Study On The Influence Of HBx Deletion Mutation (HBx-d382) On The Proliferation Ability Of L02 Cells And Its Mechanism

Objective:Hepatocellular carcinoma (HCC) is a malignant tumor arising from hepatocytes; it is one of the most common malignancies worldwide. Epidemiological studies have showed that there is a strong relationship between the chronic hepatitis B virus infection and development of hepatocellular carcinoma, almost all of the HBV-associated HCCs harbor chromosomally integrated HBV DNA. In most integrated subviral HBV genomes, the HBx gene can be transcribed and translated into HBx protein, its significance in the hepatocarcinogenesis is be widely studied. our previous study has found that HBx gene integration and mutation occur frequently in HCC samples, and isolated two HBx deletion mutations naming HBx-d431 and HBx-d382(HBx gene nt 382-400 deletion),among which the HBx-d382 occurs more frequently in HCC samples, we guess it may be implicated in liver carcinogenesis, and then we have successfully constructed the eukaryotic expression vector for the expression of HBx-d382.In this study, we will establish a L02 cell line stably expressing HBx deletion mutation (HBx-d382) and further study the influence of HBx-d382 on cell proliferation,G1 cell cycle control and microRNA expression profile of L02 cells.Methods:The recombinant plasmid encoding the HBx deletion mutation (pcDNA3.0/HBx-d382) was tested by PCR amplification,double digestion and DNA sequencing, and then introduced into L02 cells by liposome transfection. Positive clones were selected by G418. The genome integration of the HBx gene was test by PCR amplification, and the HBx expression was confirmed by reverse transcription PCR (RT-PCR) and western blot. Cell proliferation changes were measured by MTT assay and soft agar clone formation assay, and the cell cycles were tested by flow cytometry; the mRNA and protein expression of cyclin D1, cylin G1, E2F1 were detected by Real-time Quantitative PCR and Western blot respectively; microRNA expression profiles were analyzed by microRNA microarray.Results:(1)The recombinant plasmid pcDNA3.0/HBx-d382 was successfully identified by PCR amplification,double digestion and DNA sequencing. (2)Positive clone that selected by G418 harbored chromosomally integrated HBx-d382; RT-PCR and Western blot confirmed the HBx expression at the mRNA and protein lever respectively.(3)This cell line showed enhanced proliferation and anchorage-independent growth ability when tested by MTT assay and soft agar clone formation assay; and the proportion of S and G2 phrase cells was increased simultaneously. (4)Real-time Quantitative PCR showed that the mRNA expression of cyclin D1, cylin G1, E2F1 were increased in L02/HBx-d382 cells compared with L02 cells transfected with the pcDNA3 empty plasmid(102/pcDNA3.0) (p<0.05); Western blot confirmed that the protein expression of cyclin D1, cylin G1, E2F1 were increased in L02/HBx-d382 cells compared with L02/pcDNA3.0 cells.(5) Based on the microRNA microarray, mir-1, mir-338-3p, mir-551b, miR-455-3p, and miR-200c were down-regulated, miR-501, miR-595, miR-1307, miR-1180, miR-497, miR-1246 and miR-623 were up-regulated in L02/HBx-d382 cells compared with the L02/ pcDNA3.0 cells.Conclusion:L02 cell line stably expressing HBx deletion mutation (HBx-d382) is established successfully. HBx-deletion mutation(HBx-d382) can enhance the proliferation and anchorage-independent growth ability of L02 cells, and promotes cell cycle progression from G1 to the S phase, which may due to the increase gene expression of cyclin D1, cylin G1, E2F1 that are associated with G1 cell cycle control. This virus gene can also influence the microRNA expression profile in the 102 cells, which may be a new molecular biological mechanism of HBx-d382 induced hepatocytes malignant transformation.