Studies On The Mechanism Of Hepatocyte LincRNA-p21 In Regulating Liver Fibrosis

Liver fibrogenesis arises as a consequence of hepatocyte damage and is a gradual process involving the interplay between hepatocytes and other hepatic cell populations including HSCs and inflammatory cells.Despite the fact that HSCs play a pivotal role in hepatic fibrogenesis,hepatocytes are the dominant cell type residing in the liver and actively orchestrate the profibrogenic responses.In different animal models,as well as various liver diseases,liver fibrosis is associated with significant hepatocyte injury.Increased cell death in hepatocytes promotes inflammatory cell recruitment and the secretion of profibrogeneic and inflammatory cytokines including TGF-β,IL-1β,TNFa and CCL2.Among them,TGF-β is the key fibrogenic cytokine stimulating HSC activation and collagen deposition.Whereas TGF-β signaling in hepatocytes promotes hepatocyte damage,profibrogenic and inflammatory cytokine production,leads to the induction of apoptosis and growth inhibition,and thus contributes to hepatic fibrogenesis.Accumulating data show that lncRNAs are involved in regulating gene expression through a variety of mechanisms and participate in the regulation of a variety of cellular events including apoptosis,proliferation and differentiation.So far,the disregulation of lncRNAs has been shown in various disease states.In liver,many lncRNAs have already been known to contribute to liver regeneration,neoplasia and other liver diseases.However,the involvement of lncRNA in liver fibrosis remains unknown.An increasing body of experimental evidence suggests that lncRNA transcripts can regulate microRNA activity by acting as either competitive endogenous RNAs,thereby alleviating the negative effect of microRNAs on their respective mRNA targets.In our previous work,we isolated hepatocytes from the CCl4-induced fibrotic liver and conducted transcriptome RNA sequencing(RNA-seq).Among the highly expressed lncRNAs,lncRNA-p21 was characterized by RT-PCR,qRT-PCR and Northern-blot.Putative miRNA binding site of miR-30 located on lincRNA-p21 were predicted by the prediction algorithm.We previously reported that TGF-β signaling can reduced miR-30 production in hepatocytes.Moreover,miR-30 inhibited TGF-(3 signaling by targeting KLF11,which can potentiate TGF-β/Smad signaling through suppressing the transcription of inhibitory Smad7.Here,we found that TGF-β can significantly induced the expression of lncRNA-p21 in hepatocytes.The interaction of Smad2/3 with the lincRNA-p21 promoter region was then confirmed by luciferase assay and ChIP.Enhanced expression of lincRNA-p21 greatly reduced the miR-30 levels in hepatocytes.Conversely,knockdown of lincRNA-p21 exerted the opposite effects.To confirm the interaction between lincRNA-p21 and miR-30,the wild type and mutant lincRNA-p21 fragments were cloned downstream of the firefly luciferase,showing that miR-30 family members could significantly reduce the luciferase activity of the wild type lincRNA-p21 fragment reporter but the mutant one.Western blot and qPCR results showed that overexpression of lincRNA-p21 increased the expression of KLF11,led to the suppression of Smad7 in AML 12 cells,and therefore enhanced TGF(3/Smad signaling.Reciprocally,knockdown of lincRNA-p21 led to a decrease in KLF11 production,and thus suppressed TGFβ signaling.Finally,we confirmed that lincRNA-p21 regulated TGFβ/Smad signaling through miR-30.There is ample evidence that apoptotic hepatocytes can trigger liver fibrogenesis.We found that ectopic expression of lincRNA-p21 increased hepatocyte apoptosis,whereas,knockdown of lincRNA-p21 significantly inhibited the TGFβ-or doxorubicin-induced hepatocytes apoptosis.Moreover,knockdown of lincRNA-p21 prevented the TGF-β-induced hepatocyte death.A similar observation was made in cells treated with Dox.Finally,we injected lincRNA-p21 antisense oligonucleotide(ASO)via tail vein to reduce the expression of lincRNA-p21 in CCl4-injected mouse,showing that inhibition of lincRNA-p21 in hepatocytes greatly reduced CCl4-induced liver fibrosis.In conclusion,our results define roles of lincRNA-p21 in regulating TGF-β signaling and hepatocyte apoptosis,and thus provide useful insights into the mechanisms underlying liver fibrogenesis.