| Background: In China, Hepatocellular carcinoma is one of the malignant cancers which possess very high morbidity and mortality, and early diagnosis is extremely difficult. Adenovirus is widely used as a transfer vector for gene therapy of cancers; it should be used to effectively express the therapeutic genes, and conjugation with clinical chemotherapeutic drugs to enhance the antitumor activity with exertion of a dual advantage of gene therapy and chemotherapy. Recent investigations have demonstrated that the development of tumorigenesis was attributed to epigenetic modification and abnormal expression of miRNA, and there are reciprocal modulations between the two regulatory mechanisms. Related research will provide a new thinking for molecular mechanisms of liver cancer.Aim: In this thesis, we will focus on the exploratory study of the inhibition growth of liver cancer cells, which is caused by Ad-miR122 combination Chemistry drug adriamycin or vincristine. At the same time we will construct the lentiviral vector with c-myc, in order to establish a suitable HCC cell line for study of mutual regulation between miRNA and Epigenetic mechanisms, and also lay the foundation for further development of molecular mechanisms of hepatocellular carcinoma.Methods: miR-122, a kind of liver-specific microRNA, is expressed exclusively in hepatic tissues and will play an important role on hepatic tissues development, stress response, lipid metabolism, infection and replication of several hepatic viruses. Evidences have shown that the expression level of miR-122 in hepatic tumors is lower than normal liver tissues, suggesting there is a relationship between abnormal expression of miR-122 and tumorogenesis. miR-122 targets an anti-apoptotic gene Bcl-w and cell cycle protein CyclinG1 in human HCC lines, and thus inhibits the growth of cancer cell. For more effective inhibition of hepatocellular carcinoma, we will adopt combined treatment of Ad-miR122 with adriamycin or vincristine, and evaluate the related function by in vitro experiments in several cancer cell lines. The reciprocal modulation between miRNA and epigenetic could inducetumorigenesis. However, such studies in liver cancer are still relatively small and constrained by many factors, thus the establishment of a suitable cell line will provide the basis for this study. In this thesis we successfully created a lentiviral vector suitable for oncogene c-myc expression, and took advantage of lentiviral vector who can integrate exogenous gene to the host genomic thus achieved stable and efficient gene expression, and established hepatocellular carcinoma cell line induced by normal liver cells and provide tools for subsequent related experiments.Results:Our experiments have shown that adenovirus-mediated miR-122 combined with chemotherapeutics can achieve a more significant inhibition of tumor cell growth, and furthermore, miR-122 could down-regulate MDR-1 mRNA expression and induce cell cycle arrest of cancer cells.And plasmid identification result has shown that we have successfully constructed some lentiviral vector system suitable for oncogene c-myc expression and packaged lentivirus by calcium phosphate transfection method.Conclusions:Adenovirus-mediated miR-122 combined with adriamycin or vincristine can significantly inhibit tumor cell growth, primarily by reducing MDR-1 expression or disrupting the normal cell cycle of tumor cells and leading to G2/M block.The results have shown that we have successfully constructed some lentiviral vector with c-myc and packaged lentivirus, and normal liver cell lines are necessary to go on follow-up research. |
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