Effect Of HBx Gene On Endogenous Survivin Expression In Human Hepatoma Cells

Objective Survivin is a member of inhibitor of apoptosis protein family. HBx gene is the samllest in open reading frames of HBV, which plays the most important role in the development of HCC. To study the effect of HBx gene on survivin expression in hepatoma cells and detect survivin expression in three types tissues from HCC,liver cirrhosis as well as HBV-infected hepatitic tissues, which may be one of the possible molecular mechanisms of HBV-infected HCC, establish the basis of theory and experiment.Methods pcDNA3.1- HBx (encoding the HBx gene) and pcDNA3.1 were transfected into Human hepatoma cell line HepG2 cells respectively with Lipofectmine^?2000, and non-transfected HepG2 cells as control. To collect transfected cells with G418 after 48 hours of tansfection. The following experiments were carried out after stably transfection: First, immunocytochemistry was used to detect HBx expression. And then, immunocytochemistry was used to detect survivin expression in each group. To confirm the result, Western Blotting was used to compare the survivin expression in each group. At last, to detect immunohistochemically in tissues from 20 cases of Hepatocellular carcinoma (poor differentiation 9 cases, moderate differentiation 7 cases and well differentiation 4 cases),20 cases of HBV-infected hepatitic tissues as well as 10 cases of liver cirrhosis (pathologic grade: G2/S2～G4/S4) .Result Immunocytochemistry showed brown staining of HBx in cytoplasm of pcDNA3.1- HBx transfected HepG2 cells, but no staining was found in pcDNA3.1 transfected or non-transfected cells. The three groups showed high level survivin expression in cytoplasm with immunocytochemistry, even the group of pcDNA3.1-HBx transfected HepG2 cells, with significant differences between them (P<0.05). For further confirmation of survivin, we used Western Bloting experiment to detect the expression of survivin in each group. The result showed that survivin exprssion was remarkably higher in the presence of HBx in comparison to the other two groups, the findings indicate collectively that HBx induces upregulation of endogenous survivin expression, suggesting that HBx and survivin may be involved coordinately in carcinogenesis of hepatocellular carcinoma. Immunohistochemistry showed that survivin was highly expressed in hepatoma samples, corresponding liver cirrhosis and HBV-infected hepatitic tissues. The positive rates of survivin expression were high, with significant differences betwwen them (each P＜0.01) in HCC(17/20, 85%)(including proper positive 58.8%, moderate positive 23.5% and light positive 17.7%), hepatitic tissues(2/20, 10%) and liver cirrhosis (3/10, 30%), respectively. Only a few survivin-positive signals were observed in the nuclei of hepatoma cells; the majority of positive signals were found in the cytoplasm.Conclusion HBx expression plasmid was successfully transfected into HepG2 cells and HBx protein was expressed in the cytoplasm of the cells. HBx induces upregulation of endogenous survivin expression, suggesting that HBx and survivin may be involved coordinately in carcinogenesis of hepatocellular carcinoma. Survivin may consider as a new target for HCC pathogenesis and drug therapy due to specific expression in tissues.