Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4
Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4
Ying-Hsien Huang 0 1
Mao-Meng Tiao 0 1
Li-Tung Huang 0 1
Jiin-Haur Chuang 0 1
Kuang- Che Kuo 0 1
Ya-Ling Yang 0 1
Feng-Sheng Wang 0 1
0 1 Departments of Pediatrics, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 2 Departments of Surgery, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 3 Departments of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan, 4 Departments of Medical Research, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine , Kaohsiung , Taiwan
1 Editor: Leo A. van Grunsven, Vrije Universiteit Brussel , BELGIUM
Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver
fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs).
In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to
strongly reduce HSC activation in the context of liver fibrosis.
In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and
modulation of the profibrogenic phenotype in HSCs.
We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to
clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse
model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense
inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation
using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and
cell proliferation and migration assays.
markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type
littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the
Competing Interests: The authors have declared
that no competing interests exist.
expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In
contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function,
restored HSC migration, and accelerated HSC proliferation.
Persistent liver injury due to cholestasis and hepatitis may result in liver fibrosis that engages a
range of cell types [1, 2]. Liver fibrosis is a complex process modulated by a set of signaling
pathways. Following acute or chronic liver injury of any etiology, hepatic stellate cells (HSCs)
are activated and undergo morphologic and functional trans-differentiation, transforming
from vitamin A-storing cells into contractile myofibroblastic cells responsible for extracellular
matrix (ECM) production in the injured liver [1–3]. It is well known that the stimulation of
HSCs by transforming growth factor-β (TGF-β) is a crucial event in liver fibrogenesis because
of its impact on myofibroblast transition and ECM induction.
MicroRNAs (miRs) are single-stranded 21–22 nucleotide non-coding RNAs that are
capable of controlling gene expression at the post-transcriptional level by silencing endogenous
mRNA transcripts in a process referred to as RNA interference (RNAi) . Recent studies
have shown that the expression of miR-132 and miR-29, which consists of miR-29a, miR-29b,
and miR-29c, are significantly decreased in fibrotic livers, as demonstrated in human liver
cirrhosis as well as in two different models of liver injury induced by bile duct ligation (BDL) and
carbon tetrachloride (CCl4) . In vitro activation of HSCs led to a downregulation of all
miR29-members during eight days of culturing . Moreover, overexpression of miR-29 in murine
HSCs resulted in a downregulation of collagen expression through directly targeting the
mRNA expression of ECM genes. [5, 6] In contrast, another study reported increased fibrosis
and mortality in miR29ab1-knockout mice following the administration of CCl4 . Serum
levels of miR-29a are significantly lower in patients with advanced liver cirrhosis than in
healthy controls or patients with early fibrosis . Because liver fibrosis is an imbalance
between ECM deposition and ECM degradation, the miR-29-mediated suppression of ECM
synthesis in HSCs could hopefully drive the balance toward reduced fibrosis.
Histone deacetylase (HDAC) 4, a member of the class II HDACs, has been found to modify
acetylation reactions in histones and non-histone proteins, and has been reported to regulate
diabetes-induced fibrosis , idiopathic pulmonary fibrosis  and liver fibrosis .
Administration of HDAC inhibitors ameliorates both in experimental liver and kidney fibrosis .
In addition, inhibition of HDAC activity leads to a strong reduction of HSC activation through
the induction of miR-29 expression . Moreover, our group has demonstrated that HDAC4
interference increases the acetylation status of H3K9, which is enriched in the miR-29a
proximal promoter . In addition, our group also demonstrated that miR-29a signaling protects
against glucocorticoid-induced osteoporosis and hyperglycemia-induced renal fibrosis through
a reduction in HDAC4 signaling [12, 13]. Indeed, bioinformatic searches indicate that HDAC4
are predicated to be putative miR-29a targets (http://microrna.sanger.ac.uk and www.
microrna.org). Moreover, we have previously demonstrated that overexpression of miR-29a
significantly reduces the expression of pro-apoptotic proteins and enhances the expression of
phospho-AKT proteins, resulting in a decrease in cellular apoptosis, liver injury, and fibrosis in
cholestasis . We proposed that miR-29a interacted with HDAC signaling to regulate HSC
activation in liver fibrosis. In this study, we employed miR-29a transgenic mice (miR-29aTg
mice) to clarify the role of miR-29a in hepatic injury and fibrogenesis in an experimental BDL
liver fibrosis model.
Our animal protocol was reviewed and approved by the Institutional Animal Care and Use
Committee (IACUC) of the Chang Gung Memorial Hospital (#2012090301). FVB male mice
(National Animal Center of Academia Sinica, Taipei, Taiwan) weighing 25–35 g were
purchased from BioLASCO Taiwan Co., Ltd. All animals were housed in an animal facility at
22°C, with a relative humidity of 55%, in a 12 h light/12 h dark cycle, with food and sterile tap
water available ad libitum.
Construction and breeding of miR-29a transgenic mouse colony
The PGK promoter and miR-29a precursor fulllength sequence were cloned from the cDNA
library by PCR protocols. The cDNAs were then inserted into the pUSE empty expression
vector; and the linear human PGK-miR-29a-BGH poly-A cDNAs were cloned. The designed
constructs were transferred into fertilized eggs from FVB/N mice (BioLASCO Taiwan Co., Ltd).
The eggs were further transferred into Crl: CD1 foster mothers, as previously described .
Transgenic mice were bred in a specific pathogen-free condition and genotyped by PCR using
specific primers (forward: 5’-GAGGATCCCCTCAAGGATACCAAG- GGATGAAT-3’ and
reverse 5’-CTTCTAGAAGGAGTGTTTCTAGGTATCCGT- CA-3’) .
Animal model and experimental protocol
FVB male mice were used for all of the experiments. The mice were randomly divided into
either the “BDL” group or the “sham” group, depending on whether the mice had received a
ligation or a sham ligation of the common bile duct, as described in a previous study . The
mice were sacrificed one week after the procedure. Liver tissues were snap-frozen so that
mRNA and protein expression could be determined later. The samples were kept at −80°C
prior to biochemical analysis.
Primary HSC isolation and culture
Primary HSCs were isolated from livers of miR-29aTg mice or WT littermate by sequential
digestion of the liver with pronase and collagenase, followed by density gradient centrifugation
in 8.5% Nycodenz (Sigma-Aldrich, St. Louis, MO) as described previously [16, 17]. The purity
of the HSCs was assessed by autofluorescence of stored retinoids in HSC lipid droplets (S1
Fig). Cell viability determined by a Trypan Blue exclusion assay revealed that more than 95%
of the cells were viable. Purity of the HSC culture was found to be 95%–99% by oil red O
staining . Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 5%
newborn calf serum. After 1 day in culture, the HSCs had a quiescent phenotype and they
developed an activated phenotype after 7–14 days. The passage of the cultured cells was
conducted after reaching confluence and experiments were carried out using cells between
passages 2 and 6.We compared cell survival, profibrogenic gene expression, cell proliferation and
migration in primary activated HSCs from wild-type (WT) and miR-29aTg mice. Thereafter,
miR-29a or HDAC4 RNAi was added to the activated HSC culture. To investigate the
mechanism why cholestasis may affect HSC activation, we treated primary HSCs with one of the
hydrophobic bile acids, taurolithocholic acid (TLCA) (Sigma) for 24 hours. Four independent
in vitro experiments were performed.
HSCs were transfected with a miR-29a precursor (a miR-29a mimic, C-300504, Lafayette,
USA), miR-29a antisense oligonucleotide inhibitor (IH-300504, Lafayette, USA), or miR
control (N-00100 Thermo, Lafayette, USA) for 24 hours. Cells were seeded into plates (5 × 105
cells/well, 6-well plate for western blotting and real-time quantitative RT-PCR; 104 cells/well,
12-well plate for immunofluorescence), incubated overnight and transfected using
DharmaFECT siRNA Transfection Reagent (Lafayette, USA) as instructed . HDAC4 RNAi
(sc35541, Santa Cruz, USA), which targets HDAC4, were transfected into primary HSCs for 24
hours using the Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Carlsbad, CA),
according to the manufacturer's instructions .
RNA isolation and real-time quantitative RT-PCR
In order to quantify miR-29 in the tissue samples, we performed real-time, quantitative
RTPCR with the ABI 7700 Sequence Detection System (TaqMan; Applied Biosystems, Inc., Foster
City, CA). Total miR was isolated using the MicroRNA Isolation kit (BioChain Institute, Inc,
Hayward, CA), according to the manufacturer’s instructions. U6 gene (Applied Biosystems,
Foster City, CA) expression was used to normalize gene and miR expression. Templates were
pre-amplified using 2× TaqMan PreAmp Master Mix and 10X Megaplex PreAmp Primers,
and then PCR-amplified using 2× TaqMan Universal PCR Master Mix. Relative quantification
of gene expression was based on the comparative threshold cycle (CT) method in which the
amount of the target was determined to be 2-(ΔCT target – Δ CT calibrator) or 2-ΔΔCT. PCR products
were then electrophoresed on a 2% agarose gel in order to confirm the amount of the products.
Primers were designed to amplify collagen-1α1 (forward, 50-ACCCTGGAAACAGACGA-30;
reverse, 50-TTTGGTAAGGTTGAATGCACT-30), collagen-3α1 (forward, 50-TACCTCAACT
GGTCAGAACAGATA-30; reverse, 50-GTACTCCTTCAAATTCCTGCT-30), monocyte
chemoattractant protein-1 (MCP-1) (forward, 50-TTGACCCGTAAATCTGAAGCTA-30; reverse,
50-ATTAAGGCATCACAGTCCG-30) and GAPDH (forward, 50-CACTGCCACCCAGAA
GA-30; reverse, 50-TCCACGACGGACACATT-30). Validation experiments were performed in
duplicate, and amplification efficiencies were validated.
Liver tissues were embedded in TissueTek optimal cutting temperature (OCTTM) compound
(Sakura Finetek) and frozen at −80°C for storage. Frozen sections (4 μm thick) were prepared
using a cryostat (CM3050 S, Leica) and processed for Sirius Red staining. Cryosections were
fixed with an isotonic PBS and 4% paraformaldehyde solution for 1 h. To block non-specific
background staining, the samples were incubated in a solution containing 1% BSA for 30 min.
After washing with PBS, the slides were incubated with the primary antibodies. Anti-glial
fibrillary acidic protein (GFAP) (ab10062, abcam), anti-HDAC4 (#5392, cell signaling, MA), and
anti-a-SMA (ab5694, abcam, UK) primary antibodies were used. Fluor 488-conjugated (green)
and Alexa Fluor 595 (red)-conjugated secondary antibodies (Molecular Probes) were used.
Samples were co-stained with 4',6-diamidino-2-phenylindole (DAPI; Molecular Probes) to
facilitate visualization of the nuclei. The stained cells were mounted with a fluorescent
mounting medium (Dako Cytomation) and visualized by microscopy (Olympus). The exposure gains
and rates were consistent between samples. Fluorescent intensities were quantified on
independent color channels by using Image J analysis.
Extraction of cytoplasmic and nuclear fractions was performed using NE-PER Nuclear and
Cytoplasmic Extraction Reagents Kit (Pierce, Rockford, IL, USA) according to the
manufacturer’s protocol. Crude proteins (30 μg) were treated with sample buffer, boiled for 10 min,
separated using 8–15% sodium dodecyl sulfate–polyacrylamide gels, and transferred to a
nitrocellulose membrane. Blots were incubated with the primary antibodies against
collagen1α1 (#sc-8784-R, Santa Cruz Biotechnology, Dallas, TX), HDAC4 (#5392s, Cell Signaling,
MA), H3K9Ac (ab4441, abcam, UK), and GAPDH (GTX100118, GeneTex, SA),
phosphSmad3 (p-Smad3; ab51451, abcam, UK), Smad 3 (ab40854, abcam, UK), and GAPDH for
cytoplasmic protein control, and lamin B (ab16048, abcam, UK) for nuclear protein control. After
washing with TBST and incubating with horseradish peroxidase-coupled anti-rabbit
immunoglobulin-G antibodies (dilution, 1:10,000) at room temperature for 2 h, the blots were
developed with enhanced chemiluminescence detection (GE Healthcare Biosciences AB, Uppsala,
Sweden) and exposed to film. The signals were quantified with densitometry.
Detection of cellular migration using a wound-healing assay
Cells were seeded into ibidi culture-inserts (ibidi GmbH, Martinsried, Germany) at a
concentration of 10,000 cells per well. After allowing cells to attach overnight, the culture-insert was
gently removed using sterile forceps. Cells were incubated with scramble, miR-29a mimic,
miR-29a anti-sense inhibitor or HDAC4 RNAi. Images were taken at 0, 6, and 21 h, and
superimposed using PhotoImpact (Adobe). The number of cells that migrated into the wound space
were manually counted in three fields per well under a light microscope at 50× magnification.
Areas were quantified by image analysis using Image J analysis.
HSCs were seeded into 12-well plates at a concentration of 5,000 cells/ml. After one week of
culture, cells were rinsed with PBS, fixed in methanol, and stained with 200 μl crystal violet.
Cells were rinsed with distilled water and air dried. Once dry, cells were lysed with 2% (w/v)
sodium deoxycholate solution with gentle agitation. Then, the plates were washed with distilled
water at least three times prior to solubilizing the cell layer with 50 μl of 10% glacial acetic acid.
Absorbance was measured at 540 nm on a microplate reader (HIDEX Sense Microplate Reader,
All values in the figures and tables were expressed as mean ± standard error. Quantitative data
were analyzed using the one-way analysis of variance  when appropriate. The least
significant difference (LSD) test was used for post-hoc testing when appropriate. Correlations
between quantitative variables were assessed using Pearson’s coefficient. Two-sided p values
less than 0.05 were considered statistically significant.
Fig 1. Overexpression of miR-29a in the murine model resulted in downregulation of fibrosis and HDAC4 in the liver of mice after BDL. (A)
SiriusRed staining showed moderate fibrosis in WT mice and mild fibrosis in miR-29aTg mice, which was limited to the close vicinity of the portal area. (B) There
was a significantly greater expression of collagen-1α1 and HDAC4 in tissues from the BDL group than in tissues from the sham-operated group in WT mice.
Moreover, miR-29a overexpression significantly downregulated collagen-1α1 and HDAC4 protein expression in miR-29aTg mice with cholestasis compared
with the WT littermates. Data are expressed as the mean ± SE of six samples per group. *indicates a p < 0.05 between the groups.
Over-expression of miR-29a significantly reduced liver fibrosis, HDAC4
expression and HSC activation in cholestatic mice
To investigate the effect of miR-29a overexpression on the progression of fibrosis, we studied
the expression of ECM proteins during hepatic fibrogenesis. As showed in Fig 1A, Sirius-Red
staining showed moderate fibrosis in WT mice and mild fibrosis in miR-29aTg mice, which
was limited to the close vicinity of the portal area. The relatively moderate fibrosis observed in
both mice is due to the Hc-/- genotype (encoding complement factor C5) of the FBV mouse
strain used that typically yields less fibrosis when compared to Hc+/+ strains such as BALB/c
and C57BL/6J . Therefore, we have performed western blot analysis, we found a
signifisues from the sham group (p < 0.001; Fig 1B) in WT mice. However, we observed a weaker
mice with cholestasis compared with the WT littermates (p < 0.001). Then we further
characterized HDAC4 protein expression in the liver. As illustrated in Fig 1B, compared with the
sham-operation group, the BDL group of WT mice exhibited an increase in HDAC4 protein
HDAC4 immunoreactivity in miR-29aTg mice with cholestasis compared with the WT
littermates (p < 0.001), indicating that miR-29a might have an impact on HDAC4 expression in
GFAP is a type III intermediate filament protein originally identified in HSC-derived
myofibroblasts . It increases in the acute response to injury and decreases in the chronic response
[23–25]; and GFAP has therefore been suggested as an early marker of HSC activation. Hence,
GFAP expression levels reveal the overall number of HSC and can therefore be utilized as a
specific HSC proliferation marker. Compared with the sham-operation group, the BDL group
of WT and miR-29aTg mice had increased GFAP protein expression (p < 0.001 and p = 0.003,
respectively).(Fig 2) Moreover, miR-29a overexpression significantly downregulated GFAP
immunoreactivity in the miR-29aTg mice with cholestasis compared with the WT littermates
(p < 0.001).
Overexpression of miR-29a significantly reduced expression of
profibrogenic genes and HDAC4 in activated HSCs
The activation of HSCs is known to result in increased expression of several profibrogenic genes,
including collagen-1α1, collagen-3α1 and MCP-1. As shown in Fig 3, miR-29a overexpression
significantly downregulated the expression of collagen-1α1, collagen-3α1, and MCP-1 in activated
HSCs of miR-29aTg mice compared with WT littermates (all p < 0.001). We then treated primary
HSCs with one of the hydrophobic bile acids, taurolithocholic acid (TLCA; Sigma), to explore
whether miR-29a affects HDAC4 expression and nuclear translocation in response to cholestasis.
As shown in Fig 4, we found that there was significant upregulation and nuclear translation of
HDAC4 in the HSCs of WT mice following TLCA stimulation (p = 0.001 and p < 0.001,
respectively). HSCs of miR-29aTg mice could significantly reduce the upregulation and nuclear
translation of HDAC4 in response to TLCA stimulation in HSCs (p = 0.003 and p < 0.001, respectively).
To test the effects of miR-29a inhibition on the expression of HDAC4, primary HSCs stably
expressing a miR-29a mimic, antisense inhibitor, HDAC4 RNAi and scramble control were
used. As expected, overexpression of miR-29a significantly downregulated the expression of
α-SMA- and HDAC4- in miR-29aTg mice compared than in WT mice (Fig 5A and 5B). In
Fig 2. Overexpression of miR-29a decreased GFAP, a marker of HSC, and increased expression in the acute response to injury and
immunoreactivity in cholestasis. There was significantly higher expression of GFAP (green) in tissues from the BDL group than in tissues from the
shamoperated group in WT mice. Moreover, miR-29a overexpression significantly downregulated GFAP protein expression in miR-29aTg mice with cholestasis
compared with the WT littermates. Data are expressed as the mean ± SE of six samples per group. *indicates a p < 0.05 between the groups.
Fig 3. Comparison of collagen-1α1 (A), collagen-3α1 (B) and MCP-1 (C) expression in activated HSCs of WT and miR-29Tg mice. Data are expressed
as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
contrast, after treatment with a miR-29a anti-sense inhibitor, α-SMA and HDAC4 expression
was significantly upregulated in the HSCs of miR-29aTg mice. Western blotting confirmed the
immunofluorescence findings (Fig 6). We then tested whether HDAC4 affected histone
acetylation in activated HSCs. Our group has previously demonstrated that HDAC4 interference
increased the acetylation status of histone H3 at lysine 9 (H3K9Ac), and observed an
enrichment of H3K9Ac in the miR-29a proximal promoter in a diabetic nephropathy animal model
. Compared to controls, a miR-29a mimic or HDAC4 RNAi significantly increased the
Fig 4. Effects of TLCA on HDAC4 expression in cultured primary HSCs. TLCA increased HDAC4 expression (A) and nuclear translocation (B) in HSCs
of WT mice (white bar). HSCs of miR-29a transgenic mice (gray bar) exhibited significantly reduced upregulation and nuclear translation of HDAC4 in
response to TLCA stimulation. Data are expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
Fig 5. Comparison of α-SMA and HDAC4 expression in 10th culture day of activated HSCs of WT and
miR-29aTg mice after treatment with a miR-29a mimic and anti-sense inhibitor for 24 hours.
Expression of α-SMA (red) and HDAC4 (green) were much greater in activated HSCs of WT mice than in
miR-29aTg mice. Treatment with a miR-29a mimic in activated HSCs of WT inhibited α-SMA and HDAC4
expression as well as HDAC nuclear translocation. In contrast, treatment with miR-29a anti-sense inhibitor in
miR-29aTg mice increased α-SMA and HDAC4 expression as well as HDAC4 nuclear translocation. Data are
expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
Recent reports have demonstrated that miR-29 acts as a downstream inhibitor and therapeutic
shown in Fig 7B, a miR-29a mimic or HDAC4 RNAi inhibited both expression of both Smad3
and p-Smad3 in activated HSCs.
Gain of miR-29a function inhibits HSC migration and proliferation
In order to assess whether miR-29a may regulate HSC migration, a wound-healing assay was
performed using primary HSCs. The results of the present study showed that a miR-29a mimic
or HDAC4 RNAi inhibited the migration of primary HSCs (both p < 0.001; Fig 8). We then
conducted a cell proliferation assay to examine the effects of miR-29a on cell proliferation. As
showed in Fig 9, a miR-29a mimic or HDAC4 RNAi inhibited the proliferation of primary
HSCs (both p < 0.001). In addition, addition of a miR-29a anti-sense inhibitor significantly
increased HSC proliferation (p < 0.001).
Fig 6. Overexpression of miR-29a decreased HDAC4 expression in primary HSCs. Treatment with a miR-29a mimic and HDAC4 RNAi significantly
deceased HDAC4 expression in the HSCs of WT mice. Data are expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05
between the groups.
HSC activation and trans-differentiation responsible for ECM production are important
features in the pathogenesis of liver fibrosis. To the best of our knowledge, this is the first study to
report that miR-29a could directly modulate the profibrogenic phenotype of HSCs in a mouse
model of obstructive jaundice in miR-29aTg mice. This study marks the first attempt to shed
light on the interplay between miR-29a and HDAC4 signaling upon the activation of HSCs.
The main novel findings of this study are as follows. (1) Overexpression of miR-29a in
cholestatic mice significantly inhibited liver fibrosis. (2) It also induced a significant decrease in
GFAP expression, a type III intermediate filament protein that is expressed in activated HSCs
. (3) There was significant upregulation and nuclear translation of HDAC4 in HSCs of WT
mice in response to stimulation by the hydrophobic bile acid TLCA. Interestingly, HSCs of
miR-29aTg mice significantly reduced the upregulation and nuclear translation of HDAC4 in
Fig 7. Overexpression of miR-29a increased histone H3 at lysine 9 (H3K9Ac) and decreased Smad3 expression in HSCs. A miR-29a mimic and
HDAC4 RNAi significantly increased H3K9Ac and decreased both Smad3 and p-Smad3 expression in HSCs of WT mice. Data are expressed as the
mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
response to TLCA stimulation. (4) We also observed that overexpression of miR-29a and
HDAC4 RNAi significantly downregulated HDAC4, p-Smad3, and the acetylation of histone
H3 at lysine 9 (H3K9Ac), which is enriched in the miR-29a proximal promoter . (5)
Furthermore, miR-29a overexpression significantly downregulated expression of collagen-1α1,
Fig 8. Migration of primary activated HSCs was measured using a wound healing assay. A miR-29a mimic or HDAC4 RNAi significantly inhibited the
migration of primary HSCs of WT mice. Data are expressed as the mean ± SE of four independent experiments. *indicates a p < 0.05 between the groups.
Fig 9. Proliferation of primary activated HSCs was measured by crystal violet assay. A miR-29a mimic or HDAC4 RNAi inhibited the proliferation of
primary HSCs. In addition, treatment with a miR-29a anti-sense inhibitor significantly increased HSC proliferation. Data are expressed as the mean ± SE of
four independent experiments. *indicates a p < 0.05 between the groups.
collagen-3α1, and MCP-1 in activated the HSCs of miR-29aTg mice compared to WT
littermates. (6) Most importantly, overexpression of miR29a and HDAC4 RNAi significantly
attenuated the activated HSC migration and proliferation. Knockdown of miR-29a with an
antisense inhibitor promoted HDAC4 function and restored HSC migration and accelerated
proliferation. These phenomena rationalize our hypotheses to focus on the molecular events
underlying miR-29a protection against BDL or hydrophobic bile acid-mediated HSC
Obstructive jaundice has been shown to be associated with the transcriptional activation of
pro-inflammatory cytokines. Previously, we have demonstrated an increase in MCP-1
expression in cholestatic liver and in isolated HSCs . MCP-1 is one of the most significant
chemokines regulating the recruitment and maintenance of inflammatory infiltrates during liver
injury . Activated HSCs and biliary epithelial cells are responsible for MCP-1 production
and HSC recruitment and activation in chronic liver disease [28, 29]. Herein, we showed that
there was lower MCP-1 expression in isolated HSCs from miR-29aTg mice than in WT
Acetylation of lysine residues modulates protein-histone and histone-DNA interactions,
and thereby regulates many cellular processes . HDAC inhibitors have been extensively
studied in experimental models of cancer, where their inhibition of deacetylation has been
proven to regulate cell survival, proliferation, differentiation and apoptosis . Currently,
numerous HDAC inhibitors, including trichostatin A, valproic acid, and Largazole, have been
identified as potent inhibitors of HSC activation both in vitro and in vivo that could reduce
inflammatory activity and liver fibrosis [31–33]. In addition, inhibition of HDAC activity leads
to a strong reduction of HSC activation markers, α-SMA, lysyl oxidase and collagens, as well as
an inhibition of cell proliferation through the induction of miR-29 expression . Moreover,
our group has demonstrated that HDAC4 interference increases the acetylation status of
H3K9, which is enriched in the miR-29a proximal promoter, and reduces miR-29a
transcription in high glucose-stressed podocytes . In contrast, overexpression of miR-29a promotes
nephrin acetylation that ameliorates hyperglycemia-induced podocyte dysfunction through
inhibition of HDAC4 signaling transduction . In addition, we demonstrated that miR-29a
signaling protected against glucocorticoid-induced osteoporosis and improved osteoblast
differentiation and mineral acquisition  through reduced HDAC4 signaling .
In addition, miR-29 is also a major regulator of genes associated with pulmonary fibrosis
, renal fibrosis , as well as myocardial infarction  and aneurysm formation . It
seems that miR-29 is a key player in fibrogenesis. We first demonstrated that overexpression of
miR-29a in cholestatic mice significantly inhibited collagen-1α1 and collagen-4α1 protein
expression in miR-29aTg mice with cholestasis compared with the WT littermates .
Previously, we showed that hepatic overexpression of miR-29 leads to inhibition of hepatocellular
apoptosis and to reduction of acute liver damage . Therefore, stellate cell activation might
not be only attenuated by stellate cell specific direct effects by miR-29a overexpression, but also
by the indirect influence of less hepatocyte injury. In particular, inhibition of apoptosis of
hepatocytes upon miR-29aTg overexpression is suggested to result in diminution of stellate cell
activation due to the reduction of the inflammatory reaction in response to fewer hepatocellular
apoptotic bodies [38, 39]. Stimulation of HSCs by TGF-β is a crucial event in liver fibrogenesis
due to its impact on myofibroblastic transition and ECM induction. TGF-β secretion by
hepatocytes, Kupffer cells, and sinusoidal endothelial cells causes HSC to activate, transdifferentiate,
and secrete ECM . Recently, Roderburg et al. reported that TGF-β1- mediated
downregulation of miR-29 in HSCs , a finding supported by Bandyopadhyay et al. . In a recent study
of renal fibrosis, it was demonstrated that Smad3 mediated TGF-β1-induced the
downregulation of miR-29 by binding to the promoter of miR-29 . Furthermore, miR-29 acted as a
downstream inhibitor and therapeutic miR for TGF-β1/Smad3-mediated renal fibrosis.
Moreover, miR-29 can inhibit the TGF-β1-mediated upregulation of HDAC4 via the inhibition of
Smad3 expression in the regulation of myogenic differentiation . It is also consistent with
our findings that overexpression of miR-29a could downregulate p-Smad3 and HDAC4
expression in vivo and in vitro. Thus, miR-29a is an important regulator of the profibrogenic
phenotype of HSCs and plays as an important role of the cross-talk between HDAC4 and
TGF-β1 signaling (Fig 10). By suppressing HDAC4 action, miR-29a restores the acetylation
status of H3K9. It also can suppress Smad3 phosphorylation and thereafter inhibits the
activation of HSCs. Moreover, in our previous study, overexpression of miR-29a significantly
reduced the expression of pro-apoptotic proteins, inhibition of NF-κB activation and enhanced
phospho-AKT protein expression, thereby leading to a decrease in hepatocellular apoptosis in
cholestasis . Taken together, miR-29a is an important regulator in the maintenance of HSC
ultrastructure integrity and liver homeostasis.
Inhibition of the fibrogenic, proliferative, and migratory effects of HSCs is an emerging
experimental therapy for the prevention and regression of hepatic fibrosis. This study
Fig 10. Proposed model of miR-29a signaling protection in liver fibrosis and HSC activation. MiR-29a is an important regulator of the profibrogenic
phenotype of HSCs. By suppressing HDAC4 action, miR-29a increases H3K9 acetylation and suppresses Smad3 phosphorylation; therefore inhibiting the
activation of HSCs.
highlights an emerging view of an epigenetic mechanism that the activation of HSCs by
miR29a signaling may modulate their profibrogenic phenotype, thus supporting the use of
miR29a agonists as a potential therapy to treat liver fibrosis in the future.
S1 Fig. Oil red O staining and α-smooth muscle actin (α-SMA) in the hepatic stellate cell
(HSC). After 1 day in culture, the HSCs have a quiescent phenotype (A) and they develop an
activated phenotype that reveal the unique appearance of star-shaped and lose of lipid droplets
after 8 days of culture (B). A characteristic hallmark of activated HSCs is the expression of
αSMA (C, right) compared to quiescent phenotype (C, left).
The authors thank Yuan-Ting Chuang for her assistance in this study.
Conceived and designed the experiments: YHH JHC LTH FSW. Performed the experiments:
MMT KCK YLY. Analyzed the data: YHH MMT JHC LTH FSW. Contributed
reagents/materials/analysis tools: YHH MMT JHC LTH FSW. Wrote the paper: YHH LTH FSW.
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