MicroRNA-134 Inhibits Hepatic Stellate Cell Activation By Regulating The Expression Of TAB1

?Background and objective?Hepatic fibrosis is a pathological response to various chronic liver injury,with the main features of hepatic stellate cell(HSC)activation,extracellular matrix(ECM)over-deposition.Hepatic fibrosis is a necessary process for chronic liver injury to cirrhosis,resulting from viral hepatitis,autoimmune hepatitis,alcoholic liver disease,nonalcoholic steatohepatitis and metabolic abnormalities.Hepatic fibrosis is reversible.Therefore,the treatment of liver fibrosis is of great significance.MicroRNAs(miRNAs)are a class of endogenous non-coding RNA about 15-22nucleotides by binding to the 3'untranslated region(3'UTR)of the targeted messenger RNA(mRNA),direct degradation of mRNA or inhibition of transcription to regulate gene expression.Studies have shown that there are about 1880 kinds of mi RNAs in human body,and one third of the mRNAs are regulated by miRNAs.Therefore,miRNAs are involved in the regulation of cell proliferation,differentiation,apoptosis,metabolism and endocrine function.Abnormal expression of miRNAs is associated with many diseases,such as tumor formation,nerve regeneration and heart failure.A large number of studies have found that fibrosis of various organs(heart,kidney,lung,liver)in vivo is accompanied by changes of mi RNA expression.In liver fibrosis,many miRNAs can affect the development of hepatic fibrosis by regulating of proliferation of HSC,extracellular matrix synthesis and hepatic fibrosis-related signal pathway.Therefore,miRNA may become an important target for diagnosis and treatment of liver fibrosis.MicroRNA-134,belonging to the hsa-miR-379~hsa-mi R-656 miRNA cluster,is located in the Dlk1/Dio3 imprinted genes region of the human chromosome 14.It was first identified as a brain-specific miRNA,which involved in the regulation of synaptic fine structure,dendrites growth and spinal cord formation.MicroRNA-134 is closely related to nervous system tumors.By inducing cell cycle arrest and targeting stem cell-related gene Nanog,mi RNA-134 can regulate the tumor stem cell differention and proliferation,migration and invasion of tumor cell.Our previous studies confirmed that miRNA-134inhibited proliferation,clonal formation and metastasis and other malignant biological behavior of of HCC cells by targeting KRAS,and the expression levels of miRNA-134 were closely related to the clinical phenotype of hepatocellular carcinom.Recent data show that mi R-134 is also closely related to idiopathic pulmonary fibrosis(IPF),with a significant reduction in miRNA-134 expression level compared with healthy people.MicroRNA-134inhibits TGF-?-induced epithelial-to-mesenchymal transition(EMT)processes in non-small cell lung carcinoma(NSCLC).Liu Y et al also confirmed that miR-134 can inhibit tumor cell proliferation and EMT process in renal cell carcinoma.The recent study by Sangyoon Lee found that miR-134 coud play the anti-fibrotic role in liver through Dieckol.The above studies indicate that miRNA-134 is involved in the process of fibrosis of various organs,but the role of mi R-134 in the development and progression of hepatic fibrosis is not clear.Based on the above research,this subject intends to further study the role and mechanism of mi R-134 in the development of liver fibrosis with the expection that providing new ideas for the treatment of liver fibrosis.?Methods?1 The relationship of miRNA-134 expression level and hepatic fibrosis(1)Eight Sprague-Dawley(SD)rats,weighing 180-200 g,were purchased from the Shanghai Animal Center of the Chinese Academy of Sciences.They were randomly divided into two groups:normal control group(n=4)and model group(n=4).The rats in the control group were injected intraperitoneally with normal saline by 1ml/kg body weight twice a week for 8 weeks.The rats in the model group received intraperitoneal injection of 50%CCl4/olive oil twice a week for 8 weeks.At the end of the 8th week,all rats were anesthetized and sacrificed.The liver tissue was embedded in paraffin,and the degree of liver fibrosis was evaluated by hematoxylin-eosin(H&E)staining,Sirius red staining,and Image-Pro Plus(IPP)software was used for semi-quantitative analysis of fibrosis area.The mRNA expression levels of miR-134,?-smooth muscle actin(?-SMA)and type I collagen(Collagen type I,COLI)were detected by real time RT-PCR.Western blot was used to detect the protein expression levels of?-SMA and COL?.(2)The expression changes of miRNA-134 during HSC activationHSC-LX2 cells were treated with different concentrations of TGF-?(0 ng/ml,2 ng/ml,4 ng/ml)for 24 h,and then the expression levels of miRNA-134,?-SMA and COL?were detected by real time RT-PCR.2 Effect of miRNA-134 on the proliferation and activation of HSC(1)Effect of miR-134 on proliferation of HSCTo up-regulate miR-134 expression,HSC-LX2 cells were inoculated to 96-well plates at 3000 cells per well and transfected with control NC and mi RNA-134 mimics 24 hours later.The effect of miR-134 on the proliferation of HSC-LX2 was examined by CCK8 assay.To down-regulate miR-134 expression,HSC-LX2 cells were inoculated to 96-well plates at 3000 cells per well and transfected with control NC and miRNA-134 inhibitor 24hours later.The effect of miRNA-134 on the proliferation of hepatic stellate cells was investigated by CCK8 assay.(2)Effect of miRNA-134 on HSCactivationReal time RT-PCR and western blot were used to detect the expression changes of?-SMA,COL?in HSC-LX2 treated with miRNA-134 mimics or inhibitors.3 Study on the mechanism of miRNA-134 inhibiting the proliferation and activation of HSC(1)The target gene of miRNA-134 was predicted by computer software targetscan.After consulting literature,the target gene transforming growth factor-beta-actived kinase1(TAK1)binding protein(TAB1),which is associated with hepatic fibrosis,was selected and verified.(2)The mRNA and protein level changes of predicted target gene TAB1 were tested by Real time RT-PCR and western blot in HSC-LX2 treated with mi RNA-134 mimics or inhibitors.(3)The luciferase reporter gene was constructed according to the predicted binding site of miRNA-134 on TAB1 3'UTR,and the effect of miRNA-134 on the luciferase reporter gene was detected.The effect of miR-134 on the mutated luciferase reporter gene was examined to determine whether TAB1 was a direct target gene for miR-134 after mutating the binding site of miR-134 on TAB1 3'UTR.(4)To determine whether TAB1 mediates the inhibitory effect of miR-134 on HSC,HSC-LX2 cells were transfected with TAB1 siRNA to,The CCK8 assay was used to detect the proliferation of HSC,and the expression levels of?-SMA and COL?were confirmed by Real time RT-PCR and western blot.Then HSC-LX2 cells were co-transfected with mi R-134 inhibitor and TAB1 siRNA to.The CCK8 assay was used to detect the proliferation of HSC,and the expression levels of?-SMA and COL?were confirmed by Real time RT-PCR and western blot.4 The effect of upregulation of miRNA-134 on rat experimental liver fibrosisThe rats were randomly divided into two groups:control group(12 rats)were injected with AdGFP and the treatment group(12 rats)with AdmiRNA-134 respectively,at 5×10~9pfu through the tail vein twice a week for seven weeks.One week after the injection,both groups were given 50%CCl4/olive oil at 1ml/Kg body weight by intraperitoneal injection twice a week for six weeks.All rats were sacrificed at the end of the 6th week.The liver fibrosis was evaluated by histochemical staining.The expression of miR-134,?-SMA and COL?were detected by real time RT-PCR.5 Statistical analysisThe data were analyzed by SPSS 17.0 statistical software package.The two samples were analyzed by two-tailed two-tailed T test.P<0.05 was considered statistically different,and P<0.01 was very significant difference.?Results?1 Down-regulation of miRNA-134 expression is associated with progression of liver fibrosisIn the model group,H-E staining showed destroyed hepatic lobule structure,obvious steatosis and fibrous septum.And sirius red stainnig showed a large number of fibrous scar formation in the liver after modeling.Compared with control group,real time RT-PCR and western blot showed that the mRNA and protein expression of?-SMA,COLI were up-regulated in the model group.These results suggest that CCl4-induced liver fibrosis model is successfully constructed.Real time RT-PCR showed that miR-134 levels were significantly down-regulated in hepatic tissue after liver fibrosis.After HSC-LX2 cells were treated with TGF-?,Real-time RT-PCR showed that the expression of?-SMA and COLI was significantly up-regulated and mi RNA-134 was down-regulated.2 MicroRNA-134 inhibits the proliferation and activation of HSC(1)MicroRNA-134 inhibited HSC proliferationAfter ug-regulating the expression of miR-134 by transfecting miR-134 mimics in HSC-LX2 cells,CCK8 assay showed that miR-134 inhibited the proliferation of HSC.After down-regulating the expression of mi R-134 by transfecting miR-134 inhibitor in HSC-LX2 cells,CCK8 assay showed that the down-regulattion of miR-134 promoted HSC proliferation.(2)MicroRNA-134 inhibited the activation of HSCAfter ug-regulating the expression of miR-134 by transfecting miR-134 mimics in HSC-LX2 cells,the mRNA and protein levels of?-SMA,COLI were decreased significantly.After down-regulating the expression of miR-134 by transfecting miR-134 inhibitor in HSC-LX2 cells,the mRNA and protein levels of?-SMA,COLI were increased significantly.3 TAB1 is a direct target gene of miRNA-134(1)Target Scan software predicts that TAB1 3'UTR contains the binding sites of mi R-134.The mRNA and protein expression of TAB1 was significantly decreased after up-regulation of miR-134,and was significantly increased after down-regulation of mi R-134 in HSC-LX2 cells.(2)Upregulation of miR-134 expression significantly inhibited the activity of TAB13'UTR luciferase reporter gene,and mi R-134 had no significant effect on the activity of mutant luciferase reporter gene.This indicates that TAB1 is a direct target gene for miR-134.(3)Down-regulation of TAB1 expression significantly inhibited the proliferation and activation of HSC.The transfection of miRNA-134 inhibitor promotes proliferation and activation of HSC-LX2 cells.However,after co-transfection of miR-134 inhibitor and TAB1siRNA,the promotion of miRNA-134 inhibitor on HSC weakened,which indicated that TAB1 partially mediated the inhibitory effect of mi R-134 on the proliferation and activation of HSC cells.4 Upregulation of miRNA-134 inhibits experimental liver fibrosisCompared with AdGFP group,H&E staining showed that the liver lobular structure in AdmiR-134 treatment group was nearly normal with less steatosis.Sirius red staining showed that AdmiRNA-134 treatment significantly reduced the hepatic collagen deposition.The mRNA expression levels of?-SMA and COLI in AdmiR-134 treated group was also significantly decreased.These results suggest that upregulation of miR-134 inhibits experimental liver fibrosis.?Conclusions?1.Down-regulation of miRNA-134 expression is associated with progression of liver fibrosis;2.MicroRNA-134 inhibits the proliferation and activation of HSC;3.MicroRNA-134 inhibits HSC proliferation and activation by its direct target gene TAB1;4.Upregulation of mi RNA-134 inhibits experimental liver fibrosis.