DNA Methylation Of PTGIS Enhances Hepatic Stellate Cell Activation And Liver Fibrogenesis

Liver fibrosis,characterized by the progressively increased accumulation of Extracellular matrix?ECM?compounds in liver,is a wound-healing response to various chronic hepatic injuries including virus infection,alcohol abuse and high-fat diet.The hepatic architecture will be disturbed by the prolonged and repeated accumulation of ECM proteins by which can form fibrotic scars and nodules,leading to hepatic dysfunction ultimately.Without effective therapeutic methods,liver fibrosis can eventually develop into liver cirrhosis and hepatocellular caicinoma?HCC?,often requiring liver transplantation,pose a huge heath burden on the global community.The activation of hepatic stellate cells?HSCs?is a central event in the progression of liver fibrosis.Transforming growth factor-?1?TGF-?1?,an inflammatory cytokines,is a major pro-fibrogenic cytokine and can up-regulate?-smooth muscle actin??-SMA?and type I collagen?COL1a1?synthesis by HSCs-derived myofibroblast.Inhibiting HSC activation and promoting its apoptosis regarded as pivotal therapeutic strategy in liver fibrosis.Multiple studies proved that DNA methylation involved in liver fibrosis and HSC activation.The pilot experiments showed that the promoter of prostacyclin synthase?PTGIS?gene was hypermethylated in CCl₄-induced liver fibrosis mouse model.The CpG cites of PTGIS was predicted.The methylation of PTGIS gene in fibrosis group was verified through methylation-specific PCR?MSP?analysis.Prostacyclin synthase?PTGIS?is a member of family 8?CYP8?in the cytochrome P450superfamily.PTGIS catalyzes the conversion of PGH₂ to prostacyclin?PGI₂?.The down-regulated PTGIS expression can be restored by DNMTs-RNAi and5-aza-2-deoxycytidine?5-azadC?,an inhibitor of DNA methyltransferase?DNMTs?.Chromatin immunoprecipitation?ChIP?assay indicated that PTGIS methylation was mainly induced by DNMT1 and DNMT3b.We further investigated the function of PTGIS in liver fibrosis by Recombinant Hepatic-adeno-associated virus?rAAV8?-PTGIS overexpression.The data indicated that overexpression of PTGIS in mouse liver could alleviate liver fibrosis accompanied by elevated apoptosis-related protein expression in primary HSCs.The underlying mechanism of PTGIS in modulating liver fibrosis was detected through loss-and gain-experiments.Those results revealed that PTGIS was methylated in fibrotic mouse model and ectopia expression of PTGIS could alleviate liver fibrosis and inhibite HSC activation.This study mainly performs seven parts as followed:1.PTGIS expression in mouse model of liver fibrosis.Animal experiments:Normal C57BL/6j mice?4⁶ week old,18²2 g?were randomly devided into Vehicle group and CCl₄-treated group?n=8 mice per group?.Liver fibrosis mouse model was generated via intraperitoneal injection of carbon tetrachloride?10%CCl₄ in olive oil?biweekly at a dose of 0.01 mL/g/mouse for 6weeks.Mice in Vehicle group were treated intraperitoneal injection with the same volume of olive oil at the same time intervals.Six weeks later,mice were killed under anesthesia.Serum,liver tissues and primary hepatic stellate cells were collected for further experiments.The degree of liver injury was detected via macroscopic experiment,H&E staining and ALT/AST levels in serum.The collagen deposition and HSC activation were measured by Masson trichrome staining and immunohistochemical?IHC?staining for?-SMA respectively.The expression of PTGIS in liver fibrosis mouse model was examinated by IHC staining,Western blot analysis and RT-qPCR experiment.The double immunofluorescence analysis was used to identify the co-localization of?-SMA and PTGIS.Those results proved that the fibrosis mouse model was established successfully and PTGIS expression was downregulated in CCl₄-treated group.2.Expression changes of PTGIS in the progression of liver fibrosis.Animal experiments:4⁶ week old C57BL/6j mice?18²2 g in body weight?were randomly divided into Vehicle group and CCl₄-treated group?n=36 per group?.The mouse model of liver fibrosis was constructed as described in part 1.During the model establishment progress,6 mice were taked out from the two groups respectively.Serum,liver tissues and primary HSCs were collacted and saved in-80?refrigerator until all the samples have been collected.The detection method is as previous described in part 1.The results demonstrated that the degree of hepatic fibrosis increased with the time of modeling and the expression of PTGIS was increased in the first four weeks and then declined.3.Expression of PTGIS in TGF-?1-activated HSC-T6 cellsIn vitro experiments:HSC-T6 cells treated with TGF-?1 at the concentration of 0,5 10,15 ng/mL for 24 h or stimulated with TGF-?1?10 ng/mL?for 0,6,12,24,48 h to detect the best action time and the best action concentration of TGF-?1 in activating HSC-T6 cells.The expression of PTGIS,COL1a1 and?-SMA were detected by Western blot analysis and RT-qPCR experiment.The expression of PTGIS was further detected via immunofluorescence experiment.Those results demonstrated that the optimum action time and concentration of TGF-?1 was 24 h,10 ng/mL.The PTGIS expression was decreased in liver fibrosis model in vitro.4.Decreased PTGIS expression in fibrosis mouse model was attribute to DNA methylation and can be restored by 5-azadC and DNMTs-RNAi.There were three methylated CpG sites in PTGIS gene.The results of Methylation-specific PCR?MSP?revealed that TGF-?1 could induce aberrant gene methylation in PTGIS gene.The expression of DNMT1,DNMT3a and DNMT3b protein were elevated while PTGIS expression decreased in TGF-?1-activated HSC-T6cells and CCl₄-treated mouse model.The downregulated PTGIS expression can be restored by 5-aza-2'-deoxycytidine?5-azadC?and DNMTs-RNAi.The results of ChIP assay indicated that DNMT1 and DNMT3b directly binding with PTGIS gene in HSC-T6 cells.The results presented illustrated that the decreased levels of PTGIS were attributed to DNA methylation mediated by DNMT1 and DNMT3b.5.Liver specific overexpression of PTGIS alleviated liver fibrosisAnimal experiments:Normal C57BL/6j mice?4⁶ week old,18²2 g in body weight?were randomly divided into three groups:rAAV8-empty-treated Control group,rAAV8-empty-treated Model group and rAAV8-PTGIS-treated Model group?n=8 mice per group?.Empty viral vector?rAAV8-empty?or PTGIS?rAAV8-PTGIS?over expression viral vector were intravenously injected into CCl₄-treated mice via the tail vein one week before the first injection of 10%CCl₄.Macroscopic of liver tissues,serum ALT/AST levels and H&E staining were used to examining the degree of liver injury.Virus infection efficiency was identified via the eGFP signals.Masson trichrome staining and IHC staining were applied to identify the degree of liver fibrosis.The protein level of PTGIS was examinated by IHC staining and Western blot analysis.The results presents above indicated that liver specific overexpression of PTGIS alleviated liver fibrosis progression,decreased the deposition of?-SMA and COL1a1.6.Ectopia expression of PTGIS inhibited activation of HSCs and promoted activated HSC apoptosis in vivo and in vitro.HSC-T6 cells were cultured with pEX2-PTGIS plasmid.Transfection efficiency and expression of PTGIS,COL1a1 and?-SMA were measured by Western blot and RT-qPCR analysis.CCK8 experiment was performed to evaluate the viability of HSC-T6 cells.Cell cycle and apoptosis were monitored by Flow cytometric analysis?FCM?.The influence of PTGIS on cell cycle and apoptosis-associated protein expression were examinated via Western blot analysis.The results presented above suggested that ectopia expression of PTGIS inhibited COL1a1 and?-SMA expression,inhibited cell proliferation through G₀/G₁ arrest and promote activated HSC apoptosis.The results of in vivo and in vitro were consistent.7.PTGIS silencing enhanced the activation of HSC-T6 cells in vitro.TGF-?1-activated HSC-T6 cells were cultured with PTGIS-RNAi.PTGIS-RNAi transfection efficience,PTGIS,COL1a1 and?-SMA expression were verified via Western blot and RT-qPCR analysis.CCK8 analysis was performed to evaluate the viability of HSC-T6 cells.Cell cycle and apoptosis of activated HSC-T6 cells were analysed by Flow cytometric analysis?FCM?.The effect of PTGIS silencing on cell cycle and apoptosis were analysed by Western blot analysis.Those results illustrated that PTGIS silencing promoted COL1a1 and?-SMA expression,promoted cell cycle progression,while have no influence on cell apoptosis.