MicroRNA-145 Inhibits Hepatic Stellate Cell Activation And Proliferation By Targeting ZEB2

Hepatic fibrosis refers to one or more chronic pathogenic factors causing diffuse liver cell damage,and then making the activation of hepatic stellate cell(HSC).Liver fibrosis is characterized by increased deposition of extracellular matrix(ECM).The activation of HSC plays central role during liver fibrosis,and inhibiting the activation or proliferation can suppress the progression of hepatic fibrosis.MicroRNA has a regulation function in various tissues or organs fibrosis disease.Mi RNA can regulate a variety of anti-or pro-fibrosis factors.In turn,the occurrence of liver fibrosis can make expression change of miRNA.Recently,microRNA-145(miR-145)is reported to be closely related to the development of fibrosis disease,but its role in the progression of hepatic fibrosis and activation of HSCs remains unknown and is warranted for investigation.This study aims to explore the expression change and molecular mechanisms of miR-145 in hepatic fibrosis and in TGF-β1-induced HSC.The study includes the follow parts:(1)The expression change of ZEB2 and miR-145 during hepatic fibrogenesisIn vivo,we used CCl4 subcutaneously 4 weeks to establish mice liver fibrosis.Hematoxylin and eosin(H&E)staining and Masson staining verified fibrosis model was successfully established,which companied with significantly higher Colα1 andα-SMA levels.One-step real-time PCR assay showed that miR-145 was reduced while ZEB2 was increased,as compared with normal liver tissues,the changes of ZEB2 and miR-145 in primary HSC was consisted with the changes in liver tissues.Furthermore,we treated the HSC-T6 cells and LX-2 cells with different concentration of TGF-β1 for24 h.We found that ZEB2 was increased and miR-145 was also decreased in activated HSC.Double immunofluoresence staining showed that ZEB2 expression was colocalized with α-SMA,suggesting that HSC is the main source of ZEB2.(2)The effect of miR-145 in TGF-β1-induced HSC activation and proliferationTo explore the effect of miR-145 on HSCs activation and proliferation,HSC-T6 cells were transiently transfected with miR-145 mimics,miR-145 inhibitor or miR controls,and treated with 10ng/ml TGF-β1.The western blot assay indicated that over-expression of miR-145 significantly suppressed and knockdown of miR-145 promoted the protein level of α-SMA and collagen-I after TGF-β1 stimulation.Cell cycle analysis showed that over-expression of miR-145 in HSCs could inhibit TGF-β1-induced cell proliferation.In contrast,knockdown of mi R-145 in HSCs could promote cell proliferation(3)The effect of ZEB2 in TGF-β1-induced HSC activation and proliferationIn order to investigate the role of ZEB2 in TGF-β1-induced HSCs,siRNA specific for rat ZEB2 was used to knockdown the ZEB2 expression.Then,we treated the cells with TGF-β1(10ng/ml)and analyzed the effect of ZEB2 silencing on TGF-β1-induced HSCs activation and proliferation.Our results revealed that deregulation of ZEB2 could result in down-regulation of the protein expression of Col-I and α-SMA.Cell cycle analysis suggested that knockdown of ZEB2 may suppress HSC proliferation.(4)The target effect between miR-145 and ZEB2Bioinformatics approaches found that the 3’-UTR of ZEB2 contains putativebinding sites for miR-145.In our study,we found that the expression of ZEB2 could be decreased by miR-145 mimics and the ZEB2 level was increased in HSC-T6 cells after transfected with miR-145 inhibitor.To further test if ZEB2 is a target of mi R-145,the3’-UTR was cloned into a luciferase expression vector to evaluate its response to miR-145.Cotransfected the luciferase reporter with the mi R-145 mimic into HSC-T6 cells showed the 3’-UTR conveyed decreased expression.(5)The effect of miR-145 and ZEB2 in Wnt/β-catenin pathwayWe transfected HSC-T6 cells with miR-145 mimics,miR-145 inhibitor or ZEB2 si RNA,Western blot detected the expression of related protein of Wnt/β-catenin pathway.We found that over-expression of the mi R-145 in HSC-T6 cells led to a decrease in protein expression of β-catenin and its downstream target genes,cyclinD1 and c-Myc.Meanwhile,we observed an increase in protein level of these genes in the presence of miR-145 inhibitor.To further explore whether the effect of miR-145 on Wnt/β-catenin pathway related to ZEB2,we transfected cells with ZEB2 siRNA and detected the protein expression of β-catenin,cyclinD1 and c-Myc.As expected,all were decreased.Taken it together,these data indicated that the negative regulation of miR-145 on Wnt/β-catenin signaling partly through ZEB2 and in turn to influence the activation and proliferation of HSC-T6.