The Functional Role And Relevant Mechanism Of SUN2 On Hepatic Stellate Cells In Hepatic Fibrosis

Hepatic fibrosis,characterized by excessive accumulation of extracellular matrix?ECM?in liver,is a pathological damage-repair process response to multiple etiological factors.Persisting fibrogenesis may induce architecture distorted and function impaired of liver.Emerging evidences demonstrated that hepatic stellate cells?HSCs?switch to fibrogenic myofibroblasts-like cells,which were the principal source of ECM during hepatic fibrosis.Suppressing HSCs activation regarded as useful therapeutic strategie in liver fibrogenesis.Besides,DNA methylation is involved in HSCs activation.In our current study,RRBS analysis revealed hypermethylation of SUN2 gene in primary HSCs isolated from hepatic fibrosis mice,reminding us to hypothesize that SUN2 may have potential function in hepatic fibrosis.SUN2?SAD1/UNC84 domain containing protein-2?plays a critical role in location and migration of cell nucleus.It has been elucidated that SUN2,as an anti-cancer member,participates in AT/RT,breast cancer and lung cancer by regulating biological processes in cancer cells,including cell cycle and apoptosis.However,the functional role and relevant mechanism of SUN2 in hepatic fibrosis remain speculative.This study mainly performs five parts as followed:1.Expression of SUN2 in primary HSCs,TGF-?1-activated HSC-T6 and LX-2 cells.4-week-old C57BL/6J mice?male,weight 20-22g?were randomly allocated into vehicle group and model group?n=30 per group?.Model group generated via biweekly intraperitoneal injection of 10%solution of CCl₄ in olive oil at a dose of 0.002ml/g for 4weeks;vehicle group treated subcutaneously injection with the same volume of 100%olive oil at the same time intervals.After 4 weeks modeling,two groups of mice sacrificed,liver tissues,serum and primary HSCs were collected.Degree of liver injury was evaluated via hematoxylin eosin?HE?staining and ALT and AST tset;the collagen in liver tissues was evaluated via Masson Trichrome staining;double immunofluorescent analysis depicted representative co-localization of SUN2 with myofibroblast marker?-SMA immunoreactivity in mice liver tissue;expression of SUN2 was evaluated via Immunohistochemistry,Real-time PCR and Western blot analysis.Experimental results shown lower expression level of SUN2 was found in primary HSCs from CCl₄-treated mice.HSC-T6 cells and LX-2 cells were activated by TGF-?1,expression of SUN2 was evaluated via Immunohistochemistry,Real-time PCR and Western blot analysis.Experimental results shown lower expression level of SUN2was found in TGF-?1-activated HSC-T6 cells and LX-2 cells.2.Effects of SUN2 over-expression on CCl₄-treated hepatic fibrosis mice.Mice were exposed to AAV-SUN2-GFP through tail vein.10 days later,CCl₄-induced hepatic fibrosis model was established.The degree of liver injury was evaluated via hematoxylin eosin?HE?staining and ALT and AST tset;the collagen in liver tissues was evaluated via Masson Trichrome staining;expression level of?-SMA and Col1?1 was evaluated via Immunohistochemistry,Real-time PCR and Western blot analysis.Experimental results shown expression of?-SMA and Col1?1 were decreased after SUN2 over-expression in CCl₄-treated hepatic fibrosis mice.3.Effects of SUN2 over-expression on activation,proliferation and apoptotic of HSC-T6 cells.HSC-T6 cells were treated with GV141-SUN2 plasmid.Expression of?-SMA and Col1?1 was evaluated by Real-time PCR and Western blot analysis;MTT assay was performed to determine the viability of HSC-T6 cells;Cell cycle and apoptotic were evaluated via Flow cytometric analysis.Experimental results shown SUN2 ectopic expression may inhibit TGF-?1-activated HSC-T6 cells proliferation via arresting G1 to S phase in cell cycle procession,but not directly participated in cells apoptotic response.Furthermore,over-expression of SUN2 inhibited?-SMA and Col1?1 up-regulation induced by TGF-?1 in HSC-T6 cells.4.Effects of SUN2 over-expression on AKT pathway and cell cycle.The directly interaction between SUN2 and p-AKT was determined via co-immunoprecipitation assay;expression levels of p-AKT,cyclinD1 and c-myc were evaluated by Western blot analysis;Experimental results shown SUN2 ectopic expression may inhibit p-AKT,cyclinD1 and c-myc up-regulation induced by TGF-?1in HSC-T6 cells.In addition,phosphorylation and activation of AKT may one of crucial responsible pathway for SUN2 regulated cell activation.5.Effects of DNA methyltransferases on SUN2 expression in hepatic fibrosis.Inhibitor of DNMTs was intraperitoneally injected into mice,primary HSCs were isolated from CCl₄-treated mice.HSC-T6 cells were transfected with DNMT1-RNAi,DNMT3a-RNAi,DNMT3b-RNAi and scrambled-RNAi.CpG methylation status of SUN2 was evaluated by Methylation-Specific PCR;expression level of?-SMA and Col1?1 was evaluated by Real-time PCR and Western blot analysis.Experimental results shown expression of SUN2 up-regulated after DNA methyltransferases were suppressed,and DNMT3b may primarily responsible for regulation of SUN2 expression.