The Effect And Possible Mechanism Of Hepatitis B Virus On The Human Hepatoblastoma HepG2 Cell Line

Hepatitis B virus (HBV), a member of the Hepadnaviridae family, harbors a partially double-stranded DNA genome of 3.2 kb, which contains four open reading frames that encode: the envelope antigen (S/Pre-S), the core antigen (C/Pre-C), the viral polymerase (P), and a multi-functional transcriptional transactivator called the X protein. Individuals infected with HBV exhibit a broad spectrum of clinical manifestations: some are asymptomatic carriers, whereas others may experience acute self-limited hepatitis, chronic hepatitis, cirrhosis, or fulminant hepatitis. Increasing evidence suggests that, aside from the evoked host immune response, viral proteins may play an important role in HBV-induced hepatic injury. However, the global effects of HBV-expressed proteins on host cells are poorly understood. In this study, we generated the human hepatoblastoma HepG2 cell line harboring 1.2x unit-length of the HBV genome, and analyzed the global protein expression profiles of HepG2 cells with and without HBV replication and protein expression. In addition, we explored the relationship between infection of HBV and alteration of cell cycle of HepG2. The results may provide new insights into underlying molecular mechanisms involved in HBV replication and pathogenesis.1. Establishment of HepG2 cell lines with the replication and antigens expression of hepatitis B virus1.2×unit length of HBV genome was cloned into SalⅠsite of the eukaryotic expression vector pREP10 to construct the recombinant plasmid pREP-HBV. Control plasmid pREPSal was constructed by pREP10 self-ligation after digestion by SalⅠ. Human hepatoblastoma cell HepG2 was transfected with pREP-HBV or pREPSal.Transfected cells were selected in DMEM medium containing 10% FBS and 250μg/ml hygromycin B. pREP-HBV-harboring cell lines and pREPSal-harboring cell lines were acquired. All of the pREP-HBV-containing HepG2 cell lines showed viral replication and expression of the HBV antigens HBsAg and HBeAg, as determined by Southern blot and ELISA analyses, respectively. HBV particles presented in supernatant were visualized by electron microscopy.2. Global proteomic analysis of HBV in the human hepatoblastoma HepG2 cell line by two-dimensional difference gel electrophoresisRHBV-3 and RepSal-1 cells were selected for proteomic analyses. Cells were incubated in 2D lysis buffer, the supernatant was collected and the protein concentration was determined using a 2-D Quant Kit. The proteins were labeled with the CyDye DIGE Fluor minimal dyes according to the manufacturer's protocol. By using fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), 2,442 protein spots were visualized on the gels. Relative to HepG2-RepSal cells, 65 protein spots in HepG2-HBV cells were found to be differentially expressed when using the criteria of 1.5-fold or greater and p < 0.01; 35 of the differentially expressed proteins were expressed at higher levels in HepG2-HBV cells. Followed by MALDI-TOF-MS and database searching, a total of 50 differentially expressed proteins were identified unambiguously, and classified in functional categories related to lipid metabolism (10), protein synthesis, protein folding and metabolism (10), cell proliferation (8), cytoskeleton (5), mitochondrial function (5), carbohydrate metabolism (4), and others(8).3. Influence of HBV on human hepatoblastoma HepG2 cell cycle Flow cytometer was introduced to detect the cell cycle of RHBV-3 and RepSal-1. The results revealed that, as compared to RepSal-1 cells, most of RHBV-3 cells stayed in the G2/M phase, and polyploids cells were found. Laser scanning confocal microscope found higher rate of multi-spindles in RHBV-3 than RepSal-1 cells. 2D-DIGE proteomic results showed that some differentially expressed proteins were closely related to cell cycle progression. Real time RT-PCR was applied to evaluate the differences of mRNA expression levels between RHBV-3 than RepSal-1 cells. The results demonstrated that mRNA expression alteration of STMN1, VCP, CUL3, FBXO2 and YWHAE were consistent with the results of 2D-DIGE proteomic analysis, i.e., STMN1, VCP, FBXO2 and CUL3 were down regulated in HepG2-HBV cells, while YWHAE was up regulated. The above results suggested that HBV could promote cell growth through Ubiquitin-dependent proteolysis pathway and some other cycle related protein. The specific mechanism need further study.In summary, we successfully established HepG2 cell lines with the replication and antigens expression of hepatitis B virus, and analyzed the global protein expression profiles of HepG2 cells with and without HBV replication and protein expression by 2D-DIGE. In addition, we also explored the relationship between infection of HBV and alteration of cell cycle of HepG2. The results may provide new insights into underlying molecular mechanisms involved in HBV replication and pathogenesis.