Parallel Induction Of Cell Proliferation And Inhibition Of Cell Differentiation And Apoptosis In Hepatic Progenitor Cells Byhepatitis B Virus X Gene

Increasing evidence has shown that normal stem cells may contributeto the development and progression of cancer by acting as cancer-initiatingcells. The hepatitis B virus X (HBX) protein has been implicated in thehepatitis B virus (HBV)-associated liver carcinogenesis. However, the roleof HBX in hepatic progenitor cells (HPCs) is poorly understood. In thisstudy, we aimed to determine the role of HBX in regulating HPCproliferation、apoptosis and differentiation， we carried out the followingstudies.1,Parallel induction of cell proliferation and inhibition of celldifferentiation in hepatic progenitor cells by hepatitis B virus X gene.Mouse hepatic precursor cell（HP14-19） was infected with Ad-GFPor Ad-HBX. The expression of HBX mRNA and protein were examined byRT-PCR and western-blot respectively. Using MTT analysis, we showedthat（HP14-19） infected with adenovirus expressing HBx (Ad-HBx) grewmore rapidly compared to （HP14-19）infected with adenovirus expressinggreen fluorescent protein (Ad-GFP). To reveal the mechanism for theincreased cell number after HBX treatment, we searched for possible alterations in the cell cycle and apoptosis by flow cytometry. We found thatHBX treatment resulted in an increase in the S phase cell cycle fraction anda decrease in apoptosis. In addition, we examined the differentiation ofHPCs infected with Ad-HBX and found that the HBX expression inHP14-19cells result in increasing the expression of early progenitormarkers and decreasing expression of late hepatocyte markers. Furthermore,HBX inhibited glycogen synthesis in HP14.5cells, indicating that HBX iscapable of inhibiting terminal hepatic differentiation.2,The effect of hepatitis B virus (HBV) X protein (HBX)on cellapoptosis and apoptosis associated proteins in mouse hepatic precursorcellFlow cytometry、Tunnel and Hoechest33342staining method wereused to detected cell apoptosis.The expression of Bcl2, Mcl1,Bax mRNAand protein were indentified by real time PCR and western blotrespectively.Results: Hochest33342showed the number of apoptoticcellular features, including nuclear condensation and chromatinmarginization was significantly elevated in the HBX transfection groupthan that in the control group. Tunel and Flow cytometry showed theanti-apoptosis rate of Ad-GFP-HBX infected cell was higher than that inthe control group.The transcription and protein expression of Mcl1andBcl2after infecting with Ad-GFP-HBX were statistically higher than thosein control group, while the expression of Bax was lower than those in thecontrol group.3, The hepatitis B Virus X protein (HBx) affects the expression and location of β-catenin in Hp14-19cellsReal time PCR was used to assess the effect of HBx on the expressionof β-catenin.The expression of HBX、β-catenin、t-GSK3βand p-GSK3βproteins were detected by Western-blot.Immunocytochemistry was used toindentified the effect of HBX on the distribution of β-catenin.Results:The virus was effectively transfected into mouse hepatic precursor cell（Hp14-19）. The transcription of β-catenin after infecting with Ad-HBXwere statistically higher than those in control group. The expression oft-GSK3β protein has no difference in two group, while the expression of p-GSK3βand β-catenin proteins were statistically higher than those incontrol group. β-catenin proteins maily accumulated in cytoplasm,andhad the tendency to transfer into cell nucleus.In summary, our present study found that, hepatitis B virus X gene canpromote cell proliferation, inhibit cell differentiation and apoptosis inhepatic progenitor cells and upregulate the expression of β-catenin. Thismay be the potential mechanism of HBX-mediated liver carcinogenesis.