The Different Structure Forms Of HBx Protein Influence Their Intracellular Distribution In Hepatocellular Carcinoma HepG 2 Cells

Aims: HCC is a common malignant tumor in the world and it is often diagnosed at an advanced stage, with high fatality rate. Now it is indicated from epidemio- logy data that the incidence of HCC is increasing. And in the following decade, it will be not only in the Asia and Africa, but also in the North America and Europe, the incidence of HCC will gradually rise. Patients with HCC has poor prognosis and it is difficult to diagnose at early stage. Most HCC patients are of little chance that can be cured, because HCC is multi-resistant to chemotherapy drugs, and other therapies such as surgical resection, transplantation or percuta- neous and transarterial interventions are also of limited curative effect. The risk factors of HCC consist of hepatitis B virus(HBV), hepatitis C virus (HCV), aflatoxin B1, chemical carcinogen-diethylnitrosamine and alcohol. In China, chronic HBV infection is the predominant risk factor for hepatocarcinogenesis, and it is believed that the HBx protein encoded by HBV mediate the process. A potential mechanism that HBx protein can induce HCC may involve its intra-cellular localization. In our research, different eukaryotic expression vectors were selected . We respectively constructed recombinant plasmids containing HBX gene or their different fusion forms with GFP . Our aim is to explore the influence of the different structure forms of HBx protein on their intracellular localization.Methods:1. Flag-HBx gene was amplified from pcDNA3.0-HBx plasmid conserved in our laboratory. It was cloned into pMD-18T vectors, was sequenced.and then was subcloned into different eukaryotic expression vectors to construct recombinant plasmids: pFlag-HBx-IRES2-EGFP, pEGFP-C3- Flag-HBx and pFlag-HBx-EGFP-N3.2. The sucessfully constructed eukaryotic recombinant plasmids were transiently transfected into hepatocellular carcinoma HepG-2 cells respectively. The results were observed and photographed under CHK Olympus inverted fluorescence microscope, and then were processed by AdobePhotoshop7.0 and ImageJ Software.Results:1.Three different eukaryotic recombinant plasmids containing Flag-HBx gene fragment were verified by DNA sequencing , PCR and dual restrictive endonuclease digestion.2. Different structure forms of HBx protein influence their expression level and intracellular distribution in hepatocellular carcinoma HepG2 cells.The constructed pFlag-HBx-IRES2-EGFP plasmid can express HBx and GFP protein respectively. It is observed that GFP protein distribute evenly in the whole HepG2 cell, however the HBx protein mainly locate around the nucleus in granular pattern.In the pEGFP-C3-HBx-Flag plasmid transfection group, the GFP-HBx fusion protein is also predominantly located in the periphery of nucleus however with a small quantity in nucelus , both in aggregated coarse granular pattern .In the pHBx-Flag-EGFP-N3 transfection group, the HBx-GFP fusion protein is basically dispersed evenly in the entire HepG2 cell,which is distinctly different from the above two groups.Similarly, there are also some scattered granules in the periphery of nucleus, which is moderate among the three groups quantitatively.Conclusion:1. It provides us with significant experimental evidence to help research the role of HBx protein in the development of HCC, and especially the references for explanation of the results in vitro transfection experiments.2. The intracellular distribution results of HBx protein in pathological tissues are different from that in in vitro transfection experiments.3. In order to better simulate the natural situation of HBV infection, more recombinants mixture of HBx gene with high-frequency mutations or deletion patterns should be constructed for a low concentration transfection.