The Effect Of MiR-134on Hepatic Ifbrosis And The Mechanism

【Backgrounds and Objectives】Hepatic fibrosis is a common pathological consequence of chronic injury caused by thedamage of hepatic parenchymal cells and persistant inflammation, which is characterizedby the excess deposition of extracellular matrix (ECM) and the decrease of degradation ofECM, eventually contributing to the formation of scar tissue in the liver and the collapse ofnormal hepatic structure. This process is drived by the heterogeneous myofibroblastswhich is mainly derived from hepatic stellate cells (HSCs). As the major mesenchymal celltype in the liver architecture, differentiated HSCs (quiescent HSCs) are mainly responsiblefor the storage of vitamin A. Durning liver fibrogenesis, activation of intracellularsignaling transduction pathway that control the expression of activation-associatedmolecules such as transforming growth factor-β (TGFβ), platelet-derived growth factor(PDGF) and monocyte chemotactic protein-1(MCP-1), etc lead to the myofibroblastictrans-differentiation (activation) of HSC. Activated HSCs lose the lipid droplet, gainproliferation ability and produce large amout of collagen. Given that activation of HSCs isthe pivotal event in liver fibrogenesis, approaches targeting the activation of HSCs andpromoting the apoptosis of HSCs have been developed to treat hepatic fibrosis. However,no satisfactory clinical outcome has been achieved so far.Recently, several novel molecular targets have been reported to be implicated in thetreatment of hepatic fibrosis, in which microRNA (miRNA) represents a hot spot. miRNAsare endogeneous doble-strand non-coding RNA which function as posttranscriptionalmediatior. Recent studies have uncovered the contribution of microRNAs to thepathogenesis of many liver diseases, including viral hepatitis, alcoholic and nonalcoholic fatty liver disease, hepatic fibrosis and hepatocellular carcinoma (HCC). There are studiesreported the aberrant expression of miRNAs in the development of liver fibrosis and therole of miRNAs in the promotion and attenuation of liver fibrogenesis by regulating theactivation and apoptosis of HSCs, indicating that modulation of fibrosis-related miRNAsmay be a novel strategy for the therapeutic of hepatic fibrosis.miR-134is located in miRNAs cluster in the imprinted Dlk1/Dio3region. This region issituated in the human14q32domain, while in mouse, this miRNA gene cluster isconserved at the homologous distal12region. Previous studies have demonstrated theeffect of miR-134on the development of central nerve system, including promoting thedevelopment of neuron, the formation of dendrite spine and the synaptic plasticity. Itsaberrant expression is also closely related to the occurrence of brain tumors such asoligodendroglioma and glioblastoma. Recent researches have indicated that miRNAs onthis cluster play important roles in the development of HCC. Furthermore, it has also beenreported that transcription factor hepatocyte nuclear factor4α (HNF4α) which isdysregulated in the pathogenesis of hepatic fibrosis and HCC reverses the malignance ofHCC cells by regulating miR-134, indicating that miR-134may be an significant effectorin the pathological process of the liver. However, to date, the effect of miR-134on hepaticfibrosis is still unclear. Hence, this study aims to investigate the function of miR-134inliver fibrogenesis and its mechanism.【Methods】1. The expression of miR-134in the liver tissuses from rat hepatic fibrosis models and rathepatic stellate cells(1) The detection of miR-134in liver tissues from control rats and hepatic fibrosis modelsEighting male SD rats were randomized to three groups with six rats in each group.The first group was healty control, while the rats in the second and the third groupsreceived intraperitoneal injection of1%dimethylnitrosamine (DMN)(100μl/100g bodyweight). The rejection was perfomed three times continuously a week. Rats in the seond group were sacrificed in the second week while rats in the third group weresacrificed in the third week. Bile duct ligation operation (rats, n=24) was performedbased on the previous method in our labrotory to establish another hepatic fibrosisanimal model. Six rats receiving sham operations serve as controls. Six rats weresacrificed in each time point (week1, week2, week3) afterwards. Real time RT-PCRwas used to detect the expression of miR-134in the liver tissues from all these rats.(2) The comparison of miR-134levels in quiescent HSCs and activated HSCsPrimary rat HSCs were isolated and cultured using DMEM medium in vitro. RNA ofcells being cultured for3days (quiescent),6days,9days and12days were collected.Real time RT-PCR was used to detect the expression of miR-134, α smooth muscleactin (α-SMA) and collagen I in each group of cells.(3) The detection of miR-134level in T6cells treated with TGFβ1Rat HSCs lines T6cell was treated with varied concentration of TGF-β1(0ng/ml,2ng/ml and4ng/ml). Cell RNA was collected48h after the stimulation. Real timeRT-PCR was used to detect the expression of miR-134, α-SMA and collagen I.2. The effect of miR-134on the biological characteristics of T6cell(1) The effect of overexpression of miR-134on T6cellsT6cells were transfected using lipofectamine2000with miR-134mimic. Cell RNA andprotein were collected48h after the transfection. Real time RT-PCR was used to detectthe expression of miR-134, α-SMA and collagen I. Western blot was used to detect thelevel of α-SMA and collagen I. Cell counting kit8(CCK8) was used to evaluate theeffect of miR-134on the proliferation ability of T6cells.(2) The effect of down-regulation of miR-134on T6cellsT6cells were transfected using lipofectamine2000with miR-134inhibitor. Cell RNAand protein were collected48h after the transfection. Real time RT-PCR was used todetect the expression of miR-134, α-SMA and collagen I. Western blot was used todetect the level of α-SMA and collagen I. Cell counting kit8(CCK8) was used toevaluate the effect of miR-134inhibiton on the proliferation ability of T6cells. 3. The mechanism of miR-134on hepatic fibrosis(1) The prediction of potential target of miR-134using softwareTargetScan (www.targetscan.org/) was used to predict the potential target of miR-134.This approach indicated that signal transducers and activators of transcription5B(STAT5B) was the potential target of miR-134. We detected the effect of miR-134onthe expression of mRNA and protein of STAT5B.(2) The identification of the STAT5B as the direct target of miR-134We constructed the luciferase repoter carrying the binding site of miR-134on the3′-UTR of STAT5B and the mutant binding site. These reporters were co-transfectedwith miR-134mimic or negative control (NC) respectively to HEK-293cells. Duralrepoter assay was performed to detect the activity of the reporters. To further evaluatedthe regulation of STAT5B by miR-134, real time RT-PCR was used to measure the levelof STAT5B mRNA in T6cells treated with T6cells.4. Statistical analysisAll experiments were replicated at least three times. The data were shown as X±SD. Allstatistical analyses were performed using SPSS11.0software. One-Way ANOVA wasused to analyse the data from multiple groups. When the variance is not homogeneous anon parametric test was performed. P value was calculated. P <0.05was considered assignificant and P <0.05as very significant.【Results】1. miR-134was down-regulated in the liver tissues from rat hepatic fibrosis model andactivated HSCs(1) The level of miR-134was lower in the liver tissues from DMN model and BDL modelthan in that from control ratsReal time RT-PCR showed that the expression of miR-134in the liver tissues fromDMN model was gradually down-regulated compared with that in nomal rat livertissuses (P <0.05). The experiments also showed that the level of miR-134in the liver tissues from BDL model gradually declined compared with that from sham operationgroup (P <0.05).(2) Rat primary HSCs presented with activated status and down-regulation of miR-134after cultivated in vitroReal time RT-PCR showed that the expression of α-SMA and Collagen I began toincrease and miR-134decreased in rat primary HSCs six days after their being isolated.The level of α-SMA and Collagen I were further up-regulated and miR-134down-regulated thereafter.(3) The expression of miR-134was down-regulated in T6cells treated with recombinantrat TGFβ1Rat HSCs cell line T6cells were treated with TGFβ1in varied concentrations.Forty-eight hours after the stimulation, real time RT-PCR showed that the levels ofα-SMA and Collagen I were significantly up-regulated, indicating that T6cells wereactivated, while the expression of miR-134was significantly down-regulated in adose-dependent manner.2. miR-134inhibited the proliferation of T6cells and the expression of α-SMA andCollagen I(1) Overexpression of miR-134suppressed the biological characteristics of T6cellsT6cells were transfected with miR-134mimic and negative control (NC). Forty-eighthours after the transfection, real time RT-PCR showed that the levels of α-SMA andCollagenI were significantly down-regulated in miR-134mimic group. Western-blotshowed that the expression of both proteins was also decreased. Furthermore, theproliferation of T6cells was significantly inhibited by transfection of miR-134mimic.(2) Inhibition of miR-134promoted the expression of α-SMA and Collagen I as well as theproliferation of T6cellsReal time RT-PCR showed that the transfection of miR-134inhibiter significantlyup-regulated the levels of α-SMA and Collagen I. Growth curve showed that miR-134inhibitor significantly increased the proliferation of T6cells. 3. miR-134directly regulated STAT5B in HSCs(1) miR-134regulated the expression of STAT5BReal time RT-PCR and western-blot showed that the mRNA level and proteinexpression of STAT5B were down-reulated by the transfection of miR-134mimic andup-regulated by miR-134inhibitor.(2) STAT5B was the direct target of miR-134Dural reporter assays showed that miR-134mimic significantly suppressed theluciferase activity of reporter plasmid carrying the binding site of miR-134on the3′-UTR of STAT5B while exerted no effect on the plasmid carrying the mutant bindingsite. Moreover, the level of STAT5B mRNA was up-regulated in T6cells stimulated byTGFβ1, which was opposite with the expression of miR-134, further indicating thatmiR-134regulated STAT5B directly and functionally.【Conclusions】1. The expression of miR-134was down-regulated durning the activation of HSCs and inthe rat hepatic fibrosis models, indicating that miR-134was closely related to hepaticfibrosis.2. miR-134inhibited the expression of fibrotic-related genes and the proliferation of T6cells.3. miR-134may function in hepatic fibrosis by directly regulating STAT5B in HSCs.