Long Non-coding RNA PVT1 Participates In Liver Fibrosis Through Competitively Binding MiR-152

Background and Objective:Liver fibrosis is characterized by the accumulation of excessive extracellular matrix(ECM)in liver.Hepatic stellate cells(HSCs)are the main cell type for producing ECM in liver.The acitviation of HSC is considered as a key event in the progression of liver fibrosis.Recently,epithelial-mesenchymal transition(EMT)process,which could be regulated by Hedgehog signaling,has been reported to be involved in the activation of HSCs.Long non-coding RNAs(IncRNAs),which is previously defined as "noise",are demonstrated to play a role in the regulation of gene function and be involved in a wide range of diseases.Plasmacytoma variant translocation 1(PVT 1)is a tumor-associated IncRNA,however,its role in liver fibrosis is still not clear.The aim of our study is to detect PVT1 expression and explore the role of PVT1 in liver fibrosis.In addition,the underlying mechanism of PVT 1 in liver fibrosis is also explored.Methods:Mice received tetrachloride carbon(CCl4)treatment to induce mouse liver fibrosis,were treated with recombinant adenovirus Ad-shPVTl or Salvianolic acid B(Sal B).After Ad-shPVT1 or Sal B treatment,the expression of collagen in vivo was detected.In primary HSCs treated with PVT1 siRNA or Sal B treatment,cell proliferation and collagen expression were detected.Meanwhile,the expressions of mRNA and protein of EMT process,Hedgehog signaling and other related-genes were examined by quantitative real time PCR and Western blot,respectively.Bisulfite sequencing PCR was used to examine PTCH1 promoter methylaiton.Luciferase reporter assays and Pull down assays were used to examine the relation between PVT1 and microRNA-152(miR-152).Results:Compared with the control,the expression level of PVT1 was increased in fibrotic liver tissues as well as in activated HSCs.In HSCs,the loss of PVT1 effectively inhibited the activation of HSCs,including cell proliferation and the expression level of collagen.In line with it,the silencing of PVTI in vivo contributed to the reduction of type I collagen expression induced by CCl4 treatment.Next,the loss of PVT1 suppressed the progression of EMT process,with the up-regulation of the expression level of E-cadherin as well as the down-regulation of the expression levels of desmin and vimentin.Also,the loss of PVT1 resulted in Hedgehog signaling,with the up-regulation of the expression level of PTCH1 as well as the down-regulation of the expression levels of SMO and GLI2.In addition,Sal B has been shown to have a good effect on suppressing liver fibrosis in vivo and in vitro.In Sal B-treated HSCs,the association between the loss of Hedgehog signaling and reduced EMT process was further confirmed.PTCH1 has been reported to be a negative regulator for Hedgehog signaling.It was found that the promotor of PTCH1 was highly methylated during liver fibrosis progression.PTCH1 methylation could be suppressed by Sal B and its expression was restored by Sal B.Further studies demonstrated that miR-152 not only targets DNA methyltransferase 1 but also regulates the methylation of PTCH1 promoter.In vivo and in vitro,the silencing of PVT 1 also contributed to the suppression of the methylation of PTCH1 promotor,with an increase in the expression level of miR-152.Interestingly,miR-152 inhibitor led to the suppression of the effects casued by the loss of PVT1,including the down-regulation of cell proliferation and collagen expression,and the induction of PTCH1 demethylation and PTCH1 expression level.The association between PVT1 and miR-152 was confirmed by luciferase reporter assays.In line with it,PVT1 was demonstrated to directly bind miR-152,as shown by Pull dwon assays.We additionally demonstrated that in Sal B-treated HSCs,the loss of PVT 1 could enhance the inhibition of cell proliferation and collagen expression caused by SalB.Conclusion:PVT1 was significantly up-regulated in CCl4-induced fibrotic liver tissues and activated HSCs.By contrast,the loss of PVT1 showed an anti-fibrotic role in vitro and in vivo.We demonstrate that PVT1 epigenetically inhibits PTCH1 expression via competitively binding miR-152,resulting in the activation of Hedgehog signaling and EMT process in liver fibrosis.In liver fibrosis,we also identify a novel signal pathway of PVT1/miR-152/PTCH1.Futhermore,our studies demonstrate that Sal B plays an anti-fibrosis role via this signaling pathway.