The Hepatitis B Virus X Protein Up-regulates CD40 Expression In Hepatocarcinoma Cell

Hepatitis B virus (HBV) is a small circular DNA virus, belonging to the hepadnavirus family. The virus attacks liver cells and can cause lifelong infection, scarring of the liver, liver failure, and death. HBV is one of the major threaten to human health and it was estimated that about 350 million people worldwide are chronically infected with HBV.Because the host range of HBV is limited to humans and chimpanzees and because cell culture systems and small animal models that are susceptible to HBV infection do not exist, the current state of our understanding of the biology and pathogenesis of these infections reflects what has been learned about their natural history and immunobiology in humans and chimpanzees, by the virological and immunological analysis of related hepadnavirus and flavivirus infections in their natural hosts, and by biochemical, molecular, virological, and immunological analysis of cell lines and mouse models that express individual viral genes or reproduce the viral life cycles to various degrees. The major cause to chronic HBV infection is unable to product an effective cellular immune response which is a very complicated process. The HBV genome is a circular partially doubled-stranded DNA, consisting of four viral genes: S/preS,C/preC,P and X. HBx, that has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. HBx does not bind directly to DNA, but it is able to trans-activate transcription through multiple cis-acting elements including activating protein-1 (AP-1), AP-2, nuclear factor kappaB (NF-kappaB), c/EBP, and ATF/CREB binding sites.Co-signalling molecules are glycoproteins located in the cell surface and are essential for the communication of a T cell with virtually all other host cells. During cell–cell contact, specific recognition occurs between co-signalling molecules and triggers biochemical signalling. By expression at the appropriate time and location, co-signalling molecules positively and negatively control the priming, growth, differentiation and functional maturation of a T-cell response.First of all, gene expression profiles of HepG2 and HepG2.2.15 cell were analyzed using Operon genechip. HepG2.2.15 cell line was derived from HepG2 transfected with a plasmid four 5'-3'tandem copies of HBV genome. The cell line could stably express HBV particles and virus proteins, and was a novel in vitro model of HBV infection. After analysis with the genechip, in the HepG.2.2.15 cells, we found that the significantly down-regulated or up-regulated genes were metabolisms-related genes,apoptosis response related genes,cytoskeleton and cell adhesion related genes and oncogenes and tumor suppressor genes, demonstrating that hepotacarcinogenesis is invoved in abnormality of metabolism gene activity,inactivation of anti-oncogenes and so on. However, we pay close attention to co-stimulate molecules and found that there was only CD40 significantly up-regulated among CD40, CD80, CD86, B7RP-1, OX40, B7-H1, B7-DC, B7-H3 and B7-H4. The result of genechip analysis was further confirmed by quantitative real-time reverse transcriptase–polymerase chain reaction, flow cytometry and western blot. Similar levels of upregulation of CD40 were observed in these assays.Partial members of the tumour-necrosis factor(TNF) and the tumour-necrosis factor receptor (TNFR) superfamilies are co-signalling molecules. CD40 is a member of the TNF receptor superfamily, expressed on a plethora of cell types, including normal B lymphocytes, macrophages, endothelial cells, and dendritic cells, and this widespread expression is likely to account for its central role in the regulation of immunity and host defense,and the function of CD40 stimulation is dependent cell type/differentiation state. CD40L is predominantly expressed in activated T cells, the interaction of CD40 with its ligand (CD40L) have multile effects on cell growth, apoptosis and immune recognition:such as to maintain B lymphocytes survival, proliferation and normal Ig class switching and Ab production; deliver potent apoptotic signals to carcinoma cells, which are manifested only upon disruption of CD40-activated survival pathways; up-regulates the Ag-presenting function and immune recognition of malignant hemopoietic cells to induce a tumor specific CTL. Token together, all of this indicate that the CD40 pathway privide an important opportunity for cancer therapy.Secondly, The HBV gene fragments were amplified by PCR from the HBV genome plasmid and were inserted into eukaryotic expression vector pcDNA3.1(+), after the identification by digestion and sequencing on the recombinant eukaryotic expression vector, they were named pcDNA3.1(+)/HBs,pcDNA3.1(+)/HBc,pcDNA3.1(+)/HBe,pcDNA3.1(+)/HBp,pcDNA3.1(+)/HB-preS1,pcDNA3.1(+)/HB-preS2,pcDNA3.1(+)/HBx respectively. Then the recombinants were transfected into HepG2 cell respectively by lipofectamineTM 2000. After screening culture by G418, stable transfected HepG2 cell lines were established, and the expression of HBV gene products were identified by flow cytometry and immunocytochemical staining to confirm that the stable transfected HepG2 cell lines were established that were named HepG2HBs,HepG2HBc,HepG2HBe,HepG2HB-preS1,HepG2HB-preS2,HepG2HBp and HepG2HBx cell lines. Then we identified that it was HBV X protein which up-regulated the expression of CD40 in HepG2 cell lines by flow cytometry. We also detected the expression of CD40 by RT-PCR,qRT-PCR and flow cytometry in other hepatoma cell line PLC derived from a patient infected with HBV and C4 which the the X gene was interfered. The results was that the expression of CD40 was up-regulated in PLC cell line but there was no significantly change in C4 cell line.In summary, gene expression profiles of HepG2 and HepG2.2.15 cell were analyzed using Operon genechip. The HepG2.2.15 cell line could stably express HBV particles and virus proteins. We found that CD40 was significantly up-regulated in HepG2.2.15 cell, and the result of genechip analysis was further confirmed by qRT-PCR,FACS and western blot. Then we established stable transfected HepG2 cell lines which expressed HBV proteins respectively and identified that it was HBV X protein which up-regulated the expression of CD40 in HepG2 cell line by FACS. We also detected the expression of CD40 by RT-PCR,qRT-PCR and flow cytometry in other hepatoma cell line PLC/C4 and have a similar result. In further studies, we try to investigate the function of CD40 pathway to explain the mechanism of HBV infection and to search better therapeutic strategy.