Study On Hepatoma Carcinogenesis And Mechanism Of Different Fragments Of Hepatitis C Virus Core Protein

Hepatocellular carcinoma (HCC) is the most common malignant tumor which severely harm to human health. Chronic infection with hepatitis C virus (HCV) poses a major risk towards the development of HCC. Core protein encoded by HCV is an important structure protein and interacts with numerous host cellular proteins. The purpose of present study is to discuss the hepatoma carcinogenesis and the mechanism of different fragments of hepatitis C virus core protein in order to summarize the general picture of carcinogenesis and provide theory basement for preventing HCC and developing safety HCV vaccine.Method: (1) Two groups of recombinant plasmids containing different fragment of HCV core protein 1-191 l-59aa 50-140 130-191aa or l-140aa were constructed utilizing the pEGFP-Cl and pcDNA3.1(+) respectively. (2) L02 and HEK293T cells were transiently transfected with pEGFP-Cl recombinant plasmids, and the localization of the expressed different fragments of core protein were observed with confocal laser microscopy. (3) After L02 cells were stablely transfected with pcDNA3.1(+) recombinant plasmids, the ultrastructural changes, the anchorage-independent growth manner and the tumor development in athymic nude mice of the core protein expressing cells were observed. (4) The different expressed genes between L02Core expressing full-length core protein and the control cells L02M were screened and identified by suppression subtraction hybridization (SSH). (5) The new isoforms of MLF1 were cloned using RACE technology.Results: (1) The localization of different fragments of the core protein is different, but there are no differences between in L02 cell line and in HEK293T cell line. The full-length core protein and its fragment 130-191aa completely localized in cytoplasm,whereas truncated core protein l-59aa exclusively localized in nucleus, and other fragments existed in both cytoplasm and nucleus. (2) The ultrastructural changes of the L02 cells expressing full-length core protein were observed obviously, including irregular nuclear, increased nuclearcytoplasmic ratio and mitochondria swelling etc. The slightly ultrastructure changes were observed in fragments 50-140aa and 130-191aa expressing L02 cells too, whereas no ultrastructural changes were observed in core fragments l-59aa and l-140aa expressing L02 cells. (3) In medium containing agar, the colony formation efficiency of L02 cells expressing the full-length core protein was much higher than others and the ability of colony formation of L02 cells expressing truncated core protein l-59aa was most feeble. (4) All above-mentioned L02 cells expressing fragments of core protein except fragment l-59aa can induce tumors in the nude mice without significant discrepancy. (5) 47 known genes and 8 unknown function genes were screened by SSH and analyzed by bioinformatics. (6) The full-length cDNAs of three new isoforms of MLF1 were cloned and nucleotide sequences were analyzed.Conclusions: In the anterior segment of HCV core protein (l-59aa) transfected L02 cells, there is no transforming potency observed, but both the intermediate and posterior segments of HCV core protein expressing L02 cells have the potency of carcinogenesis in vitro and in vivo. That lay promising foundation for developing safety HCV vaccine, whereby anterior segment of core protein is the most immunogenic and generally becomes HCV vaccine. Among the high expressed genes identified by SSH, those whose function has been known were classified into six groups, in particular, involving in cell migration, proliferation, antioxidation, material transport, signal transduction and cell metabolism respectively. It means that the carcinogenesis of HCV core protein is intimately connected with these six fields.