The Study About HBX Proliferative Effect On Hepatic Oval Cells And Related Molecular Mechanism

PartⅠHBX protein directly promotes the proliferation and up-regulate cyclinDl levels in oval cells, dependent on the HBX-stimulated MEK/ERK and PI-3K/Akt pathway PartⅠ-1 HBX promote Proliferation and stimulate the DNA synthesis in HBX-transfected LE/6 oval Cells in vitroObjective:To determine whether HBX protein has a proliferative effect on the hepatic oval cells.Methods:Western blot analysis was performd to detect the expression of HBX protein in HBX-transfectd oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. Furthermore, all three oval cells were observed by fluorescence microscopy. MTT assay was preformed to determine whether HBX gene has a proliferative effect on the oval cells. EDU proliferation assay was performed to detect HBX effect on the DNA synthesis.Results:HBx protein is only expressed of LE6-EGFP-HBX cells, was determined by Western blot analysis. In cells that expressed EGFP and HBX-EGFP fusion protein, the fluorescence was distributed throughout the entire cell. The MTT results exhibited, compared to the control cells, HBX expression promoted the proliferation of HBX-EGFP-LE/6 oval cells in 24.48h and 72h, The difference was significant (p<0.01).Conclusions:HBX promote Proliferation and stimulate the DNA synthesis in HBX-transfected LE/6 oval Cells in vitro, raising the possibility that the HBX protein may directly regulate oval cell-drived liver regeneration in absence of immune response.PartⅠ-2 Deregulated expression of modulators of cell cycle progression in HBX overexpressing cellsObjective:To investigate the effect of HBX protein on the expression of G1 cell cycle regulator, including cyclinD1,p21,p27,CDK2 and CDK4.Methods:the protein levels of cyclinD1,p21,p27,CDK2 and CDK4 were determined by western blot analysis in HBX transfected rat oval cells named HBX-EGFP-LE/6, and LE6,EGFP-LE/6 control oval cells; Immunostaining for cyclinDl were also perfomed in HBX-EGFP-LE/6 cells and control oval cells.Results:Compared with the cells control, cyclinDl level was up-regulated and the expression of CDK inhibitor, p27 Kipl, was slightly declined, while that of p21WAF1/Cip1, CDK2, and CDK4 was unchanged in HBX-EGFP-LE/6 oval cell line. Conclusions:HBX expression in rat LE/6 oval cells was sufficient to module protein expression of cyclinD1, protein expression of p27 was also slightly changed by HBX, in contrast, HBX has no effect on the p21,CDK2 and CDK4 protein expression in rat oval cells.PartⅠ-3 Up-regulation of cyclinD1 in oval cells is dependent on the HBX-stimulated MEK/ERK and PI-3K/Akt sigaling pathwayObjective:To investigate the effect of HBX protein on the singaling of MAPK pathway and PI-3K/AKT pathwayMethods:the protein levels of p-ERK,p-Akt,ERK,Akt,JNK,p38,p-p38,p-JNK were determined by western blot analysis in HBX transfected rat oval cells named HBX-EGFP-LE/6, and LE6,EGFP-LE/6 control oval cells; Immunostaining for cyclinD1 were also perfomed in HBX-EGFP-LE/6 cells and control oval cells.Results:Compared with the cells control, p-ERK,p-AKT level was up-regulated and the expression of ERK,AKT,JNK,p38,p-p38,p-JNK were unchanged in HBX-EGFP-LE/6 oval cell line.Conclusions:HBX expression in rat LE/6 oval cells was sufficient to module the ERK activity and AKT activity in rat oval cells, HBX has no effect on the activity of JNK activity and p38 activity in rat oval cells.PartⅠ-4 Up-regulation of cyclinDl and Proliferative effect of HBX protein in oval cells is dependent on the HBX-stimulated MEK/ERK and PI-3K/Akt sigaling pathwayObjective:In order to provide the direct evidence that the HBX proliferative effects and the increased HBX-stimulated DNA synthesis and up-regulation of cyclinD1 are all dependent on the stimulated activities of PI-3K/Akt and MEK/ERK by HBX.Methods:after the addition of PI-3K inhibitor LY294002 (LY) (25μM) and the MEK inhibitor PD184352 (PD) (5μM) for 24 h, in indicated time point, the protein levels of cyclinDl were determined by western blot analysis in HBX transfected rat oval cells named HBX-EGFP-LE/6, and LE6,EGFP-LE/6 control oval cells; meanwhile, MTT assay were also perfomed in HBX-EGFP-LE/6 cells and control oval cells in indicated time after these drugs.Results:up-regulation of cyclinD1 expression by HBX protein was abolished by MEK inhibitor PD184352 or PI-3K inhibitor LY294002 treatment, respectively. Compared to untreated HBX-EGFP-LE/6 oval cells, the proliferative effect of HBX in PD184352/ LY294002 treated HBX-EGFP-LE/6 oval cells declined in 72h, the difference was significant (p<0.01) Compared to untreated HBX-EGFP-LE/6 oval cells, the proliferative effect of HBX in LY294002/PD184352 treated HBX-EGFP-LE/6 oval cells declined in 72h, the difference was significant (p<0.01)Conclusions These data suggest that both proliferative effect of HBX oval cells and the up-regulation in cyclinD1 by HBX in cultured oval cells are dependent on the both HBX-stimulated PI-3K/Akt and MEK/ERK pathways. PartⅡ-1 The studies of hepatitis B virus X gene insenstize oval cell to antiproliferative effect of TGFβ1 in vitroObjective:To determine whether HBX protein expression affect the oval cells'response to anti-proliferative effect of TGFβ1 in oval cells.Methods:detect the expression of TGFβRⅡin HBX-transfectd oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. In addition. Exogenous TGFβ1 was added into all three oval cell lines, MTT assay was preformed to clarify different responses to the anti-proliferative effect of TGFβ1.Results:compared to the control cells, HBX protein expression reduce the TGFβRⅡmRNA and protein levels of TGFβRⅡin HBX-EGFP-LE/6 oval cells,. The MTT results showed, HBX protein more strongly inhibit the proliferation of HBX-EGFP-LE/6 cells than those in control cells, The difference was significant (P<0.01).Conclusions:HBX protein reduce TGFβEⅡmRNA and protein levels, and insensitize oval cells to anti-proliferative effect of TGFβ1.PartⅡ-2 X Protein of Hepatitis B Virus mediate the gene expression of smad2, smad3 and smad4 in oval cell in vitroObjective:To determine whether HBX protein expression affect the oval cells'response to anti-proliferative effect of TGFβ1 in oval cells. Methods:Real-time PCR,Western blot analysis was performd to detect the expression of TGFβRⅡin HBX-transfectd oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. In addition, Exogenous TGFβ1 was added into all three oval cell lines, MTT assay was preformed to clarify different responses to the anti-proliferative effect of TGFβ1. Results:The TGFβRⅡ...