Hepatitis B Virus X Protein Epigenetically Activates SIP1 To Induce Epithelial–mesenchymal Transition And Promote HCC Aggressiveness

ObjectiveTo explore the mechanism of HBx protein induced Epithelial–mesenchymal Transition and hepatocellular carcinoma and the role of SIP1 in the process.Methods 1、The cellullar mobility of hepatocellular carcinoma cell line HepG2 cells and SMMC-7721 cells transfected with HBx plasmid was detected using transwell experiment.SIP1 and EMT related protein in cells was detected by Western blot,qPCR and immunefluorescence technique.2、HepG2-X cell line were constructed and were analyzed the level of SIP1 and EMT related protein with western blotting and immunofluorescent staining assay.3、HepG2-X cell line were transfected with siHBX or transfected with shSIP1 and were analyzed the level of related protein.4、The HepG2 cells and SMMC-7721 cells treated with HBx were then co-transfected with pRL-PK and pGL3-E-cadherin-luc or pGL3-Basic.The cells lysates were measured on a multiple function enzyme analyzer for luciferase activity.5、After HepG2 cells were treated with pHBx for 12,24,36 and 48h,respectively,q-PCR was utilized to measure E-cadherin mRNA.According to the level of E-cadherin mRNA,the optimal time point was selected,and the binding level of SIP1 to promoter region in E-cadherin gene at this time point was measured using the Ch IP assay,which includes 3 regulatory sites: E-box region,2000bp(-2kb)at upstream regulatory region,2000bp(+2kb)at downstream regulatory region.6、The HepG2 cells treated with HBx and shSip1 were then co-transfected with pRL-PK and pGL3-E-cadherin-luc or pGL3-E-cad-MUT-luc.The cells lysates were measured for luciferase activity.7 、 After HepG2 and HepG2-X cells were stimulated by different concentration of TSA for 72h(the culture medium was replaced every 24h).The protein expression level of HDAC1 was detected to screen the optimal TSA concentration.After HepG2 and HepG2-X were stimulated by TSA for totally 72 h,respectively,Western-blot was applied to detect the protein expression level of HDAC1 and E-cadherin.8、The Hep G2 cells were co-transfected with pRL-PK and pGL3-E-cadherin-luc or p GL3-Basic with or without HBx treatment.The cells lysates were measured on a multiple function enzyme analyzer for luciferase activity.9、After treatment with pHBx /pcDNA3.1 and shControl / shSIP1 in the HepG2 cells,ChIP assays were used to detect the histone modification level of transcriptional regulatory region in E-cadherin gene after HBx stimulation and Sip repression.10、The interaction between HBx、SIP1、HDAC1 and their common localization in hepatocellular carcinoma was measured using the Co-IP assay as well as immunofluorescent staining assay.11、The effect of HBx overexpression and SIP1 silence on the vability,apoptotic rates and cell cycle of HepG2 and SMMC-7721 cells was detected by CCK8 Apoptosis Floe Cytometry,respectively.12、The HepG2 cells and two group of HepG2-X were treated with shControl / shControl / sh SIP1 respectively and subcutaneously injected into mouse.Four weeks later,the tumor mass was evaluated by western blot to detect the protein expression in the tissues.Then,the tumor masses were cut into little pieces and carefully transplanted into livers of randomly selected 6~8 weeks old nude mice to establish hepatic orthotopic transplantion tumor model.After the mice died,their livers,diaphragm,lungs were removed and prepared for subsequent HE to detect the tumor metastases.Results 1、HBx improved cell mobility in Hep G2 cells and SMMC-7721 cells.The mRNA and protein expression of SIP1 and Vimentin was increased,which was contrary to E-cadherin.2、HepG2-X cell line were successfully constructed and the level of SIP1 protein was increased.Compared with mock-HepG2 cell,HepG2-X cell had enhanced cellular mobility,increased SIP1 level and decreased E-cadherin expression;While HBx silencing in Hep G-X cells showed an opposite change.3、SIP1 silencing in HBx-expressing HepG2 cell reduced cellur mobility,increased E-cadherin expression and repressed Vimintin protein.4、HBx increased the promoter activity of E-cadherin in SMMC-7721 and HepG2 cells.The effect of HBx on E-cadherin promoter activity can be affected by TSA,SIP1over-expression as well as SIP1 knocking down.5、Results from ChIP assays showed HBx increased the combination of SIP1 to the E-cadherin promoter near the start region,while no differences were detected in the distant region of E-cadherin promoter far from the start region.HBx over-expression can enhance combination between HDAC1 and E-cadherin promoter,while konocking down of SIP1 can diminish the combination.6、HBx induced E-cadherin repression can somehow be revived by TSA.7、There was interaction between HBx、SIP1as well as HDAC1 and their common localization in cellular nucleus was detected by immunofluorescent staining assay.8、HBx overexpression increased cellular vability and repressed apoptotic rate of HepG2 and SMMC-7721 cells,while SIP1 silence have the opposite effect;HBx promoted cell cycle from G1 to S phase,while SIP1 silence prevented it in G1 phase.9、The tumor masses from mice of Hep G2-X+shControl group were larger averagely than the other two group.The hepatic orthotopic tumor-transplantion model was successfully established.The tumor metastasis from the transplanted hepatic tumor to diaphragm or mesentery tissue can only be detected in mice from Hep G2-X+shControl group which had more obvious intrahepatic metastatic than the other two groups.ConclusionHepatitis B Virus X protein epigenetically activates SIP1 to induce epithelial–mesenchymal transition and promote HCC aggressiveness.