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MicroRNA-130a and -130b enhance activation of hepatic stellate cells by suppressing PPARγ expression: A rat fibrosis model study
Biochemical and Biophysical Research Communications 465 (2015) 387e393
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MicroRNA-130a and -130b enhance activation of hepatic stellate cells by suppressing PPARg expression: A rat fibrosis model study Le Lu a, Jinlong Wang a, Hongwei Lu a, Guoyu Zhang b, Yang Liu a, Jiazhong Wang a, Yafei Zhang a, Hao Shang a, Hong Ji a, Xi Chen a, Yanxia Duan a, Yiming Li a, * a b
Department of General Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, No.157, West 5th Road, Xi'an, Shaanxi 710004, China West Hospital Ward 1, Shaanxi Provincial People's Hospital, No.256, Youyi Road(west), Xi'an, Shaanxi 710068, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 July 2015 Accepted 3 August 2015 Available online 6 August 2015
Hepatic stellate cells (HSCs) are the primary sources of extracellular matrix (ECM) in normal and fibrotic liver. Peroxisome proliferator-activated receptor gamma (PPARg) maintains HSCs in a quiescent state, and its downregulation induces HSC activation. MicroRNAs (miRNAs) can induce PPARg mRNA degradation, but the mechanism by which miRNAs regulate PPARg in rat HSCs is unclear. This study aimed to investigate some miRNAs which putatively bind to the 30 -untranslated region (30 -UTR) of PPARg mRNA, and increase expression of ECM genes in rat HSCs. In carbon tetrachloride injection (CCl4) and common bile duct ligation (CBDL) liver fibrosis models, miRNAs miR-130a, miR-130b, miR-301a, miR-27b and miR340 levels were found to be increased and PPARg expression decreased. Overexpression of miR-130a and miR-130b enhanced cell proliferation by involving Runx3. MiR-130a and miR-130b decreased PPARg expression by targeting the 30 -UTR of PPARg mRNA in rat HSC-T6 cells. Transforming growth factor-b1 (TGF-b1) may mediate miR-130a and miR-130b overexpression, PPARg downregulation, and ECM genes overexpression in cell culture. These findings suggest that miR-130a and miR-130b are involved in downregulation of PPARg in liver fibrosis. © 2015 Elsevier Inc. All rights reserved.
Keywords: miR-130a miR-130b PPARg TGF-b1 Hepatic stellate cells Liver fibrosis
1. Introduction Portal hypertension is a frequent cause of death in patients with cirrhosis [1]. Liver fibrosis and cirrhosis are the main causes of portal hypertension. Hepatic stellate cells (HSCs) are the primary source of extracellular matrix (ECM) in normal and fibrotic liver. When the liver is injured, quiescent HSCs undergo ‘activation’ and transdifferentiation from non-proliferating cells to myofibroblasts, which proliferate and produce ECM [2]. Peroxisome proliferator-activated receptors g (PPARg) are transcriptional factors that are abundant in normal HSCs, where they regulate lipid metabolism and adipocyte differentiation [3]. Recently, PPARg has been identified as a key molecular switch involved in HSC activation and phenotype alteration. PPARg expression is dramatically diminished during HSC activation and enables HSCs transdifferentiation to a myofibroblast phenotype [4]. However, little has been reported on the direct cause of downregulated expression or inactivation of PPARg.
MicroRNAs (miRNAs) regulate gene expression by binding to the 30 -untranslated region (30 -UTR) of the target gene mRNAs to repress protein translation or induce mRNA degradation [5]. Several hundred miRNAs have been identified, and more than 60% of mammalian genes are known to be targeted and regulated by miRNAs [6]. A variety of miRNAs are involved in the regulation of HSC proliferation, differentiation, senescence, apoptosis and autophagy [7]. However, the role of miRNAs in regulating PPARg gene in the HSCs of liver fibrosis and cirrhosis is unclear. The aim of the current study was to investigate miRNAs, which putatively bind the 30 -UTR of PPARg mRNA in rat HSCs and increase ECM gene expression in vitro. We focused on two miRNAs miR-130a and miR-130b that are overexpressed in activated HSCs in rat and target the 30 -UTR of PPARg mRNA. 2. Materials and methods 2.1. Prediction of miRNAs using bioinformatics
* Corresponding author. E-mail address: [email protected] (Y. Li). http://dx.doi.org/10.1016/j.bbrc.2015.08.012 0006-291X/© 2015 Elsevier Inc. All rights reserved.
NCBI database (http://www.ncbi.nlm.nih.gov/nuccore/) was used to search sequences of PPARg genes from several species. The
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Targetscan (http://www.targetscan.org/), microRNA (http://www. microrna.org/) and miRBase (http://www.mirbase.org/) databases were used to select miRNAs that may sequence-specifically target the 30 -UTR of PPARg. 2.2. Animals and HSC isolation Male SpragueeDawley rats weighing between 200 and 300 g were purchased from the Laboratory Animal Center, Xi'an Jiaotong University Health Science Center (Xi'an, Shaanxi, China). All procedures were reviewed and approved by the Ethics Committee, Xi'an Jiaotong University Health Science Center. In order to clarify the exact role of miRNAs in PPARg, the two rat models of liver fibrosis were used: carbon tetrachloride injection (CCl4) and common bile duct ligation (CBDL). CCl4 was administered by intraperitoneal injection of (Sinopharm Chemical Reagent Co. Ltd. Shanghai, China; 0.6 ml/kg of body weight) in olive oil, twice a week for six weeks. Controls received only olive oil. Ligationtransection of the common bile duct was performed on rats as described previously [8]. Sham-operated rats served as controls. HSC isolation was performed as described previously [9]. 2.3. Materials and reagents Oligonucleotides, miR-mimic (miR), miR inhibitor (anti-miR), miR negative control (miR-CTL) and anti-miR negative control (anti-miR-CTL) were purchased from Biomics Biotechnologies Co., Ltd (Nantong, Jiangsu, China). The pMIR-REPORT™ miRNA Expression Reporter Vector was ordered from Ambion (Austin, TX, USA). Transforming growth factor-b1 (TGF-b1) was obtained from Sigma (St. Louis, MO, USA). 2.4. Cell cultures HSC-T6, an immortalized rat HSC line, purchased from KeyGEN (Nanjing, Jiangsu, China), was maintained in Dulbecco's modified Eagle's medium (DMEM, Hyclone, NY, USA) containing 10% fetal calf serum (FCS, Tianhang Biotechnology; Hangzhou, Zhejiang, China) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; KeyGEN, Nanjing, Jiangsu, China). Culture conditions were set at 37 C under 5% CO2 and 95% atmospheric air at constant humidity (Thermo, USA). Culture medium was changed every 2e3 days. 2.5. Cell proliferation Rat HSC-T6 cells were seeded in 96-well tissue culture plates by adding 150 ml/well of a suspension of 5 104 cells. Cell viability was determined using the MTT (MTT, Sigma) assay according to the manufacturer's instructions. 2.6. RNA isolation and real-time PCR Total RNA was isolated from HSCs using TRIzol (Invitrogen, Carlsbad, CA, USA). For analysis of miRNA and mRNA levels, realtime polymerase chain reaction (PCR) was performed with SYBR® PrimeScript™ miRNA RT-PCR Kit and SYBR® Premix EX Taq™ II (Tli RNaseH Plus) (TaKaRa Bio, Osaka, Japan) according to the manufacturer's instructions. Rat U6 snRNA and GAPDH mRNA were included separately as an internal references. The primers were used in this study are shown in Supplementary Table 1. The relative expression levels of miRNAs and mRNAs were calculated by the 2DDCT method [10].
2.7. Western blot analysis HSCs were lysed using RIPA buffer (APPLYGEN, Beijing, China) according to the manufacturer's manual. Protein concentration was estimated with an ultraviolet spectrophotometer (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed using antibodies against PPARg (1:500), alpha-smooth muscle actin (a-SMA, 1:1000), a1 type I collagen (Col1A1, 1:1000), tissue inhibitor of metalloproteinases-1 (TIMP-1, 1:1000), b-actin (1:1000) (Proteintech Group, Chicago, USA) and goat anti-rabbit antibody (1:3000, Bioworld, Minnesota, USA) as previously described [11]. The rat b-actin protein was used as an internal reference. 2.8. Dual luciferase assays The sequence cDNA containing PPARg30 -UTR and PPARg 30 -UTR mutant (PPARg30 -UTR-Mut) were inserted into a dual luciferase reporter vector of pMIR-REPORT (Supplementary Materials and Methods). For the luciferase assays, 200 ng vector (pMIR-REPORT, pMIR-REPORT-PPARg30 -UTR- WT or pMIR-REPORT-PPARg30 -UTRMut), 20 ng renilla luciferase control plasmid (pRL-TK), and 10 pmol oligonucleotides (miR-130a, miR-130b or miR-CTL) were cotransfected in rat HSC-T6 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h, firefly luciferase activity was determined using the Dual Luciferase Assay system (Promega, Madison, WI) according to the manufacturer's instructions. The experiments were performed in quadruplicate. 2.9. Statistical analysis All experiments were repeated at least three times. Data are expressed as mean ± SD. Two samples were compared using twotailed Student's t-test and p < 0.05 was considered as statistically significant. 3. Results 3.1. Predicted miRNA binding to the 30 -UTR of PPARg mRNA miRNAs were selected by microRNA database (2623) and Targetscan database (66) without regard to species. Many miRNAs were excluded (2675) based on non-mammalian animal, duplicate sequence, conserved microRNA and conserved seed pairing [12]. Fourteen miRNAs, mir-130b, mir-130a, mmu-mir-130c, mir-3666, mir-454, mir-4295, mir-301a, has-mir-301b, mmu-mir-301b, mir27a, mir-27b, mir-128, mir-721 and rno-miR-340 were ultimately determined to putatively bind the 30 -UTR of PPARg mRNA by Rattus norvegicus PPARg mRNA (NCBI Reference Sequence: NM_001145366.1 (http://www.ncbi.nlm.nih.gov/nuccore/NM_ 001145366.1)) and miRBase databases (Supplementary Fig. 1A). Some of the selected miRNAs are shown (Supplementary Fig. 1B). 3.2. Detection of miRNA binding to the 30 -UTR of PPARg mRNA in rat liver fibrosis models Rat liver fibrosis models of CCl4 and CBDL were prepared. Compared with miRNAs in quiescent HSCs from normal rat liver (Oil group and Control group), the mRNA expression of a-SMA, Col1A1 and TIMP-1 was increased (Fig. 1A). The expression of five miRNAs miR-130a, miR-130b, miR301a, miR-27b and miR-340 was increased, especially miR-130a and miR-130b, in activated HSCs from the two models (Fig. 1B). The decreased PPARg expression was revealed using the real-time PCR (Fig. 1C) and Western blot analysis (Fig. 1D and 2E) in two models.
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3.3. Overexpression of miR-130a and miR-130b enhances proliferation of HSCs As the levels of miR-130a and miR-130b were shown to be increased in rat activated HSCs, we further verified the effects of them on cell proliferation in HSCs. MiR-130a, miR-130b or miRCTLs was transiently transfected into the rat HSC-T6 cells. As shown in Supplementary Fig. 2A and 2B, in a concentrationdependent manner, overexpression of miR-130a or miR-130b gradually increased cell proliferation from 1 day to 5 days compared with the miR-CTL. A similar increased cell growth was observed under serum-deprived media (Supplementary Fig. 2C). In liver fibrosis, cell proliferation is increased and apoptosis decreased. B-cell 1ymphoma/leukemia-2 (Bcl-2) overexpression has been shown to play an important role in proliferation of activated HSCs [13,14]. Runt-related transcription factor 3 (Runx3) is the target gene of miR-130a. It regulates the expression of Bcl-2 during the apoptosis and autophagy in endothelial progenitor cells and hepatocellular carcinoma cells [15,16]. Therefore, we examined the expression of Runx3 and Bcl-2 mRNA in rat HSC-T6 cells treated with miR-130a, miR-130b, anti-130a or anti-130b. We found that the expression of Runx3 mRNA was decreased and Bcl-2 was increased in rat HSC-T6 cells treated with miR-130a or miR-130b (Supplementary Fig. 2D), while Runx3 was increased and Bcl-2 was decreased treated with anti-130a or anti-130b (Supplementary Fig. 2E). These findings suggest a potential role of Runx3 downregulation and Bcl-2 upregulation in miR-130a or miR-130b-mediated proliferation of HSCs. 3.4. MiR-130a and miR-130b specifically inhibits PPARg protein expression by targeting the 30 -UTR of PPARg mRNA In order to determine whether miR-130a and miR-130b directly targeted the 30 -UTR of PPARg mRNA, we prepared wild-type and mutant-type luciferase reporter pMIR-PPARg-30 -UTR-WT and pMIR-PPARg-30 -UTR-Mut, in which four nucleotides were changed to disrupt the putative interaction between PPARg mRNA and miR130a/b (Fig. 2A). In a concentration-dependent manner, miR-130a or miR-130b from 0.1 to 100 pmol decreased levels of pMIRPPARg-30 -UTR-WT luciferase activity. These miR-130a or miR-130b concentrations showed no significant effect on the levels of pMIRPPARg-30 -UTR-Mut luciferase activity (Fig. 2B, C). As shown in Fig. 2D, compared with the miR-CTL group, miR-130a or miR-130b suppressed the luciferase activity of the pMIR-PPARg-30 -UTR-WT by about 50%e60% in rat HSCs-T6 cells. In contrast, they did not significantly decrease the luciferase activity of the pMIR-PPARg-30 UTR-Mut probably due to altered putative binding sites of miR130a/b (p > 0.30). This finding suggested that miR-130a/b was able to bind the wild-type sequence, but not the mutated site. To confirm that miR-130a, miR-130b or miR-130a/b regulated PPARg protein, the PPARg expression was measured by real-time PCR and Western blotting analysis. In rat HSC-T6 cells transfected with miR-130a, miR-130b or miR-130a/b, the PPARg mRNA and protein levels were significantly decreased after 2 days, compared with the miR-CTL group (Fig. 2 E, F, and G). The levels in rat HSC-T6 cells transfected with anti-miR-130a, anti-miR-130b or anti-miR130a/b were significantly increased after 2 days, compared with the anti-miR-CTL group (Fig. 2 H, I, and J). However, the decrease in PPARg levels after exposure to miR-130a/b was two times lower
Fig. 1. Expression of miRNAs and PPARg in rat liver fibrosis models of CCl4 and CBDL. (A and B) Relative RNA expression of a-SMA, Col1A1, TIMP-1 and five miRNAs was examined by real-time PCR in HSCs of the two models. (C and D) PPARg and a-SMA
expression in the HSCs was analyzed by real-time PCR and Western blot analysis. (E) Quantification of the protein levels of a-SMA and PPARg. wks: weeks. Oil: Olive oil rat group. Control: sham-operated rat group. All results represent mean ± SD (n ¼ 6 per group).*p < 0.05, compared with Oil group or Control group.
Fig. 2. MiR-130a or miR-130b specifically inhibits PPARg by targeting 30 -UTR of PPARg. (A) Schematic diagram of miR-130a and miR-130b target sequence in the 30 -UTR of PPARg and its mutated version. Four nucleotides were changed to disrupt the putative interaction between PPARg mRNA and miR-130a/b in mutant PPARg-30 -UTR. (B and C) Doseresponse of miR-130a or miR-130b levels and luciferase activity of pMIR-PPARg-30 -UTR. (D) The pMIR-REPORT, pMIR-PPARg-30 -UTR-WT or pMIR-PPARg-30 -UTR-Mut vector was co-transfected with miRs into rat HSC-T6 cells, and the luciferase assay was performed, 48 h after transfection. (E, F, H and I) Rat HSC-T6 cells were transfected with miRs or antimiRs. PPARg mRNA and protein expression was analyzed by real-time PCR and Western blot analysis, 48 h later. (G and J) Quantification of the protein levels of PPARg by the miRs or anti-miRs. The levels were normalized to b-actin loading control. All results represent mean ± SD (n ¼ 3 per group).*p < 0.05, compared with miR-CTL group or anti-CTL group. WT: pMIR-PPARg-30 -UTR-WT. Mut: pMIR-PPARg-30 -UTR-Mut. U.T: untreated group.
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compared with miR-130a or miR-130b exposure, which suggested that miR-130a and miR-130b compete for binding with 30 -UTR of PPARg mRNA (Fig. 2E, G). These data strongly suggest that miR130a and miR-130b negatively regulate PPARg expression through direct targeting of the 30 -UTR of PPARg mRNA in rat HSCs. 3.5. MiR-130a and miR-130b positively regulates the expression of ECM in HSCs The above evidence suggested that PPARg is a direct gene target for miR-130a and miR-130b. In order to verify whether overexpression of miR-130a and miR-130b upregulated the expression of ECM genes in rat HSCs, miR-130a/b, anti-miR-130a/b, miR-CTL or anti-miR-CTL was transfected into rat HSC-T6 cells. The ECM genes (a-SMA, Col1A1 and TIMP-1) expression was measured after 24 h. After treatment of rat HSC-T6 cells with miRs, mRNA and protein levels of ECM genes were significantly increased (Fig. 3 A, B and C). After treatment with the anti-miRs, they were significantly decreased (Fig. 3D, E and F). The above evidence suggests that miR130a and miR-130b were positively correlated with expression of ECM genes and anti-miR-130a and anti-miR-130b were negatively correlated with expression of ECM in rat HSCs. Given that miR-130a and miR-130b negatively regulate PPARg expression through direct targeting of the 30 -UTR of PPARg mRNA, we conclude that miR-130a and miR-130b may upregulate the expression of ECM genes by downregulation of PPARg in rat HSCs. However, no significant cytoplasmic lipid droplets were found in rat HSC-T6 cells transfected with anti-miRs, which suggested that the results were not sufficient to activate HSC phenotypic switch to quiescent HSCs. 3.6. TGF-b1 downregulates the expression of PPARg by upregulating miR-130a and miR-130b In HSCs of liver fibrosis, the expression of miR-130a and miR130b is significantly increased and TGF-b plays a dominant role in the synthesis and deposition of collagens. We hypothesized that TGF-b1 may upregulate the expression of miR-130a and miR-130b in activated HSCs. In rat HSC-T6 cells treated with TGF-b1 (10 ng/ ml, 48 h), we found that the expression of miR-130a and miR-130b was significantly increased and the mRNA and protein levels of PPARg were significantly decreased (Fig. 4A, D and E). The a-SMA, Col1A1 and TIMP-1 levels were increased after TGF-b1 treatment by real-time PCR (Fig. 4B) and Western blot analysis (Fig. 4D, E). The levels of a-SMA, Col1A1 and TIMP-1 were decreased and PPARg were increased after TGF-b1 combined with anti-miR-130a or antimiR-130b treatment as determined by real-time PCR (Fig. 4C). However, protein levels were not found to be changed (data not shown). We concluded that in rat HSC-T6 cells treated with TGF-b1, the levels of miR-130a and miR-130b were significantly upregulated and PPARg was decreased. Furthermore, the expression of ECM genes was upregulated, which further activated HSCs and increased collagen synthesis. 4. Discussion It has been shown previously that PPARg is a ligand-dependent transcription factor in HSCs [3]. The expression of PPARg has been found to be increased by its ligands or synthetic ligands in activated
Fig. 3. MiR-130a and miR-130b positively regulated the expression of ECM in HSCs. (A and D) Expression of a-SMA, Col1A1 and TIMP-1 mRNA was analyzed by real-time PCR, 48 h following the treatment of miRs or anti-miRs. (B, C, E and F) Protein expression
levels of a-SMA, Col1A1 and TIMP-1 were determined by Western blot analysis and quantification analysis, 48 h following the treatment of miRs or anti-miRs. The levels were normalized to b-actin loading control. All results represent mean ± SD (n ¼ 3 per group).*p < 0.05, compared with miR-CTL group or anti-CTL group. U.T: untreated group.
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HSCs [17]. However, the mechanism of downregulation or inactivation of PPARg has yet to be clearly established. This current study demonstrates that in rat liver fibrosis, miR-130a and miR-130b acted as negative regulatory factors of PPARg, downregulated PPARg by targeting 30 -UTR of PPARg mRNA, enhancing activation of HSCs and increasing secretion of ECM. In the two rat liver fibrosis models, levels of miR-130a and miR130b were found to be significantly increased (Fig. 1B). Although miR-130a and miR-130b may negatively regulate expression of PPARg by binding to the 30 -UTR of PPARg mRNA in hepatocellular carcinoma, scleroderma and adipose tissue [18e20], it has not been reported to the best of our knowledge that they negatively regulate PPARg in the HSCs of liver fibrosis. Therefore, we focused on miR130a and miR-130b in this study. Interestingly, the miR-130a and miR-130b levels were found to vary in different liver diseases. They were downregulated in liver cells of hepatitis B, liver metabolic diseases and liver cancer [21e23], while they were upregulated in HCV-infected hepatocytes, cholangiocytes and activated HSCs [24e27]. In the current study, miR-130a and miR-130b increased 4.98 and 4.23, 4.59 and 3.75 times in activated HSCs in the rat liver fibrosis models of CCl4 and CBDL. The result was similar to published studies suggesting that in primary rat HSCs cells, miR-130a increased 3.427 times and miR-130b increased 2.046 times after incubation for 3e10 days [26,27]. In different cell types, miR-130a and miR-130b engage in crosstalk with TGF-b1/Smad signaling pathway. TGF-b1 combined with TGF-bR activates Smad2/3, and then combines with Smad4, which increases the expression of nuclear transcription factors and induces liver fibrosis [28]. TGF-b1 upregulates the expression of miR-130a and miR-130b in systemic sclerosis (SSc) skin fibroblasts and renal mesangial cells [19,29] and downregulates their expression in human gastric cancer cells [30]. Conversely, miR-130a and miR-130b regulate the expression of target genes by TGF-b1 signaling in granulocytes and non-small cell lung cancer (NSCLC) cells [31,32]. In the current study, in rat HSC-T6 cells treated with TGF-b1, the expression of miR-130a and miR-130b was significantly upregulated, and that of PPARg was decreased. Furthermore, the expression of a-SMA, Col1A and TIMP-1 was upregulated, which activated HSCs and increased collagen synthesis. Changes in TGF-b1 levels may explain the overexpression of miR-130a and miR-130b. In addition to increasing collagen production, miR-130a or miR130b also showed a moderate effect in promoting the proliferation of activated HSCs. In the current study, miR-130a or miR-130b mediated proliferation of HSCs by downregulation of Runx3 and upregulation of Bcl-2. Runx3 is the target gene of not only miR-130a, but also miR-130b. Additional studies are needed to further define the mechanism of miR-130a and miR-130b-mediated activation of HSCs in liver fibrosis. This study has a few limitations. The molecular mechanisms of liver fibrosis in human livers and experimental animals may not be identical. Fibrosis in human livers usually progresses slowly, over the course of decades, while liver fibrosis in experimental animals progresses quickly, for several weeks [27]. Therefore, miRNAs in liver fibrosis in experimental animals cannot be equated to human liver fibrosis. To establish the role of miRNAs as a direct cause of
Fig. 4. TGF-b1 upregulates ECM genes by miR-130a and miR-130b. (A) Real-time PCR analysis showing expression of miR-130a and miR-130b and expression of PPARg mRNA in HSC-T6 treated with TGF-b1 (10 ng/ml, 48 h). (C) The levels of PPARg, a-SMA,
Col1A1 and TIMP-1 were decreased after TGF-b1 combined with anti-miR-130a or anti-miR-130b treatment by real-time PCR. (B and D) real-time PCR showing expression levels of a-SMA, Col1A and TIMP-1 (B) and Western blot analysis revealing protein expression of a-SMA, Col1A and TIMP-1 (D), in rat HSC-T6 treated with TGF-b1 (10 ng/ ml, 48 h). (E) Quantification of the protein levels of PPARg, a-SMA, Col1A and TIMP-1 treated with TGF-b1. The levels were normalized to b-actin loading control. All results represent mean ± SD (n ¼ 3 per group).*p < 0.05, compared with TGF-b1 () group.
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PPARg inhibition in HSCs of human livers, gene chip technology should be used to comprehensively screen potential miRNAs. Studies on the time course of interactions are planned for the future. Additional studies investigating miRNAs and target genes in human HSC cell lines and human primary HSC cells are needed in order to understand the relationship between miRNAs and PPARg, and their roles in hepatic fibrosis and cirrhosis. In conclusion, miR-130a and miR-130b were found to specifically regulate PPARg expression through the 30 -UTR of PPARg mRNA, suggesting a role in the progression of liver fibrosis. In addition, TGF-b1 may cause overexpression of miR-130a and miR130b in liver fibrosis, increasing cell proliferation and differentiation by targeting Runx3 in vitro. Further studies are needed to confirm and extend these initial findings. Conflict of interest The authors have no conflicts to disclose.
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Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (NSFC No. 81170454). We are grateful to the Medjaden Bioscience Limited in editing and proofreading the manuscript.
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Transparency document
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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.08.012.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
MicroRNA-130a and -130b enhance activation of hepatic stellate cells by suppressing PPARg expression: A rat fibrosis model study Le Lu a, Jinlong Wang a, Hongwei Lu a, Guoyu Zhang b, Yang Liu a, Jiazhong Wang a, Yafei Zhang a, Hao Shang a, Hong Ji a, Xi Chen a, Yanxia Duan a, Yiming Li a, * a b
Department of General Surgery, The Second Affiliated Hospital of Xi'an Jiaotong University, No.157, West 5th Road, Xi'an, Shaanxi 710004, China West Hospital Ward 1, Shaanxi Provincial People's Hospital, No.256, Youyi Road(west), Xi'an, Shaanxi 710068, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 July 2015 Accepted 3 August 2015 Available online 6 August 2015
Hepatic stellate cells (HSCs) are the primary sources of extracellular matrix (ECM) in normal and fibrotic liver. Peroxisome proliferator-activated receptor gamma (PPARg) maintains HSCs in a quiescent state, and its downregulation induces HSC activation. MicroRNAs (miRNAs) can induce PPARg mRNA degradation, but the mechanism by which miRNAs regulate PPARg in rat HSCs is unclear. This study aimed to investigate some miRNAs which putatively bind to the 30 -untranslated region (30 -UTR) of PPARg mRNA, and increase expression of ECM genes in rat HSCs. In carbon tetrachloride injection (CCl4) and common bile duct ligation (CBDL) liver fibrosis models, miRNAs miR-130a, miR-130b, miR-301a, miR-27b and miR340 levels were found to be increased and PPARg expression decreased. Overexpression of miR-130a and miR-130b enhanced cell proliferation by involving Runx3. MiR-130a and miR-130b decreased PPARg expression by targeting the 30 -UTR of PPARg mRNA in rat HSC-T6 cells. Transforming growth factor-b1 (TGF-b1) may mediate miR-130a and miR-130b overexpression, PPARg downregulation, and ECM genes overexpression in cell culture. These findings suggest that miR-130a and miR-130b are involved in downregulation of PPARg in liver fibrosis. © 2015 Elsevier Inc. All rights reserved.
Keywords: miR-130a miR-130b PPARg TGF-b1 Hepatic stellate cells Liver fibrosis
1. Introduction Portal hypertension is a frequent cause of death in patients with cirrhosis [1]. Liver fibrosis and cirrhosis are the main causes of portal hypertension. Hepatic stellate cells (HSCs) are the primary source of extracellular matrix (ECM) in normal and fibrotic liver. When the liver is injured, quiescent HSCs undergo ‘activation’ and transdifferentiation from non-proliferating cells to myofibroblasts, which proliferate and produce ECM [2]. Peroxisome proliferator-activated receptors g (PPARg) are transcriptional factors that are abundant in normal HSCs, where they regulate lipid metabolism and adipocyte differentiation [3]. Recently, PPARg has been identified as a key molecular switch involved in HSC activation and phenotype alteration. PPARg expression is dramatically diminished during HSC activation and enables HSCs transdifferentiation to a myofibroblast phenotype [4]. However, little has been reported on the direct cause of downregulated expression or inactivation of PPARg.
MicroRNAs (miRNAs) regulate gene expression by binding to the 30 -untranslated region (30 -UTR) of the target gene mRNAs to repress protein translation or induce mRNA degradation [5]. Several hundred miRNAs have been identified, and more than 60% of mammalian genes are known to be targeted and regulated by miRNAs [6]. A variety of miRNAs are involved in the regulation of HSC proliferation, differentiation, senescence, apoptosis and autophagy [7]. However, the role of miRNAs in regulating PPARg gene in the HSCs of liver fibrosis and cirrhosis is unclear. The aim of the current study was to investigate miRNAs, which putatively bind the 30 -UTR of PPARg mRNA in rat HSCs and increase ECM gene expression in vitro. We focused on two miRNAs miR-130a and miR-130b that are overexpressed in activated HSCs in rat and target the 30 -UTR of PPARg mRNA. 2. Materials and methods 2.1. Prediction of miRNAs using bioinformatics
* Corresponding author. E-mail address: [email protected] (Y. Li). http://dx.doi.org/10.1016/j.bbrc.2015.08.012 0006-291X/© 2015 Elsevier Inc. All rights reserved.
NCBI database (http://www.ncbi.nlm.nih.gov/nuccore/) was used to search sequences of PPARg genes from several species. The
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Targetscan (http://www.targetscan.org/), microRNA (http://www. microrna.org/) and miRBase (http://www.mirbase.org/) databases were used to select miRNAs that may sequence-specifically target the 30 -UTR of PPARg. 2.2. Animals and HSC isolation Male SpragueeDawley rats weighing between 200 and 300 g were purchased from the Laboratory Animal Center, Xi'an Jiaotong University Health Science Center (Xi'an, Shaanxi, China). All procedures were reviewed and approved by the Ethics Committee, Xi'an Jiaotong University Health Science Center. In order to clarify the exact role of miRNAs in PPARg, the two rat models of liver fibrosis were used: carbon tetrachloride injection (CCl4) and common bile duct ligation (CBDL). CCl4 was administered by intraperitoneal injection of (Sinopharm Chemical Reagent Co. Ltd. Shanghai, China; 0.6 ml/kg of body weight) in olive oil, twice a week for six weeks. Controls received only olive oil. Ligationtransection of the common bile duct was performed on rats as described previously [8]. Sham-operated rats served as controls. HSC isolation was performed as described previously [9]. 2.3. Materials and reagents Oligonucleotides, miR-mimic (miR), miR inhibitor (anti-miR), miR negative control (miR-CTL) and anti-miR negative control (anti-miR-CTL) were purchased from Biomics Biotechnologies Co., Ltd (Nantong, Jiangsu, China). The pMIR-REPORT™ miRNA Expression Reporter Vector was ordered from Ambion (Austin, TX, USA). Transforming growth factor-b1 (TGF-b1) was obtained from Sigma (St. Louis, MO, USA). 2.4. Cell cultures HSC-T6, an immortalized rat HSC line, purchased from KeyGEN (Nanjing, Jiangsu, China), was maintained in Dulbecco's modified Eagle's medium (DMEM, Hyclone, NY, USA) containing 10% fetal calf serum (FCS, Tianhang Biotechnology; Hangzhou, Zhejiang, China) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin; KeyGEN, Nanjing, Jiangsu, China). Culture conditions were set at 37 C under 5% CO2 and 95% atmospheric air at constant humidity (Thermo, USA). Culture medium was changed every 2e3 days. 2.5. Cell proliferation Rat HSC-T6 cells were seeded in 96-well tissue culture plates by adding 150 ml/well of a suspension of 5 104 cells. Cell viability was determined using the MTT (MTT, Sigma) assay according to the manufacturer's instructions. 2.6. RNA isolation and real-time PCR Total RNA was isolated from HSCs using TRIzol (Invitrogen, Carlsbad, CA, USA). For analysis of miRNA and mRNA levels, realtime polymerase chain reaction (PCR) was performed with SYBR® PrimeScript™ miRNA RT-PCR Kit and SYBR® Premix EX Taq™ II (Tli RNaseH Plus) (TaKaRa Bio, Osaka, Japan) according to the manufacturer's instructions. Rat U6 snRNA and GAPDH mRNA were included separately as an internal references. The primers were used in this study are shown in Supplementary Table 1. The relative expression levels of miRNAs and mRNAs were calculated by the 2DDCT method [10].
2.7. Western blot analysis HSCs were lysed using RIPA buffer (APPLYGEN, Beijing, China) according to the manufacturer's manual. Protein concentration was estimated with an ultraviolet spectrophotometer (Bio-Rad, Hercules, CA, USA). Western blot analysis was performed using antibodies against PPARg (1:500), alpha-smooth muscle actin (a-SMA, 1:1000), a1 type I collagen (Col1A1, 1:1000), tissue inhibitor of metalloproteinases-1 (TIMP-1, 1:1000), b-actin (1:1000) (Proteintech Group, Chicago, USA) and goat anti-rabbit antibody (1:3000, Bioworld, Minnesota, USA) as previously described [11]. The rat b-actin protein was used as an internal reference. 2.8. Dual luciferase assays The sequence cDNA containing PPARg30 -UTR and PPARg 30 -UTR mutant (PPARg30 -UTR-Mut) were inserted into a dual luciferase reporter vector of pMIR-REPORT (Supplementary Materials and Methods). For the luciferase assays, 200 ng vector (pMIR-REPORT, pMIR-REPORT-PPARg30 -UTR- WT or pMIR-REPORT-PPARg30 -UTRMut), 20 ng renilla luciferase control plasmid (pRL-TK), and 10 pmol oligonucleotides (miR-130a, miR-130b or miR-CTL) were cotransfected in rat HSC-T6 cells with Lipofectamine 2000 (Invitrogen, Carlsbad, CA). After 48 h, firefly luciferase activity was determined using the Dual Luciferase Assay system (Promega, Madison, WI) according to the manufacturer's instructions. The experiments were performed in quadruplicate. 2.9. Statistical analysis All experiments were repeated at least three times. Data are expressed as mean ± SD. Two samples were compared using twotailed Student's t-test and p < 0.05 was considered as statistically significant. 3. Results 3.1. Predicted miRNA binding to the 30 -UTR of PPARg mRNA miRNAs were selected by microRNA database (2623) and Targetscan database (66) without regard to species. Many miRNAs were excluded (2675) based on non-mammalian animal, duplicate sequence, conserved microRNA and conserved seed pairing [12]. Fourteen miRNAs, mir-130b, mir-130a, mmu-mir-130c, mir-3666, mir-454, mir-4295, mir-301a, has-mir-301b, mmu-mir-301b, mir27a, mir-27b, mir-128, mir-721 and rno-miR-340 were ultimately determined to putatively bind the 30 -UTR of PPARg mRNA by Rattus norvegicus PPARg mRNA (NCBI Reference Sequence: NM_001145366.1 (http://www.ncbi.nlm.nih.gov/nuccore/NM_ 001145366.1)) and miRBase databases (Supplementary Fig. 1A). Some of the selected miRNAs are shown (Supplementary Fig. 1B). 3.2. Detection of miRNA binding to the 30 -UTR of PPARg mRNA in rat liver fibrosis models Rat liver fibrosis models of CCl4 and CBDL were prepared. Compared with miRNAs in quiescent HSCs from normal rat liver (Oil group and Control group), the mRNA expression of a-SMA, Col1A1 and TIMP-1 was increased (Fig. 1A). The expression of five miRNAs miR-130a, miR-130b, miR301a, miR-27b and miR-340 was increased, especially miR-130a and miR-130b, in activated HSCs from the two models (Fig. 1B). The decreased PPARg expression was revealed using the real-time PCR (Fig. 1C) and Western blot analysis (Fig. 1D and 2E) in two models.
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3.3. Overexpression of miR-130a and miR-130b enhances proliferation of HSCs As the levels of miR-130a and miR-130b were shown to be increased in rat activated HSCs, we further verified the effects of them on cell proliferation in HSCs. MiR-130a, miR-130b or miRCTLs was transiently transfected into the rat HSC-T6 cells. As shown in Supplementary Fig. 2A and 2B, in a concentrationdependent manner, overexpression of miR-130a or miR-130b gradually increased cell proliferation from 1 day to 5 days compared with the miR-CTL. A similar increased cell growth was observed under serum-deprived media (Supplementary Fig. 2C). In liver fibrosis, cell proliferation is increased and apoptosis decreased. B-cell 1ymphoma/leukemia-2 (Bcl-2) overexpression has been shown to play an important role in proliferation of activated HSCs [13,14]. Runt-related transcription factor 3 (Runx3) is the target gene of miR-130a. It regulates the expression of Bcl-2 during the apoptosis and autophagy in endothelial progenitor cells and hepatocellular carcinoma cells [15,16]. Therefore, we examined the expression of Runx3 and Bcl-2 mRNA in rat HSC-T6 cells treated with miR-130a, miR-130b, anti-130a or anti-130b. We found that the expression of Runx3 mRNA was decreased and Bcl-2 was increased in rat HSC-T6 cells treated with miR-130a or miR-130b (Supplementary Fig. 2D), while Runx3 was increased and Bcl-2 was decreased treated with anti-130a or anti-130b (Supplementary Fig. 2E). These findings suggest a potential role of Runx3 downregulation and Bcl-2 upregulation in miR-130a or miR-130b-mediated proliferation of HSCs. 3.4. MiR-130a and miR-130b specifically inhibits PPARg protein expression by targeting the 30 -UTR of PPARg mRNA In order to determine whether miR-130a and miR-130b directly targeted the 30 -UTR of PPARg mRNA, we prepared wild-type and mutant-type luciferase reporter pMIR-PPARg-30 -UTR-WT and pMIR-PPARg-30 -UTR-Mut, in which four nucleotides were changed to disrupt the putative interaction between PPARg mRNA and miR130a/b (Fig. 2A). In a concentration-dependent manner, miR-130a or miR-130b from 0.1 to 100 pmol decreased levels of pMIRPPARg-30 -UTR-WT luciferase activity. These miR-130a or miR-130b concentrations showed no significant effect on the levels of pMIRPPARg-30 -UTR-Mut luciferase activity (Fig. 2B, C). As shown in Fig. 2D, compared with the miR-CTL group, miR-130a or miR-130b suppressed the luciferase activity of the pMIR-PPARg-30 -UTR-WT by about 50%e60% in rat HSCs-T6 cells. In contrast, they did not significantly decrease the luciferase activity of the pMIR-PPARg-30 UTR-Mut probably due to altered putative binding sites of miR130a/b (p > 0.30). This finding suggested that miR-130a/b was able to bind the wild-type sequence, but not the mutated site. To confirm that miR-130a, miR-130b or miR-130a/b regulated PPARg protein, the PPARg expression was measured by real-time PCR and Western blotting analysis. In rat HSC-T6 cells transfected with miR-130a, miR-130b or miR-130a/b, the PPARg mRNA and protein levels were significantly decreased after 2 days, compared with the miR-CTL group (Fig. 2 E, F, and G). The levels in rat HSC-T6 cells transfected with anti-miR-130a, anti-miR-130b or anti-miR130a/b were significantly increased after 2 days, compared with the anti-miR-CTL group (Fig. 2 H, I, and J). However, the decrease in PPARg levels after exposure to miR-130a/b was two times lower
Fig. 1. Expression of miRNAs and PPARg in rat liver fibrosis models of CCl4 and CBDL. (A and B) Relative RNA expression of a-SMA, Col1A1, TIMP-1 and five miRNAs was examined by real-time PCR in HSCs of the two models. (C and D) PPARg and a-SMA
expression in the HSCs was analyzed by real-time PCR and Western blot analysis. (E) Quantification of the protein levels of a-SMA and PPARg. wks: weeks. Oil: Olive oil rat group. Control: sham-operated rat group. All results represent mean ± SD (n ¼ 6 per group).*p < 0.05, compared with Oil group or Control group.
Fig. 2. MiR-130a or miR-130b specifically inhibits PPARg by targeting 30 -UTR of PPARg. (A) Schematic diagram of miR-130a and miR-130b target sequence in the 30 -UTR of PPARg and its mutated version. Four nucleotides were changed to disrupt the putative interaction between PPARg mRNA and miR-130a/b in mutant PPARg-30 -UTR. (B and C) Doseresponse of miR-130a or miR-130b levels and luciferase activity of pMIR-PPARg-30 -UTR. (D) The pMIR-REPORT, pMIR-PPARg-30 -UTR-WT or pMIR-PPARg-30 -UTR-Mut vector was co-transfected with miRs into rat HSC-T6 cells, and the luciferase assay was performed, 48 h after transfection. (E, F, H and I) Rat HSC-T6 cells were transfected with miRs or antimiRs. PPARg mRNA and protein expression was analyzed by real-time PCR and Western blot analysis, 48 h later. (G and J) Quantification of the protein levels of PPARg by the miRs or anti-miRs. The levels were normalized to b-actin loading control. All results represent mean ± SD (n ¼ 3 per group).*p < 0.05, compared with miR-CTL group or anti-CTL group. WT: pMIR-PPARg-30 -UTR-WT. Mut: pMIR-PPARg-30 -UTR-Mut. U.T: untreated group.
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compared with miR-130a or miR-130b exposure, which suggested that miR-130a and miR-130b compete for binding with 30 -UTR of PPARg mRNA (Fig. 2E, G). These data strongly suggest that miR130a and miR-130b negatively regulate PPARg expression through direct targeting of the 30 -UTR of PPARg mRNA in rat HSCs. 3.5. MiR-130a and miR-130b positively regulates the expression of ECM in HSCs The above evidence suggested that PPARg is a direct gene target for miR-130a and miR-130b. In order to verify whether overexpression of miR-130a and miR-130b upregulated the expression of ECM genes in rat HSCs, miR-130a/b, anti-miR-130a/b, miR-CTL or anti-miR-CTL was transfected into rat HSC-T6 cells. The ECM genes (a-SMA, Col1A1 and TIMP-1) expression was measured after 24 h. After treatment of rat HSC-T6 cells with miRs, mRNA and protein levels of ECM genes were significantly increased (Fig. 3 A, B and C). After treatment with the anti-miRs, they were significantly decreased (Fig. 3D, E and F). The above evidence suggests that miR130a and miR-130b were positively correlated with expression of ECM genes and anti-miR-130a and anti-miR-130b were negatively correlated with expression of ECM in rat HSCs. Given that miR-130a and miR-130b negatively regulate PPARg expression through direct targeting of the 30 -UTR of PPARg mRNA, we conclude that miR-130a and miR-130b may upregulate the expression of ECM genes by downregulation of PPARg in rat HSCs. However, no significant cytoplasmic lipid droplets were found in rat HSC-T6 cells transfected with anti-miRs, which suggested that the results were not sufficient to activate HSC phenotypic switch to quiescent HSCs. 3.6. TGF-b1 downregulates the expression of PPARg by upregulating miR-130a and miR-130b In HSCs of liver fibrosis, the expression of miR-130a and miR130b is significantly increased and TGF-b plays a dominant role in the synthesis and deposition of collagens. We hypothesized that TGF-b1 may upregulate the expression of miR-130a and miR-130b in activated HSCs. In rat HSC-T6 cells treated with TGF-b1 (10 ng/ ml, 48 h), we found that the expression of miR-130a and miR-130b was significantly increased and the mRNA and protein levels of PPARg were significantly decreased (Fig. 4A, D and E). The a-SMA, Col1A1 and TIMP-1 levels were increased after TGF-b1 treatment by real-time PCR (Fig. 4B) and Western blot analysis (Fig. 4D, E). The levels of a-SMA, Col1A1 and TIMP-1 were decreased and PPARg were increased after TGF-b1 combined with anti-miR-130a or antimiR-130b treatment as determined by real-time PCR (Fig. 4C). However, protein levels were not found to be changed (data not shown). We concluded that in rat HSC-T6 cells treated with TGF-b1, the levels of miR-130a and miR-130b were significantly upregulated and PPARg was decreased. Furthermore, the expression of ECM genes was upregulated, which further activated HSCs and increased collagen synthesis. 4. Discussion It has been shown previously that PPARg is a ligand-dependent transcription factor in HSCs [3]. The expression of PPARg has been found to be increased by its ligands or synthetic ligands in activated
Fig. 3. MiR-130a and miR-130b positively regulated the expression of ECM in HSCs. (A and D) Expression of a-SMA, Col1A1 and TIMP-1 mRNA was analyzed by real-time PCR, 48 h following the treatment of miRs or anti-miRs. (B, C, E and F) Protein expression
levels of a-SMA, Col1A1 and TIMP-1 were determined by Western blot analysis and quantification analysis, 48 h following the treatment of miRs or anti-miRs. The levels were normalized to b-actin loading control. All results represent mean ± SD (n ¼ 3 per group).*p < 0.05, compared with miR-CTL group or anti-CTL group. U.T: untreated group.
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HSCs [17]. However, the mechanism of downregulation or inactivation of PPARg has yet to be clearly established. This current study demonstrates that in rat liver fibrosis, miR-130a and miR-130b acted as negative regulatory factors of PPARg, downregulated PPARg by targeting 30 -UTR of PPARg mRNA, enhancing activation of HSCs and increasing secretion of ECM. In the two rat liver fibrosis models, levels of miR-130a and miR130b were found to be significantly increased (Fig. 1B). Although miR-130a and miR-130b may negatively regulate expression of PPARg by binding to the 30 -UTR of PPARg mRNA in hepatocellular carcinoma, scleroderma and adipose tissue [18e20], it has not been reported to the best of our knowledge that they negatively regulate PPARg in the HSCs of liver fibrosis. Therefore, we focused on miR130a and miR-130b in this study. Interestingly, the miR-130a and miR-130b levels were found to vary in different liver diseases. They were downregulated in liver cells of hepatitis B, liver metabolic diseases and liver cancer [21e23], while they were upregulated in HCV-infected hepatocytes, cholangiocytes and activated HSCs [24e27]. In the current study, miR-130a and miR-130b increased 4.98 and 4.23, 4.59 and 3.75 times in activated HSCs in the rat liver fibrosis models of CCl4 and CBDL. The result was similar to published studies suggesting that in primary rat HSCs cells, miR-130a increased 3.427 times and miR-130b increased 2.046 times after incubation for 3e10 days [26,27]. In different cell types, miR-130a and miR-130b engage in crosstalk with TGF-b1/Smad signaling pathway. TGF-b1 combined with TGF-bR activates Smad2/3, and then combines with Smad4, which increases the expression of nuclear transcription factors and induces liver fibrosis [28]. TGF-b1 upregulates the expression of miR-130a and miR-130b in systemic sclerosis (SSc) skin fibroblasts and renal mesangial cells [19,29] and downregulates their expression in human gastric cancer cells [30]. Conversely, miR-130a and miR-130b regulate the expression of target genes by TGF-b1 signaling in granulocytes and non-small cell lung cancer (NSCLC) cells [31,32]. In the current study, in rat HSC-T6 cells treated with TGF-b1, the expression of miR-130a and miR-130b was significantly upregulated, and that of PPARg was decreased. Furthermore, the expression of a-SMA, Col1A and TIMP-1 was upregulated, which activated HSCs and increased collagen synthesis. Changes in TGF-b1 levels may explain the overexpression of miR-130a and miR-130b. In addition to increasing collagen production, miR-130a or miR130b also showed a moderate effect in promoting the proliferation of activated HSCs. In the current study, miR-130a or miR-130b mediated proliferation of HSCs by downregulation of Runx3 and upregulation of Bcl-2. Runx3 is the target gene of not only miR-130a, but also miR-130b. Additional studies are needed to further define the mechanism of miR-130a and miR-130b-mediated activation of HSCs in liver fibrosis. This study has a few limitations. The molecular mechanisms of liver fibrosis in human livers and experimental animals may not be identical. Fibrosis in human livers usually progresses slowly, over the course of decades, while liver fibrosis in experimental animals progresses quickly, for several weeks [27]. Therefore, miRNAs in liver fibrosis in experimental animals cannot be equated to human liver fibrosis. To establish the role of miRNAs as a direct cause of
Fig. 4. TGF-b1 upregulates ECM genes by miR-130a and miR-130b. (A) Real-time PCR analysis showing expression of miR-130a and miR-130b and expression of PPARg mRNA in HSC-T6 treated with TGF-b1 (10 ng/ml, 48 h). (C) The levels of PPARg, a-SMA,
Col1A1 and TIMP-1 were decreased after TGF-b1 combined with anti-miR-130a or anti-miR-130b treatment by real-time PCR. (B and D) real-time PCR showing expression levels of a-SMA, Col1A and TIMP-1 (B) and Western blot analysis revealing protein expression of a-SMA, Col1A and TIMP-1 (D), in rat HSC-T6 treated with TGF-b1 (10 ng/ ml, 48 h). (E) Quantification of the protein levels of PPARg, a-SMA, Col1A and TIMP-1 treated with TGF-b1. The levels were normalized to b-actin loading control. All results represent mean ± SD (n ¼ 3 per group).*p < 0.05, compared with TGF-b1 () group.
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PPARg inhibition in HSCs of human livers, gene chip technology should be used to comprehensively screen potential miRNAs. Studies on the time course of interactions are planned for the future. Additional studies investigating miRNAs and target genes in human HSC cell lines and human primary HSC cells are needed in order to understand the relationship between miRNAs and PPARg, and their roles in hepatic fibrosis and cirrhosis. In conclusion, miR-130a and miR-130b were found to specifically regulate PPARg expression through the 30 -UTR of PPARg mRNA, suggesting a role in the progression of liver fibrosis. In addition, TGF-b1 may cause overexpression of miR-130a and miR130b in liver fibrosis, increasing cell proliferation and differentiation by targeting Runx3 in vitro. Further studies are needed to confirm and extend these initial findings. Conflict of interest The authors have no conflicts to disclose.
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Acknowledgments This study was supported by grants from the National Natural Science Foundation of China (NSFC No. 81170454). We are grateful to the Medjaden Bioscience Limited in editing and proofreading the manuscript.
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Transparency document
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Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.08.012.
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