Ministry of Education, Culture, Sports, Science and Technology (M.K). The
funders had no role in study design, data collection and analysis, decisio n to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: ymurakami@genome .med.kyoto-u.ac.jp
¤ Current address: Department of Human Genetics and Public Health, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan
Chronic viral hepatitis is a major risk factor for hepatocellular
carcinoma (HCC) . Worldwide 120–170 million persons are
currently chronically Hepatitis C Virus (HCV) infected . Due to
repetitive and continuous inflammation, these patients are at
increased risk of developing cirrhosis, subsequent liver decompen-
sation and/or hepatocellular carcinoma. However, the current
standard of care; pegylated interferon and rivabirin combination
therapy is unsatisfied in the patients with high titre of HCVRNA
and genotype 1b. Activated human liver stellate cells (HSC) with
chronic viral infection, can play a pivotal role in the progression of
liver fibrosis . Activated HSC produce a number of profibrotic
cytokines and growth factors that perpetuate the fibrotic process
through paracrine and autocrine effects.
MicroRNAs (miRNAs) are endogenous small non-coding RNAs
that control gene expression by degrading target mRNA or
suppressing their translation . There are currently 940
identifiable human miRNAs (The miRBase Sequence Database -
Release ver. 15.0). miRNAs can recognize hundreds of target
genes with incomplete complementary; over one third of human
genes appear to be conserved miRNA targets . miRNA is
associated several pathophysiologic events as well as fundamental
cellular processes such as cell proliferation and differentiation.
Aberrant expression of miRNA can be associated with the liver
diseases . Recently reported miRNAs can regulate
the activation of HSCs and thereby regulate liver fibrosis. miR-
29b, a negative regulator for the type I collagen and SP1, is a key
regulator of liver fibrosis . miR-27a and 27b allowed culture-
activated rat HSCs to switch to a more quiescent HSC phenotype,
PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16081
with restored cytoplasmic lipid droplets and decreased cell
In this study, we aimed to reveal the association between
miRNA expression patterns and the progression of liver fibrosis by
using a chronic liver inflammation model in mouse. We also
sought to identify the miRNA expression profile in chronic
hepatitis (CH) C patients according to the degree of liver fibrosis,
and to clarify how miRNAs contribute to the progression of liver
fibrosis. We observed a characteristic miRNA expression profile
common to both human liver biopsy specimens and mouse CCL
specimens, comprising the key miRNAs which are associated with
the liver fibrosis. This information is expected to uncover the
mechanism of liver fibrosis and to provide a clearer biomarker for
diagnosis of liver fibrosis as well as to aid in the development of
more effective and safer therapeutic strategies for liver fibrosis.
The expression level of several mouse miRNAs was
increased by introducing mouse liver fibrosis
In order to identify changes in the miRNA expression profile
between advanced liver fibrosis and non-fibrotic liver, we intra-
peritoneally administered CCL
in olive oil or olive oil alone twice a
week for 4 weeks and then once a week for the next 4 weeks. Mice
were sacrificed at 4, 6, or 8 weeks and then the degree of mouse liver
fibrosis was determined by microscopy (Figure S1). miRNA
expression analysis was performed from the liver tissue collected
at the same time. Histological examination revealed that the degree
of liver fibrosis progressed in mice that received CCL
mice receiving olive oil alone (Figure 1A). Microarray analysis
revealed that in CCL
mice, the expression level of 11 miRNAs was
consistently higher than that in control mice (Figure 1B).
miRNA expression profile in each human liver fibrosis
We then established human miRNAs expression profile by using
105 fresh-frozen human chronic hepatitis (CH) C liver tissues
without a history of anti-viral therapy, classified according to the
grade of the liver fibrosis (F0, F1, F2, and F3 referred to
METAVIR fibrosis stages)(Figure 2, Table S2). Fibrosis grade F0
was considered to be the negative control because these samples
were derived from patients with no finding of liver fibrosis. In
zebrafish, most highly tissue-specific miRNAs are expressed during
embryonic development; approximately 30% of all miRNAs are
expressed at a given time point in a given tissue . In mammals,
the 20–30% miRNA call rate has recently been validated .
Such analysis revealed that the diversity of miRNA expression
level among specimens was small. Therefore, we focused on
miRNAs with a fold change in mean expression level greater than
1.5 (p,0.05) in the two arbitrary groups of liver fibrosis.
Expression of several miRNAs was dramatically different
among grades of fibrosis. In the mice study 11 miRNAs were
related to the progression of liver fibrosis (mmu-let-7e, miR-125-
5p, 199a-5p, 199b, 199b*, 200a, 200b, 31, 34a, 497, and 802). In
the human study 10 miRNAs were extracted, and the change in
their expression level varied significantly between F0 and F3
(F0,F3: hsa-miR-146b, 199a, 199a*, 200a, 200b, 34a, and 34b,
F0.F3: hsa-miR-212, 23b, and 422b). The expression level of 6
miRNAs was significantly different between F0 and F2 (F0,F2:
hsa-miR-146b, 200a, 34a, and 34b, F0.F2: hsa-miR-122 and
23b). 5 extracted miRNAs had an expression level that was
significantly different between F1 and F2 (F1,F2: hsa-miR-146b,
F1.F2: hsa-miR-122, 197, 574, and 768-5p). The expression level
of 9 miRNAs changed significantly between F1 and F3 (F1,F3:
hsa-miR-146b, 150, 199a, 199a*, 200a, and 200b, F1.F3: hsa-
miR-378, 422b, and 768-5p). The miRNAs related to liver fibrosis
were extracted using two criteria: similar expression pattern in
both the human and the mice specimens and shared sequence
between human and mouse. We compared the sequences of
mouse miRNAs as described on the Agilent Mouse MiRNA array
Version 1.0 (miRbase Version 10.1) and human miRNAs as
described on the Agilent Human MiRNA array Version 1.5
(miRbase Version 9.1). The sequences of mmu-miR-199a-5p,
mmu-miR-199b, mmu-miR-199b, mmu-miR-200a, and mmu-
miR-200b in mouse miRNA corresponded to the sequences of
hsa-miR-199a, hsa-miR-199a*, hsa-miR-199a, hsa-miR-200a,
and hsa-miR-200b in human miRNA, respectively (Table S3).
Validation of the microarray result by real-time qPCR
The 4 human miRNAs (miR-199a, miR-199a*, miR-200a, and
miR-200b) with the largest difference in fold change between the
F1 and F3 groups were chosen to validate the microarray results
using stem-loop based real-time qPCR. The result of real-time
qPCR supported the result of that microarray analysis. The
expression level of these 4 miRNAs was significantly different
between F0 and F3 and spearman correlation analysis also showed
that the expressions of these miRNAs were strongly and positively
correlated with fibrosis grade (n = 105, r = 0.498(miR-199a),
0.607(miR-199a*), 0.639(miR-200a), 0.618(miR-200b), p-val-
ues,0.0001) (Figure 3).
Over expression of miR-199a, 199a*, 200a, and 200b was
associated with the progression of liver fibrosis
In order to reveal the function of miR-199a, miR-199a*, miR-
200a, and miR-200b, we investigated the involvement of these
miRNAs in the modulation of fibrosis-related gene in LX-2 cells.
The endogenous expression level of these 4 miRNAs in LX2 and
normal liver was low according to the microarray study (Figure
S2). Transforming growth factor (TGF)b is one of the critical
factors for the activation of HSC during chronic inflammation
 and TGFb strongly induced expression of three fibrosis-
related genes include a matrix degrading complex comprised of a1
procollagen, matrix remodeling complex, comprised of metallo-
proteinases-13 (MMP-13), tissue inhibitors of metalloproteinases-1
(TIMP-1) in LX-2 cells (Figure 4A). Furthermore, overexpression
of miR-199a, miR-199a*, miR-200a and miR-200b in LX-2 cells
resulted significant induction of above fibrosis-related genes
compared with control miRNA (Figure 4B). Finally we validated
the involvement of TGFb in the modulation of these miRNAs. In
LX-2 cells treated with TGFb, the expression levels of miR-199a
and miR-199a* were significantly higher than in untreated cells;
the expression levels of miR-200a and miR-200b were significantly
lower than in untreated cells. Thus, our in vitro analysis suggested
a possible involvement of miR-199a, 199a*, 200a, and 200b in the
progression of liver fibrosis.
Our comprehensive analysis showed that the aberrant expres-
sion of miRNAs was associated with the progression of liver
fibrosis. We identified that 4 highly expressed miRNAs (miR-199a,
miR-199a*, miR-200a, and miR-200b) that were significantly
associated with the progression of liver fibrosis both human and
mouse. Coordination of aberrant expression of these miRNAs may
contribute to the progression of liver fibrosis.
Prior studies have discussed the expression pattern of miRNA
found in liver fibrosis samples between previous and present study.
In this report and prior mouse studies and the expression pattern of
miRNA and Liver Fibrosis
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3 miRNAs (miR-199a-5p, 199b*, 125-5p) was found to be similar
while the expression pattern of 11 miRNAs (miR-223, 221, 24, 877,
29b, 29a, 29c, 30c, 365, 148a, and 193) was partially consistent with
fibrosis grade . In low graded liver fibrosis, the low expression
pattern of 3 miRNAs (miR-140, 27a, and 27b) and the high
expression pattern of 6 miRNAs in rat miRNAs (miR-29c*, 143,
872, 193, 122, and 146) in rat miRNA was also similar to our mouse
study (GEO Series accession number GSE19865)   .
The results in this study and previously completed human
studies reveal that the expression level of miR-195, 222, 200c, 21,
and let-7d was higher in high graded fibrotic liver tissue than in
low graded fibrotic liver tissue. Additionally, the expression level of
miR-301, 194, and 122 was lower in the high graded fibrotic liver
tissue than in low graded fibrotic liver tissue   (GEO
Series accession number GSE16922). This difference in miRNA
expression pattern may be contributed to (1) the difference of
microarray platform, (2) difference of analytic procedure, and (3)
the difference of the species (rat, mouse, and human).
The miR-199 and miR-200 families have are circumstantially
related to liver fibrosis. TGFb-induced factor (TGIF) and SMAD
Figure 1. The change of liver fibrosis in mouse model. A. Representative H&E-stained, Azan-stained, Ag-stained, and EVG-stained histological
sections of liver from mice receiving olive oil alone or CCL
in olive oil. Magnification is 610. B. The expression level of mmu-miRNA in mouse liver
with olive oil or CCL
at 4W, 6W, and 8W respectively, by microarray analysis.
miRNA and Liver Fibrosis
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miRNA and Liver Fibrosis
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specific E3 ubiquitin protein ligase 2 (SMURF2), both of which
play roles in the TGFb signaling pathway, are candidate targets of
miR-199a* and miR-200b, respectively, as determined by the
Targetscan algorithm. The expression of miR-199a* was silenced
in several proliferating cell lines excluding fibroblasts . Down
regulation of miR-199a, miR-199a* and 200a in chronic liver
injury tissue was associated with the hepatocarcinogenesis .
miR-199a* is also one of the negative regulators of the HCV
replication . According to three target search algorithms
(Pictar, miRanda, and Targetscan), the miRNAs that may be
associated with the liver fibrosis can regulate several fibrosis-
related genes (Table S4). Aberrant expression of these miRNAs
may be closely related to the progress of the chronic liver disease.
Epithelial-mesenchymal transition (EMT) describes a reversible
series of events during which an epithelial cell loses cell-cell
contacts and acquires mesenchymal characteristics . Although
EMT is not a common event in adults, this process has been
implicated in such instances as wound healing and fibrosis. Recent
reports showed that the miR-200 family regulated EMT by
targeting EMT accelerator ZEB1 and SIP1 . From our
observations, overexpression of miR-200a and miR-200b can be
connected to the progression of liver fibrosis.
The diagnosis and quantification of fibrosis have traditionally
relied on liver biopsy, and this is still true at present. However, there
are a number of drawbacks to biopsy, including the invasive nature
of the procedure and inter-observer variability. A number of staging
systems have been developed to reduce both the inter-observer
variability and intra-observer variability, including the METAVIR,
the Knodell fibrosis score, and the Scheuer score. However, the
reproducibility of hepatic fibrosis and inflammatory activity is not as
consistent . In fact, in our study, the degree of fibrosis of the two
arbitrary fibrosis groups was classified using the miRNA expression
profile with 80% or greater accuracy (data not shown). Thus,
miRNA expression can be used for diagnosis of liver fibrosis.
In this study we investigated whether common miRNAs in
human and mouse could influence the progression of the liver
fibrosis. The signature of miRNAs expression can also serves as a
tool for understanding and investigating the mechanism of the
onset and progression of liver fibrosis. The miRNA expression
profile has the potential to be a novel biomarker of liver fibrosis.
Figure 3. The expression level of miR-199 and 200 families in human liver biopsy specimen by real-time qPCR. Real-time qPCR
validation of the 4 miRNAs (miR-199a, miR-199a*, miR-200a, and miR-200b). Each column represents the relative amount of miRNAs normalized to the
expression level of U18. The data shown are the means+SD of three independent experiments. Asterisks indicates to a significant difference of
p,0.05 (two-tailed Student-t test), respectively.
Figure 2. Liver fibrosis in human liver biopsy specimen. A. B. C. D. and E. miRNAs whose expression differs significantly between F0 and F3, F0
and F1, F0 and F2, F1 and F2, and F1 and F3, respectively. Relative expression level of each miRNA in human liver biopsy specimen by microarray.
Data from microarray were also statistically analyzed using Welch’s test and the Bonferroni correction for multiple hypotheses testing. Fold change, p-
value are listed in Table S2.
miRNA and Liver Fibrosis
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Moreover miRNA expression profiling has further applications in
novel anti-fibrosis therapy in CH.
Materials and Methods
105 liver tissues samples from chronic hepatitis C patients
(genotype 1b) were obtained by fine needle biopsy (Table S1).
METAVIR fibrosis stages were F0 in 7 patients, F1 in 57, F2 in 24
and F3 in 17. Patients with autoimmune hepatitis or alcoholic liver
injury were excluded. None of the patients were positive for hepatitis
B virus associated antigen/ antibody or anti human immunodefi-
ciency virus antibody. No patient r eceived interferon therapy or
immunomodulatory therapy prior to the enrollment in this study. We
also obtained normal liver tissue from the Liver Transplantation Unit
of Kyoto University. All of the patients or their guardians provided
written informed consent, and Kyoto University Graduate School
and Faculty of Medicine’s Ethics Committee approved all aspects of
this study in accordance with the Helsinki Declaration.
RNA preparation and miRNA microarray
Total RNA from cell lines or tissue samples was prepared using
a mirVana miRNA extraction Kit (Ambion, Austin, TX, USA)
according to the manufacturer’s instruction. miRNA microarrays
were manufactured by Agilent Technologies (Santa Clara, CA,
USA) and 100 ng of total RNA was labeled and hybridized using
the Human microRNA Microarray Kit protocol for use with
Agilent microRNA microarrays Version 1.5 and Mouse micro-
RNA Microarray Kit protocol for use with Agilent microRNA
microarrays Version 1.0. Hybridization signals were detected with
a DNA microarray scanner G2505B (Agilent Technologies) and
the scanned images were analyzed using Agilent feature extraction
software (v22.214.171.124). Data were analyzed using GeneSpring GX
7.3.1 software (Agilent Technologies) and normalized as follows: (i)
Values below 0.01 were set to 0.01. (ii) In order to compare
between one-color expression profile, each measurement was
divided by the 75th percentile of all measurements from the same
species. The data presented in this manuscript have been
deposited in NCBI’s Gene Expression Omnibus and are accessible
through GEO Series accession number GSE16922 (human) and
accession number GSE19865 (mouse).
Real-time qPCR for human miRNA
For detection of the miRNA level by real-time qPCR, TaqManH
microRNA assay (Applied Biosystems) was used to quantify the
relative expression level of miR-199a (assay ID. 002304), miR-
199a* (assay ID. 000499), miR-200a (assay ID. 000502), miR-200b
(assay ID. 002251), and U18 (assay ID. 001204) was used as an
internal control. cDNA was synthesized using the Taqman miRNA
RT Kit (Applied Biosystems). Total RNA (10 ng/ml) in 5ml of
nuclease free water was added to 3 ml of 56 RT primer, 1061.5
of reverse transcriptase buffer, 0.15
ml of 100 mM dNTP, 0.19 mlof
RNase inhibitor, 4.16
ml of nuclease free water, and 50U of reverse
transcriptase in a total volume of 15
ml. The reaction was performed
for 30 min at 16uC, 30 min at 42uC, and 5 min at 85uC. All
reactions were run in triplicate. Chromo 4 detector (BIO-RAD) was
used to detect miRNA expression.
Animal and Chronic Mouse Liver Injury Model
Each 5 adult (8-week-old) male C57BL/6J mice were given a
biweekly intra-peritoneal dose of a 10% solution of CCL
oil (0.02 ml/g/ mouse) for the first 4 weeks and then once a week
Figure 4. The relationship between expression level of miR-199 and 200 families and expression level of three fibrosis related
genes. A. Administration of TGFb in LX2 cells showed that the expression level of three fibrosis related genes were higher than that in non-treated
cells. The data shown are the means+SD of three independent experiments. Asterisk was indicated to the significant difference of p ,0.05 (two-tailed
Student-t test). B. The expression levels of 3 fibrosis related genes in LX2 cells with overexpressing miR-199a, 199a*, 200a, or 200b, respectively were
significantly higher than that in cells transfected with control miRNA (p,0.05; two-tailed Student t-test).
miRNA and Liver Fibrosis
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for the next 4 weeks. At week 4, 6 or 8, the mice were sacrificed.
Partial livers were fixed, embedded in paraffin, and processed for
histology. Serial liver sections were stained with hematoxylin-
eosin, Azan staining, Silver (Ag) staining, and Elastica van Gieson
(EVG) staining, respectively. Total RNA from mice liver tissue was
prepared as described previously. All animal procedures concern-
ing the analysis of liver injury were performed in following the
guidelines of the Kyoto University Animal Research Committee
and were approved by the Ethical Committee of the Faculty of
Medicine, Kyoto University.
Cell lines and Cell preparation
The human stellate cell lines LX-2, was provided by Scott L.
Friedman. LX-2 cells, which viable in serum free media and have
high transfectability, were established from human HSC lines .
LX-2 cells were maintained in D-MEM (Invitrogen, Carlsbad, CA,
USA) with 10% fetal bovine serum, plated in 60 mm diameter
dishes and cultured to 70% confluence. Huh-7 and Hela cells were
also maintained in D-MEM with 10% fetal bovine serum. HuS-E/2
immortalized hepatocytes were cultured as described previously
. LX-2 cells were then cultured in D-MEM without serum with
0.2% BSA for 48 hours prior to TGFb1 (Sigma-Aldrich, Suffolk,
UK) treatment (2.5 ng/ml for 20 hours). Control cells were
cultured in D-MEM without fetal bovine serum.
LX-2 cells were plated in 6-well plates the day before
transfection and grown to 70% confluence. Cells were transfected
with 50 pmol of SilencerH negative control siRNA (Ambion) or
double-stranded mature miRNA (Hokkaido System Science,
Sapporo, Japan) using lipofectamine RNAiMAX (Invitrogen).
Cells were harvested 2 days after transfection.
cDNA was synthesized using the Transcriptor High Fiderity
cDNA synthesis Kit (Roche, Basel, Switzerland). Total RNA (2
ml of nuclease free water was added to 1 mlof50mM
random hexamer. The denaturing reaction was performed for
10min at 65uC. The denatured RNA mixture was added to 4
56 reverse transcriptase buffer, 2
ml of 10 mM dNTP, 0.5 mlof
ml RNase inhibitor, and 1.1 ml of reverse transcriptase
(FastStart Universal SYBR Green Master (Roche) in a total volume
ml. The reaction ran for 30 min at 50uC (cDNA synthesis), and
five min at 85uC (enzyme denaturation). All reactions were run in
triplicate. Chromo 4 detector (BIO-RAD, Hercules, CA, USA) was
used to detect mRNA expression. The primer sequences are follows;
MMP13 s; 59-gaggctccgagaaatgcagt-39, as; 59-atgccatcgtgaagtctggt-
39, TIMP1 s; 59-cttggcttctgcactgatgg-39, as; 59-acgctggtataaggtggtct-
39, a1-procollagen s; 59-aacatgaccaaaaaccaaaagtg-39, as; 59-catt-
gtttcctgtgtcttctgg-39, and b-actin s; 59-ccactggcatcgtgatggac-39, as;
59-tcattgccaatggtgatgacct-39. Assays were performed in triplicate,
and the expression levels of target genes were normalized to
expression of the b-actin gene, as quantified using real-time qPCR
as internal controls.
Statistical analyses were performed using Student’s t-test; p
values less than 0.05 were considered statistically significant.
Microarray data were also statistically analyzed using Welch’s test
and Bonferroni correction for multiple hypotheses testing.
Figure S1 Time line of the induction of chronic liver fibrosis.
Upward arrow indicated administration of olive oil or CCL
Downward arrow indicates when mice were sacrificed.
Figure S2 Comparison of the expression level of miR-199 and
200 familes in several cell lines and human liver tissue.
Endogenous expression level of miR-199a, 199a*, 200a, and
200b in normal liver and LX2 cell as determined by microarray
analysis (Agilent Technologies). Endogenous expression level of
same miRNAs in Hela, Huh-7 and, immortalized hepatocyte:
HuS-E/2 by previously analyzed data .
Table S1 Clinical characteristics of patients by the grade of
Table S2 Extracted human miRNAs related to liver fibrosis.
Table S3 Corresponding human and mouse miRNAs.
Table S4 Hypothetical miRNA target genes according to in
Concei ved and designed the experiments: YM KS. Performed the
experiments: YM HT YH NK. Analyzed the data: MT MK. Contributed
reagents/materials/analysis tools: YM HT YH NK. Wrote the paper: YM
MT AT FM NK TO.
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miRNA and Liver Fibrosis
PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16081