The Effect Of HBX Protein On The Proliferation Of Normal Hepatic Cell And The Activity Of Telomerase

Background:A large number of epidemiological surveys show there is a close relationship between the chronic infection of HBV and hepatocellular carcinoma, and HBV chronic infection is one of the major risk factor for the hepatocellular carcinoma. However, it is not clear that HBV causes liver cancer of the mechanism. With the developing of the research about hepatocellular carcinoma and HBV, it was found that the X-gene of HBV and its expression product HBX protein play a significant role in the formation of HBV-related hepatocellular carcinoma. The HBV gene consists of the4opening reading frame (ORF),The X gene is the minimum in the opening reading frame, which encodes the protein containing145-154amino acids and the molecular weight about17kDa. The HBX protein is a multifunctional regulatory protein involved in regulation of the cell proliferation and apoptosis, cell cycle, gene transcription and cell signal transduction and so on, thus it has the effect on the produce and development of hepatocellular carcinoma. The infinite proliferation of the hepatoma cells is one of the main difficulties for us to control the liver cancer, while the telomerase activation is the basis of unlimited proliferation. It has great significance to study the transformation function of the role of HBX on normal liver cells and its effect on telomerase activity, and new ideas for the treatment of liver cancer based on the telomerase will be coming.Objective:Observe the influence of hepatitis B virus X protein (HBX protein) on L02hepatocyte’s form and multiplication capacity, and its influence on hTERT mRNA L02cell express and telomerase activity.Methods:1.Normal hepatic cell L02was cultured in vitro cell culture.2.Constructed PEGFP-N1-HBX plasmid.3.Liposomes LipofectamineTM2000mediated stably transfected L02cell with the plasmid of PEGFP-N1-HBX.4.The expression of EGFP in L02cell after stability transfected was examined by fluorescence microscope.5.The expression of HBX protein in L02cells after stability transfected was detected by Western blotting analysis.6.Cellular morphology of L02cell which stability transfected PEGFP-N1-HBX plasmid were observated by electron microscope.7.The effect of HBX protein on the proliferation of L02cells was detected by MTT assay.8.The effect of HBX protein on the expression of hTERT mRNA in L02cells was detected by RT-PCR.9.The effect of HBX protein on the activity of telomerase in L02cells was detected by TRAP-PCR argentation.Results:1.We successfully constructed PEGFP-N1-HBX recombinant plasmid contains HBV X gene.2.The expression of EGFP fluorescence was observed in L02cells which stably transfected PEGFP-N1-HBX plasmid and PEGFP-N1plasmid,while the expression of HBX protein was detected in L02cells which stably transfected PEGFP-N1-HBX plasmid.3. L02cells with PEGFP-N1-HBX plasmid are of different sizes, whose forms are obviously naive, and had the trends of malignization.4. The multiplication capacity of L02cells, stably transfected by PEGFP-N1-HBX plasmid, increased obviously (P<0.05).5. The hTERT mRNA express of L02cells, stably transfected by PEGFP-N1-HBX plasmid, increased obviously, and the telomerase activity is activated.Conclusions:L02cells, stably transfected by PEGFP-N1-HBX plasmid, express the target protein HBX protein. HBX protein can induce normal hepatocyte’s vicious transformation, increase the express of hTERTmRNA in cells, and activate cell telomerase’s activity.