Impact Of Hepatitis B Virus X Gene Mutation On The Role Of Androgen Receptor In Liver Cancer

Research ObjectiveConstruction of a Hep G2 cell line stably expressing human androgen receptor,were transfected with null vector,wild type plasmid and HBx mutant plasmid to Hep G2 cells,through measuring the interaction of cell cycle,cell apoptosis,cell migration and research on HCC related HBx mutation and AR on the cell phenotype influence.Methods1.Construction of human AR exon gene,green fluorescent protein gene(GFP)and puromycin gene(PM)recombinant expression plasmid(p CDH-CMV-MCS-EF1-GFPPuro);2.The recombinant expression plasmid was packaged and infected Hep G2 cells and screened with puromycin to construct a Hep G2 cell line stably expressing human AR;3.The lentiviral null vector,lentiviral vector carrying HBx wild type full-length gene and lentiviral vector carrying HBx gene of A1727G+A1762T/G1764 combined mutation or 3 ’terminal deletion mutation,transiently transfected Hep G2-GFP-AR cells.Migration,cell cycle and apoptosis were detected by flow cytometry to study the effect of HCC related HBx mutation and AR on the malignant degree of hepatocellular carcinoma.Results1.The lentiviral expression vector p CDH-CMV-MCS-EF1-GFP-Puro-AR was successfully constructed.2.Cell lines stably high expressing human androgen receptor were screened by Hep G2 cells infected with lentivirus which carrying AR expression of gene;Through flow cytometry to verify the expression of GFP,after passage tenth,the cell’s expression level of GFP was 97.7%;Western-blot assay showed that the expression of AR in the fourteenth generation of monoclonal cell lines,clone 2 was higher than other clones.3.Clone 2 with high expression of AR were selected for subsequent functional experiments.Transwell experimental results show: HBx transfection group after joining DHT,the OD value of cells in the lower chamber(0.45 ± 0.02)was significantly higher than that without DHT(0.33 ± 0.03),p< 0.001;Compared with the Hep G2-GFP-AR cells(null-group)(0.33 ± 0.03)of the non transfected HBx gene,transfected with HBx A1727G+A1762T/G1764 combined mutation(167-group)(0.43 ± 0.01,p=0.001)or 3 ’end deletion mutation(CT-group)(0.45 ± 0.03,p < 0.001)plasmid,OD value was significantly higher in the lower chamber.Hep G2-GFP-AR cells were transfected with HBx combination mutation A1727G+A1762T/G1764(167-group)or 3 ’end deletion mutation(CT-group)plasmid after joining DHT,the OD value of cells in the lower chamber was significantly decreased(167-group vs 167+ group,0.43 ± 0.01 vs 0.36 ± 0.02,p=0.009;CT-vs group CT + group,0.45 ± 0.03 vs 0.35 ± 0.04,p=0.001)Cell cycle results revealed: There was no significant difference in total cell count between G2+S cells in each group.Experimental results of cell apoptosis: There was no significant difference in cell apoptosis between groups.Conclusion1.In this study,we successfully constructed a lentiviral vector carrying human AR exon gene,named p CDH-CMV-MCS-EF1-GFP-Puro-AR.Using this vector,lentivirus infected Hep G2 cells,and successfully constructed a Hep G2 cell lines which stable expression of AR,named Hep G2-GFP-AR.2.Through the further functional experiments by AR high expression cell line Hep G2-GFP-AR,it was found that in Hep G2-GFP-AR cells with high expression of AR,the migration ability of the transfected empty vector group was significantly enhanced after added DHT,The transfection of HBx combined mutation or 3 ’terminal deletion mutation can also enhance the migration ability of cells;However,the effect of HBx A1727G+ A1762T/G1764 mutation or 3 ’-terminal deletion mutation on the migration ability of tumor cells after DHT entry may be inhibition.In this study,we didn’t found that HBx mutant and AR had significant effects on the proliferation and apoptosis of Hep G2 cells.