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Virus Oncogene Hbx And Cellular Oncogene RMP Cooperatively Promote The Growth Of Hepatocellular Carcinoma And Its Molecular Mechanism

Posted on:2014-11-24Degree:MasterType:Thesis
Country:ChinaCandidate:Q WangFull Text:PDF
GTID:2254330398496862Subject:Cell biology
Abstract/Summary:PDF Full Text Request
Objective:To explore the molecular mechanism of the virus oncogene HBx and cellular oncogene RMP cooperatively promote the growth of hepatocellular carcinoma.Methods:1. CCK-8assay was used to detect proliferation of5groups of cell lines.2. qRT-PCR and western blot was used to detect the mRNA and protein expression of HBx and RMP of each cell line.3. Flow cytometry was applied to detect the change of cell apotosis and cell cycle.4. qRT-PCR and western blot was used to detect the expression of the genes and protein associated with cell apotosis and cell cycle.5. Comet assay was used to detect the change of DNA damage after the irradiation of60Co y ray.6. The xenografts of hepatocellular carcinoma in nude mices were applied to observe the effects of HBx and RMP on xenografts formation.7. HE staining was applied to observe the morphological changes of transplant tumor cells, immunohistochemical staining was applied to detect the expression of HBx、RMP、AFP、Bcl-2、Bax and Bad in the xenografts.8. Immunohistochemical staining was applied to detect the expression of HBx and RMP in tissues of hunman hepatocellular carcinoma and adjacent to tumor.9. qRT-PCR and western blot was used to discuss the chang of the expression of endogenous RMP due to the HBx dose effect.10. Confocal laser scanning microscopy was used to observe the co-localization of HBx and RMP.11. Co-Immunoprecipitation was used to discuss the interaction of HBx and RMP.Results: 1. CCK-8assay showed that RMP and HBx co-expression promoted the poloferation of HepG2cells2. The mRNA and protein of HBx and RMP were successfully co-expressed in HepG2cell line.3. The results of flow cytometry demonstrated that after the irradiation of60Co γ ray the rate of G2phase arrest and apoptosis was reduced after the co-expression of RMP and HBx.4. qRT-PCR and western blot showed that the expression of Bcl-2、Cyclin B and CDK1was up-regulated and the expression of P53、Caspase3、Bax and p21was down-regulated.5. Comet assay demonstrated that RMP reduced the DNA damage induced by the irradiation of60Co y ray.6. The co-expression of RMP and HBx can promote the growth of the xenografts of hepatocellular carcinoma in nude mices.7. HE staining showed that HBx and RMP cooperatively promoted the proliferation of hepatoma cells, immunohistochemical staining showed that AFP、Bcl-2was up-regulated and Bax、Bad was down-regulated in the xenografts.8. Immunohistochemical staining showed that the expression of HBx and RMP was increased in the tissue of hunman hepatocellular carcinoma than the tissue adjacent to tumor.9. The HBx dose effect demonstrated that there was on significant interaction between HBx and RMP on mRNA and protein expression level.10. Confocal laser scanning microscopy showed that HBx and RMP mainly located in the cytoplasm and had a part of localization overlaped.11. Co-Immunoprecipitation demonstrated that HBx and RMP can form a protein complex partly.Conclusion:The study confirmed that HBx and RMP can cooperatively promote the proliferation of HepG2cells, ruduce the rate of apotosis and the G2/M phase arrest after the irradiation of60Co y ray through the change of key factors related to cell apotosia and cycle. They can promote the growth of the xenografts of hepatocellular carcinoma in nude mices and make hepatoma cells survive. The two factors may play an important role during the development of hepatocellular carcinoma.There was on significant interaction between HBx and RMP on mRNA and protein expression level, but they may form a protein complex partly and co-locate in the cytoplasm. These results showed that this kind of relationship between HBx and RMP may contribute a lot to the development of hepatocellular carcinoma.
Keywords/Search Tags:RMP, HBx, cell apoptosis, cell cycle, DNA damage
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