NF-κB-induced Gp96Up-regulation Promotes Hepatocyte Growth, Cell Cycle Progression And Transition

Chronic hepatitis B is a global problem. There are many studies that have shown that the incidence of liver cancer is closely associated with sustained liver injury and chronic inflammation. It is essential to research the hepatocellular carcinoma development mechanism induced by chronic inflammation because that ninety percent of hepatocellular carcinoma development was associated with chronic inflammation. Our previous studies have showed that the heat shock protein gp96expression levels in hepatitis B patients were significantly higher than normal. However, the mechanism of gp96up-regulation and the role of gp96in the HCC progression were still unclear.Nuclear transcription factor κB (NF-κB) plays an important role in chronic hepatitis B-induced hepatocellular carcinoma. In this study, we first detect the mRNA and protein levels of gp96by real time-PCR and Western blot after the eukaryotic expression vector of NF-κB subunit p65transfacted into cells. We found that the mRNA and protein levels of gp96were significantly up-regulation. Then we analysis the promoter sequence of gp96by bioinformatic analysis, we found there is the NF-κB binding sites in gp96promoter by nline prediction software. The result of the dual luciferase reporter assay that transfection of p65subunit of NF-kB significantly increased the gp96promoter activity of a wild type but not mutant in NF-κB binding site, and enhanced gp96expression. These prove NF-κB activates transcription of gp96by the binding sites and induces protein expression.Further, we study the role of gp96in hepatocellular carcinoma occur. We up-regulate gp96protein levels by transfecting of the gp96eukaryotic expression vector or knock-down the protein levels by RNA interference. We have determined the levels of proliferation, cell cycle and apoptosis by CCK-8kit and flow cytometry. The results suggest that gp96promotes hepatocyte proliferation, inhibits apoptosis, and induces G0/G1to S phase cell cycle progression. To determine the role of gp96in normal liver cells transformated into cancer cells by detected the EMT indicators and colony-forming assay. The results prove that gp96induces epithelial-mesenchymal transition and increases colony formation ability of hepatocytes.In conclusion, our studies indicate a positive the mechanism of NF-λB activating gp96expression. And these results also imply that gp96promotes hepatocyte proliferation, inhibits apoptosis, and induces G0/G1to S phase cell cycle progression. Moreover, gp96induces epithelial-mesenchymal transition and increases colony formation ability of hepatocytes. Our results therefore provide insights in chronic HBV infection-induced gp96expression, and indicate that elevated gp96may contribute to HCC development during chronic inflammation.