The Effects On Apoptosis And Cell Cycle In Hepatocyte Lines By Stable Expression Of Hepatitis B Virus X Protein

Aim: To investigate the effects of hepatitis B virus (HBV) X gene on apoptosis and cell cycle in hepatocyte line HL-7702 and discuss the possible mechanisms in the pathway.Methods: The reconstituted plasmid pcDNA3-X was established through recombination DNA technique. The reconstituted plasmid pcDNA3-X and empty vector pcDNA3 were transfected into HL-7702 cells and selected by G418 to construct two new cell line which were named HL-7702-HBx and HL-7702-con respectively. RT-PCR and Western-blot analysis were used to confirm that HBV X gene expressed steadily in HL-7702-HBx cells. Then apoptosis and cell cycle of the two cells were detected by DNA ladder, flow cytometric analysis and electronic microscope observation. Gene expressions in these two cells were subsequently evaluated by using Human apoptosis and cell cycle gene arrays. And some of results were further confirmed by Real-time PCR and Western-blot analysis.Results: RT-PCR and Western-blot analysis showed that a new stable cell line(HL-7702-HBx) expressing HBV X gene was constructed successfully. Besides, DNA ladder, flow cytometric analysis and electronic microscope observation revealed that there were increased apoptosis and accumulation of S phase in HL-7702-HBx cells, unlike HL-7702-con cells, which showed no apoptosis and distinct accumulation. Gene arrays analysis indicated that some DNA repair genes (XRCC1, DDB1, etc) and DNA damage checkpoint related genes(Cdc47, RAD17,etc) involved in this way. Both cDNA arrays analysis and Real-time RT-PCR showed that mRNA of XRCC1, Cdc47, RAD17 were up-regulated by HBx. Unexpectedly, western-blot analysis revealed that HBx can not promote the protein expression of these three genes, but inhibit their protein expression.Conclusions: Through inhibiting DNA repair and down-regulating DNA damage checkpoint, HBx protein promoted apoptosis and accumulation of S phase in HL-7702 cells, which may be responsible for the development of HBV-related hepatitis, fibrosis and carcinoma. And many complicated post-transcriptional mechanisms may be involved in this procession.