Hepatitis B Virus X Protein Promoted HepG2 Cell Proliferation By Activating The NF-κB Signaling Pathways

Objective:To investigate the mechanism of HBx in Hep G2 cells promoting cell proliferation;To explore whether HBx promoted Hep G2 cell proliferation by activating NF-κB signaling pathways;To investigate the effect of the cell proliferation by inhibiting NF-κB.Methods:Hep G2 cells by the transfection of recombinant HBV X gene lentivirus vectors(Hep G2/HBx) as the experimental group;Hep G2 cells by the transfection of empty lentivirus vectors(Hep G2/Mock) as the negative control;Hep G2 cells without transfection(Hep G2) as the blank control.Hep G2/HBx,Hep G2/Mock and Hep G2 cells were recovered and cultured respectively.We extracted the intracellular protein and nuclear protein by Western blotting to detect HBx expression.RT-PCR detected the NF-kappa B P65 and P50 m RNA level of the above cells;Western blotting tested the nucleoprotein of NF-kappa B P65,NF-kappa B P50 and Cyclin D1.Cell-counting kit-8 assay was used to detect the influence of the different concentrations(0,1.5625,3.125,6.25,12.5,including 25 μM) PDTC on the proliferation of Hep G2/HBx,Hep G2/Mock and Hep G2 cells.Western blotting detected the nucleoprotein expression of the NF-kappa B P65,NF-kappa B P50 and Cyclin D1 in Hep G2/HBx cells that were treated with the best inhibition concentrations 3.125 μM and 12.5 μM PDTC.Results:(1)Western blotting indicated that HBx were found in the cytoplasm and nuclei protein of Hep G2/HBx cells, but not Hep G2/Mock cells and Hep G2 cells.(2)RT-PCR indicated that the m RNA expression of the NF-κB P65 and P50 genes inHep G2/HBx cells were not different of the controls(P > 0.05); Western blotting indicated the nuclei protein expression of NF-κB P65, NF-κB P50 and Cyclin D1 in Hep G2/HBx cells were higher relative to the controls(P < 0.05).(3) CCK8 test showed that PDTC inhibited Hep G2/HBx,Hep G2/Mock and Hep G2 cells proliferation,but this inhibition of Hep G2/HBx cells were more obviously significant than the controls(P < 0.05).(4)After Hep G2/HBx cells were treated with PDTC,the nucleoprotein expression of NF-κB P65, NF-κB P50 and Cyclin D1 were inhibited significantly, statistically(P < 0.05).Conclusions : HBx could promote Hep G2 cell proliferation by activating the NF-κB/Cyclin D1 signaling pathway.PDTC could inhibit the proliferation of Hep G2/HBx cells by reducing the protein expression of NF-κB and Cyclin D1.