Effect On The Expression Of GSK3B In L02 And HEPG2 Cells Stably Transfected By HBx

Objective The liver cell lines L02 and HepG2 were infected with HBx retroviral respectively, constructed the liver cells L02 and HepG2 which can express HBx protein stably. To investigate the influence of HBx on cell proliferation, cell apoptosis, cell cycle control, signaling molecule glycogen synthase kinase3β(GSK3β),and to discuss the molecular mechanism of HBV-associated hepatocellular carcinoma.Methods To construct the HBx retroviruses, then infected L02 and HepG2 cells, the expression of HBx was identified by RT-PCR and Western blot; MTT was used to detect the cell proliferation of the two stable cell lines L02 and HepG2; Flow cytometry was used to detect cell apoptosis and cell cycle distribution. Furthermore, the change of total GSK3β, p-GSK3β,β-catenin and CyclinD1 protein were measured by WB.Results HBx retroviruse was constructed successfully, the L02 and HepG2 resistant cells were obtained after infected by the retroviruses and bolting by antibiotic, the expression of HBx has been detected in RNA and protein levels between two lines; MTT and Flow cytometry showed that HBx enhanced the cell proliferation, and the cell ratio was lower than controlled group in G1 period, but higher in G2 period, and the cell apoptosis ratio was lower compared with the negative group respectively in L02-HBx cell line(p<0.05);The cell proliferation stepped down, and the cell ratio was higher in S period but lower in G2 period, and the cell apoptosis ratio was higher than control groups in HepG2-HBx cell line(p<0.05);The total GSK3βmRNA and protein had no significant, p-GSK3β,β-catenin and CyclinD1 protein increased in L02-HBx cell line(p<0.05); The total GSK3βmRNA and protein increased, but p-GSK3β,β-catenin and CyclinD1 protein had no obvious change between groups in HepG2-HBx cell line(p<0.05).Conclusion 1. Retroviruses can infect L02 and HepG2 cells in vitro effectively; The L02-HBx and HepG2-HBx cells were established successfully, which can express HBx protein steadily. 2. HBx can enhance the cell proliferation, accelerate cell cycle and inhibited cell apoptosis in L02 cell line. 3. HBx inhibited the cell proliferation, block cell cycle in S period and accelerate cell apoptosis in HepG2 cell line. 4. HBx may contribute to activate the Wnt signaling passageway, enhance the phosphorylationthe of GSK3β, then induce to the accumulation ofβ-catenin protein and activate downstream relative target genes, promote cell proliferation in L02 cell line accordingly. 5. HBx can promote the expression of GSK3βin HepG2 cells, may be involved in induction of apop tosis in other signaling pathways.