Wild Type Competes To Bind KPNA2 With Sw172* Mutant HBsAg To Downregulate C-myc Expression

Objective: This study aims to investigate the carcinogenesis mechanism of HBV s W172* drug resistant mutant and preliminarily explore a new idea to prevent hepatocellular carcinoma resulted from HBV multidrug resistant truncated variant due to antiviral therapy with nucleos(t)ide analogs.Methods: 1.Pre-S/S segments was obtained by designing sister PCR primers and used recombinant plasmid p IRES2-pre-S/S(WT)as the template to replace the green fluorescent protein(EGFP)gene in p IRES2-EGFP.Pre-S/St segments obtained from p IRES2-pre-S/St(s W172*)inserted into the replaced plasmid to construct double gene co-expression vector p IRES2-pre-S/St-pre-S/S(WT+s W172*).2.Hep G2 cells were transiently transfected with control,WT and s W172* groups.Exogenous co-immunoprecipitation(Co-IP)determined whether WT and s W172* interacted with KPNA2.Semi-exogenous co-ip determined KPNA2 interacted with SHBs.3.Hep G2 cells were transiently transfected with control,WT,s W172* and WT+s W172* groups.Immunofluorescence staining to detect subcellular localization of WT,s W172* and KPNA2 in Hep G2 cells.4.Hep G2 cells were transiently transfected with control,WT,s W172* and WT+s W172* groups,nuclear/cytoplasmic fractionation,followed by western blotting to detect subcellular localization of HBs Ag in Hep G2 cells.5.Hep G2 cells were transiently transfected with control,WT,s W172* and WT+s W172* groups,c-Myc m RNA and protein expression were analyzed by RT-q PCR and Western Blot assay.6.Hep G2 cells were treated with or without 1?M,5?M and 10?M Ivermectin,then Hep G2 cells were transiently transfected with s W172*.c-Myc m RNA and protein expression were analyzed by RT-q PCR and Western Blot assay.7.Hep G2 cells were treated with or without 1?M,5?M and 10?M Ivermectin,then Hep G2 cells were transiently transfected with s W172*.Nuclear/cytoplasmic fractionation,followed by western blotting to detect subcellular localization of HBs Ag in Hep G2 cells.Results: 1.DNA sequencing confirmed that double genes co-expression vector p IRES2-pre-S/St-pre-S/S was constructed successfully.2.HepG2 cells were transiently transfected with control,WT and sW172* groups,exogenous co-immunoprecipitation revealed that wild-type HBs Ag and s W172* mutant could both interact with KPNA2,Semi-exogenous co-ip revealed that KPNA2 could only interact with SHBs(WT)and SHBs(s W172*).3.Hep G2 cells were transiently transfected with control,WT,s W172* and WT+s W172* groups.Immunofluorescence obviously verified that the s W172* mutants bound to KPNA2 were located in the cytoplasm as well as the nucleus,but the wild-type HBs Ag only in the cytoplasm.4.Hep G2 cells were transiently transfected with control,WT,s W172* and WT+s W172* groups.Nuclear/cytoplasmic fractionation revealed that the protein levels of the s W172* mutant in the nuclear were determined,but the wild-type HBs Ag not.5.Hep G2 cells were transiently transfected with control,WT,s W172* and WT+s W172* groups.c-Myc m RNA and protein expression in WT+s W172* group were significantly decreased than these in s W172* groups.6.Hep G2 cells were treated with or without 1?M,5?M and 10?M Ivermectin,then Hep G2 cells were transiently transfected with s W172*.Compared to DMSO-treated group,m RNA and protein levels of c-Myc with Ivermectin pre-treatment significantly were significantly reduced.7.Hep G2 cells were treated with or without 1?M,5?M and 10?M Ivermectin,then Hep G2 cells were transiently transfected with s W172*.Nuclear extracts were analyzed for s W172* mutant expression by Western Blot.In cell nucleus proteins,the s W172* mutant level with Ivermectin pre-treatment was lower than that with DMSO.Conclusion: Wild type competes to bind KPNA2 with s W172* mutant HBs Ag to downregulate c-Myc expression.