The Function And Regulatory Mechanism Of YAP In Hepatic Fibrosis And Reversion

Yes-associated protein?YAP?is a significant downstream protein in the Hippo signaling pathway with important functions in cell proliferation,apoptosis,invasion and migration.YAP also plays a role in the progression of various hepatic diseases.However,the contribution of YAP to hepatic fibrosis progression and reversion and the underlying mechanism have not been investigated.In hepatic fibrosis development and reversion,the proliferation and apoptosis of activated hepatic stellate cells?HSCs?play a critical role.Here we investigated the function of YAP in the proliferation and apoptosis of activated HSCs.In hepatic fibrosis,the Wnt/?-catenin signaling pathway is activated,affecting the activation and proliferation of hepatic stellate cells,and YAP is closely related to the Wnt/?-catenin signaling pathway.Therefore,we also investigated whether YAP could participate in the activation and proliferation of HSCs by affecting the Wnt/?-catenin signaling pathway.In order to study the YAP role in hepatic fibrosis progression and reverse,we injected 10%CCl₄ through intraperitoneal injection in mice for 4 weeks,2 times per week to establish mice model of hepatic fibrosis.Mice in the reversal group recovered spontaneously for 6 weeks after stopping CCl₄ injection to established mice liver fibrosis reversal model.We demonstrated the success of the mice model by detecting Alanine transaminase?ALT?,Aspartate Aminotransferase?AST?changes and pathological tests such as Hematoxylin Eosin?HE?,Masson-trichrome staining?Masson?and Sirius scarlet staining.We extracted proteins from mouse liver tissues and RNA from primary cells,and detected the changes of YAP by Western Blot and Real-Time Quantitative PCR?RT-PCR?,and found that the expression of YAP increased in hepatic fibrosis mice and decreased in reversed mice.The proteins of HSC-T6 cells stimulated by transforming growth factor?1?TGF-?1?and activated cells reversed by adipocyte differentiation cocktail?MDI?were extracted,and changes in YAP were detected by Western Blot and RT-PCR.The results showed that YAP expression was increased in activated cells,but decreased in reversed cells.In order to explore the function of YAP,we used YAP-siRNA to silence activated HSC-T6 cells.Western Blot,CCK-8 assays,cell cycle experiment and apoptosis experiment proved that after silencing YAP,the activation and proliferation of HSC-T6 cells were inhibited,but the apoptosis of activated cells was promoted.In order to further explore the role of YAP in MDI-treated cell reversion,we overexpressed YAP via SV40-YAP plasmid in MDI-treated cells,and Western Blot showed that overexpression of YAP inhibited the reversion of MDI to activated HSC-T6 cells.We further explored the function of YAP by using the inhibitor of YAP,Verteporfin?VP?.Through Western Blot,cell proliferation experiment and apoptosis experiment,we found that VP inhibited the activation,proliferation and promoted apoptosis of HSC-T6 cells,further confirming the role of YAP.On the mechanism,we found Wnt/?-catenin signaling pathways downstream target genes?C-myc and cyclinD1?protein expression were suppressed after YAP being silenced.In addition,after overexpression of YAP in the reversed cells treated with MDI,we found that the protein expressions of?-catenin,C-myc and cyclinD1 were increased,which reversed the reversal effect of MDI on the Wnt/?-catenin signaling pathway,proving that YAP could positively regulate the Wnt/?-catenin signaling pathway to affect the activation and proliferation of hepatic stellate cells.In conclusion,our results demonstrated that silencing of YAP contributed to the recovery of hepatic fibrosis,indicating that YAP may be an effective target for HSCs activation and reversion.