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High level expression of hepatitis C virus core protein (HCV-C) was detected in hilar cholangiocarcinoma tissues in our previous studies. This protein played an important role in the process of cancer cell inversion and proliferation, by some direct and indirect effects on certain genes. Based on this observation, we investigated the effect of HCV-C on human normal biliary epithelial (hBE) cell transformation and tumor development. Plasmid pHCV-C encoding the gene of HCV core protein was constructed and transfected into hBE cells. The expression and the biological effect of HCV-C in HCV-C gene-modified hBE cells were determined in vitro and in vivo. The clone formation rates of hBE cells transfected with pHCV-C, pcdna3.1 and mock-transfected cells were 36, 2.5 and 1.5%, respectively. Tumor developed in 7 of 7 nude mice after incubated with pHCV-C transfected hBE cells, while no tumor appeared in mice injected with pcdna3.1- and mock-transfected hBE cells. To investigate the possible mechanism of malignant transformation, we further studied the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in pHCV-C transfected hBE cells. The elevated expression of hTERT was confirmed by RT-PCR, immunocytochemistry (ICC) and Western blot analysis, which in turn elevated the telomerase activity, confirmed by TRAP-ELISA. These results indicated that HCV-C protein could participate in malignant transformation of human normal biliary epithelial cells and induce cholangiocarcinoma tumorigenesis, and the activation of telomerase was one of the possible mechanisms.

Aim: To explore the expression effect of mutated IkappaBalpha transfection on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells by inhibiting the activity of nuclear transcription factor-kappaB (NF-kappaB)., Methods: We used the mutated IkappaBalpha plasmid to transfect QBC(939)HCVC+ cells and QBC939 cells, and electrophoretic gel mobility shift assay (EMSA) to detect the binding activity of NF-kappaB DNA and the effect of the transfecting mutated IkappaBalpha plasmid on multidrug resistance gene (MDR-1) in hilar cholangiocarcinoma cells and its expression protein (P-GP)., Results: Plasmid DNA was digested by restriction enzymes Xbal and Hand III, and its product after electrophoresis showed two bands with a big difference in molecular weight, with a size of 4.9 kb and 1.55 kb respectively, which indicated that the carrier was successfully constructed and digested with enzymes. The radioactivity accumulation of QBC(939)HCVC+ and QBC939 cells transfected with mutated IkappaBalpha plasmid was significantly lower than that of the control group not transfected with mutated IkappaBalpha plasmid. Double densimeter scanning showed that the relative signal density between the tansfection group and non-transfection group was significantly different, which proved that the mutated IkappaBalpha plasmid could inhibit the binding activity of NF-kappaB DNA in hilar cholangiocarcinoma cells. Compared to control group not transfected with m IkappaBalpha plasmid, the expression level of MDR-1mRNA in the QBC939 and QBC939HCVC+ cells transfected with mutated IkappaBalpha plasmid was lower. The expression intensity of P-GP protein in QBC939 and QBC939HCVC+ cells transfected with mutated IkappaBalpha was significantly lower than that of the control group not transfected with mutated IkappaBalpha plasmid., Conclusion: The mutated IkappaBalpha plasmid transfection can markedly reverse the multidrug resistance of hilar cholangiocarcinoma cells. Interruption of NF-kappaB activity may become a new target in gene therapy for hilar cholangiocarcinogenesic carcinoma.

Objective: To study the effect of hepatitis C virus core protein (HCV-C) on human normal biliary epithelial cells (BEC) transformation and tumor development., Methods: BEC cells were transfected with plasmid pcDNA HCV-C (expressing HCV-C) by lipofectamine and selected in G418. The expression of HCV-C gene and protein was determined by PCR and immunohistochemical staining, respectively. Biological effect of transfected cells was observed through cell proliferation assay, anchor independent growth, and tumor development in nude mice. The expression of HCV-C protein in the induced tumor was evaluated by immunohistochemistry., Results: HCV-C was strongly expressed in BEC cells transfected with plasmid pcDNA HCV-C and the positive signal was located in cytoplasm. The HCV-C expression protein in the induced cytoplasm. Cell proliferation assay showed that the population doubling time in the pcDNA HCV-C transfected cells was much shorter than that in the pcDNA3 and non-transfected cells (14 h, 28 h, 30 h respectively). The cloning efficiencies of transfected cells with pcDNA HCV-C, pcDNA3 and non-transfected cells were 36%, 2.5% and 1.5%, respectively (P < 0.01). Tumor developed in nude mice inoculated with pcDNA HCV-C transfected cells after the inoculation. HE staining showed bile duct carcinoma character and immunohistochemistry confirmed HCV-C expression in the tumor tissue. The positive control group also showed tumor development, while no tumor mass obtained in the nude mice inoculated with pcDNA3 and non-transfected cells even 36 days after the injection., Conclusion: HCV-C protein showed human normal biliary epithelial cells transformation and tumorigenic features.