| Objective To explore the effect of ?-catenin on Hepatitis B Virus replication and expression and its mechanism.Methods1.Hepatoma carcinoma cell lines Hep G2 and Hep G2.2.15 which is stably expressing HBV were cultured under routine conditions,Hep G2 were transfected with pc DNA3.1-HBV1.3 or pc DNA3.1-HBV1.1 to establish the transiently expressing HBV cell lines.The protein levels of HBV core and ?-catenin in transfected Hep G2 and Hep G2.2.15 were detected by Western Blot and immunofluorescent stain assay,respectively.2.Hep G2.2.15 were transfected with si-?-catenin to slience or p GFP-?-catenin to overexpress ?-catenin and the the levels of HBV DNA replication intermediates,3.5kb m RNA and core protein were measured using q PCR and Western Blot assay.3.The plasmids p TOP-Flash and p RL-TK were cotransfected to Hep G2.2.15,Dual luciferase reporter assay was applied to verify the activity of Wnt/?-catenin signal pathway after various concentrations of Li CL treatment.Western Blot assay was conducted to detect the level of?-catenin.On the basis of ?-catenin overexpression,q PCR and Western Blot assay were used to detect the expression of HBV DNA replication intermediates,3.5kb m RNA and core protein under various concentrations and time points.4.Hep G2.2.15 cells were cotransfected with p TOP-Flash and p RL-TK and then treated with various doses of i CRT14,Dual luciferase reporter assay was utilized to test the activity of Wnt/?-catenin signal pathway.q PCR and Western Blot assay were used to detect the expression of HBV DNA replication intermediates,3.5kb m RNA and core protein.5.Hep G2 cells were transfected with si NC/?-catenin.72 h later,plasmisd p GL3-Xp,p GL3-Cp,p GL3-Sp1 or p GL3-Sp2 were transfected into those cells along with p RL-TK respectively.Dual luciferase reporter assay was used to detect the activity of promoters.6.Hep G2 cells were transfected with p GL3-Cp or p GL3-Enhancer ?along with p RL-TK,then the cells were treated with Li CL or i CRT14 for48 h,The activity of core promoter and Enhancer ? were measured by Dual luciferase reporter assays.Results1.Compared to the mock transfected cells,The hepatoma carcinoma cells transiently or stablely expressing HBV exert an improved expression of?-catenin.Besides,Immunofluorescence staining also showed the main location of ?-catenin changed from cell membrane to nuclear membrane.2.Silencing the ?-catenin in Hep G2.2.15 facilitated HBV replication,while overexpression of ?-catenin showed an inhibition effect on HBV core protein level.3.Li CL activated Wnt/?-catenin signal pathway and up-regulated?-catenin expression in Hep G2.2.15.Under the treatment of various concentrations of Li CL and different time point of the same dose,the HBV replication level decreased significantly.4.i CRT14 showed a suppressive effect on Wnt/?-catenin signal pathway in Hep G2.2.15,The HBV replication level was significantly up-regulated in a concentration-dependent manner when the cells were treated with i CRT14.5.Silencing of ?-catenin in Hep G2 cells resulted in significant increased activity of HBV core promoter whereas the activities of other three promoters changed little,In addition,Li CL inhibited the activities of CP and Enhancer ?,and i CRT14 has the opposite effect.Conclusion?-catenin may inhibit HBV replication and expression through affecting the activity of HBV core promoter. |
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