The Important Role Of Tmem88 In Hepatic Stellate Cell And The Relevant Mechanism

Hepatic fibrosis is a damage-repair response caused by a variety of factors lead to liver injury(HBV or HCV infection,alcohol abuse,high fat diet,etc.).The activation of hepatic stellate cells(HSC)is the central issue in the development and progression of hepatic fibrosis.Transmembrane protein 88(Tmem88)is a two transmembrane protein with inhitory effect on Wnt/?-catenin pathway in the development of cardiomyocyte and Xenopus embryos.Moreover,it has been reported that the expression of ?-catenin is increased in activated hepatic stellate cells and ?-catenin activation plays an important role in hepatic stellate cell activation and proliferation.Therefore,we assumed that Tmem88 could reverse hepatic stellate cells activation and proliferation by inhibiting Wnt/?-catenin activation.To test this hypothesis,firstly,we examined the expression of Tmem88 in human liver fibrosis tissues,mice primary hepatic stellate cells and activated HSC-T6 cells;secondly,by regulating the expression level of Tmem88 in HSC-T6 cells,the role of Tmem88 in HSC-T6 cells activation,proliferation and apoptosis was investigated;thirdly,since the Tmem88 expression might be associated with DNA hypermethylation,we further investigated the effect of DNA methyltransferases on Tmem88 expression.The main content of this paper as followed:1.Tmem88 expression was decreased in human liver fibrosis tissues,mice primary hepatic stellate cells and activated HSC-T6 cells.The tissues of human liver cancer were collected several patients undergoing partial liver resection.H&E and Masson staining were used to examine the pathological changes of liver tissues.According to the Liver Cancer Study Group of Japan,the degree of fibrosis was classified as normal liver(Control)and mild to moderate fibrosis(Fibrosis).Western Blot and immunohistochemistry were used to detect Tmem88 expression in liver tissues.The results showed that Tmem88 expression was significantly decreased human liver fibrotic tissue.24 C57BL/6 mice(male,20 ~ 22g)were randomly divided into Control group and CCL4 group(12/group).Mice in CCL4 group were injected subcutaneously in mice back with 20% CCL4 olive oil solution,twice/week for 4 weeks;mice in Control group received the same dose of olive oil.Serum,liver tissues and primary HSCs was collected from sacrified mice.H&E and Masson staining were used to observe pathological changes in liver tissue structure,and serum ALT and AST activities were examined to verify the establishment of mice liver fibrosis model.Immunofluorescence double staining,immunohistochemistry,QPCR and Western Blot were used to detect Tmem88 co-localization and expression.The results showed that the model of mice with liver fibrosis had been established successfully,Tmem88 mainly located in hepatic stellate cells and Tmem88 expression was down-regulated in primary hepatic stellate cells of mice with liver fibrosis.HSC-T6 cells were activated with 10ng/ml TGF-?1 for 24 h.QPCR and Western Blot were used to detect Tmem88 expression in activated HSC-T6 cells.The results showed that Tmem88 expression was significantly decreased in TGF-?1-induced HSC-T6 cells.2.The effect of Tmem88 overexpression on HSC-T6 cell activation,proliferation and apoptosisFirstly,to determine the role of Tmem88 overexpression in HSC-T6 cell activation,Real-time PCR and Western Blot were used to examine the expression of marker proteins(Col1?1 and ?-SMA)in activated HSC-T6 cells.Secondly,to determine the role of Tmem88 overexpression in HSC-T6 cell proliferation,MTT assay was used to detect the viability of HSC-T6 cells and flow cytometry was used to examine the cell cycle distribution.Thirdly,Western Blot was used to detect the effect of Tmem88 overexpression on Wnt/?-catenin pathway activation in HSC-T6 cells.Finally,to determine the role of Tmem88 overexpression in HSC-T6 cells apoptosis,flow cytometry was used to detect cell apoptosis changes,Western Blot was used to detect the expression of apoptosis related proteins(Caspase3,Bcl-2 and Bax)in HSC-T6 cells.The results showed that Tmem88 overexpression could reverse the TGF-?1-induced activation and proliferation of HSC-T6 cells by blocking Wnt/?-catenin pathway activation,arrest HSC-T6 cells in G0/G1 period,and increase apoptosis in HSC-T6 cells.3.The effect of Tmem88 silence on HSC-T6 cell activation,proliferation and apoptosis.Firstly,to determine the role of Tmem88-si RNA in HSC-T6 cells activation,Real-time PCR and Western Blot were used to detect the expression of activation marker proteins(Col1?1 and ?-SMA)in HSC-T6 cells;Secondly,Western Blot was used to detect the effect of Tmem88-si RNA on Wnt/?-catenin activation in HSC-T6cells;Finally,in order to determine Tmem88-si RNA on apoptosis of HSC-T6 cells,Western Blot was used to detect the expression of HSC-T6 apoptosis related proteins(Caspase3,Bcl-2 and Bax).The results showed that Tmem88-si RNA could promote TGF-?1-induced HSC-T6 cell activation,enhanced the activation of Wnt/?-catenin pathway,and inhibit the expression of HSC-T6 cells in apoptosis-related proteins.4.The effect of DNA methyltransferases on Tmem88 expressionA study suggested that Tmem88 expression was associated with the level of promoter hypomethylation in ovarian cancer.Moreover,RRBS found that the methylated level of Tmem88 DNA in primary hepatic stellate cells of mice with liver fibrosis is 158 times as much as that of Control mice.Thus,we assumed that Tmem88 expression is related to the status of DNA methylation.To test this hypothesis,firstly,we added DNA methylation inhibitor 5-Azad C(2umol/L)to treate TGF-?1 activated HSC-T6 cells.Western Blot was used to detect the expressions of Tmem88 and activation marker proteins(Col1?1 and ?-SMA);Secondly,Western Blot was used to detect the expressions of DNA methyltransferases(Dnmt1,Dnmt3 a and Dnmt3b)in mice primary heaptic stellate cells and activated HSC-T6 cells;Finally,Western Blot analysis was used to detect Tmem88 expression in activated HSC-T6 cells transfected with Dnmts-si RNA.The results showed: 5-Azad C could restore Tmem88 expression in activated HSC-T6 cells;the protein level of Dnmt1,Dnmt3 a Dnmt3b were significantly upregulated in primary hepatic stellate cells of mice with liver fibrosis and activated HSC-T6 cells;Dnmt3a-si RNA could increase the expression Tmem88 in activated HSC-T6 cells.