Expression And Tumor Inhibition Of SIRT3in The Hepatocellular Carcinoma

Hepatocellular carcinoma is one of the most common maliganant tumor in the world and its mortality ranks third just behind gastric cancer, esophageal cancer. China is a high incidence of hepatccellular carcinor and the number of people died of hepatocellular carcinoma accounts for50%of its deaths worldwide. In the past twenty years, the level of the treatment of hepatocellular carcinoma has made great progress in China, but the morbidity and mortality is still in second place of cancer. The SIRT3which is one of the Sirtuins, silent infermatioa regulator is a major mitochondrial deacetylase and it was found as a cancer tumor suppress gene rencently. At persent, there is no reseach for the role of SIRT3in1hepatocellular carcinoma, and the reseach of the roles and functions of SIRT3in the hepatocellular carcinoma is of great significance. It would have a great significance for the diagnosis and treatment of hepatocellular carcinoma.Chapter I The expression and clinical significance of SIRT3in the hepatocellular carcinomaObjectiveTo observe expression of SIRT3in the hepatocellular carcinoma and analyze its significance.MethodsImmunohistochemistry and Western-blotting was performed to investigate the expression of SIRT3in10cases of normal liver tissue and32cases of primary liver cancer and its para-cancer tissue.Results(1) The immunohistochemical results:There was expression of STRT3observed in hepatocellular carcinoma tissue, with the rate of59.38%(19/32), while no strong expression was observed. There was expression and strong expression observed in para-cancer tissues with the rate of96.88%(31/32) and56.23%(10/10), while total expression and strong expression with the rate of70%(7/10) in normal tissue. A significant difference was found between the positive expression rate and average number of positive ratings of SIRT3in liver cancer and para-cancer, normal tissues (P<0.05). However, there was no significant difference between pare-cancer and normal tissues.(2) The Western-blotting results:the expression of SIRT3protein (SIRT3/GAPDH) in hepatocellular carcinoma tissues was significant lower than their pare-cancer tissues and normal tissues (P<0.05), and significant difference was not found between SIRT3protein expression level in pare-cancer and normal tissues(P>0.05).(3) The relation between SIRT3and clinical date:The expression of SIRT3in32cases of hepatocellular carcinoma showed significant relation hepatocellular carcinoma with portal vein tumor thrombus and cellular differentiation degree(p<0.05), and it was notassociated withsignificant relation with age, gender, AFP, tumor capsuleandthe size of tumor (p>0.05).Conclusion(1) SIRT3are highly expressed in normal liver and para-cancer tissues; SIRT3is lowly or not expressed in hepatocellular carcinoma tissues; Low expression or absence of SIRT3may play a role in the occurrence of liver cancer development process Chapter â…¡ The construction and identification of SIRT3gene eukaryotic expression vectorObjectiveTo build the SIRT3gene eukaryotic expression vector, transfect HepG2cells and determine the best test time point.MethodsThe SIRT3gene full-length sequence which was gotten by RT-PCR with the mRNA from normal liver tissue as a template was cloned to pZsGreen-C1vector and analyzed for sequence and digestion. The HepG2cells were divided into three groups:pZsGreen-cl-SIRT3HepG2group (pZsGreen-c1-SIRT3transfection group), pZsGreen-c1HepG2(experimental control group),HepG2group (blank control group), the recombinant plasmid was transfected to HepG2by using the liposomal transfection. The SIRT3expression of each group was identified Oh,24h,48h,72h and96h after transfection by Western-blotting and which the next best experimental time was determined.ResultsThe pZsGreen-c1-SIRT3eukaryotic expression vector was built and it was transfected to HepG2cells. The expression levels of SIRT3mRNA and SIRT3protein of pZsGreen-c1-SIRT3HepG2cell lines was higher than pZsGreen-C1HepG2group and HepG2group after48h (P<0.01)ConclusionThe pZsGreen-c1-SIRT3eukaryotic expression vector is built and identified. ObjectiveTo observe the proliferation and apoptosis of pZsGreen-c1-SIRT3HepG2cells, and to study the initial mechanism.MethodsRT-PCR and Western-bolting was used to detect the expression of SIRT3in HepG2and LO2cell lines. The groups, proliferation o pZsGreen-c1-SIRT3HepG2,pZsGreen-cl HepG2and HepG2cell lines was observed by the MTT and the apoptosis was detected by the AnnexinV/PI. RT-PCR and Western-bolting was used to detect the expression of SIRT3,Fas,Bax and P53in pZsGreen-cl-SIRT3HepG2, pZsGreen-c1HepG2and HepG2cell lines, while the MnSOD conten was detected by WST-1methods.Results(1) There was differences between expression of SIRT3, of IIepG2and Lo2cell lines by RT-PCR and Western-blotting(P<0.05).(2) MTT assay:There were no significant differences between each OD value after0h,24h and48h cultured in the regular and each cell of cells of pZsGreen-cl-SIRT3HepG2,pZsGreen-c1HepG2and HepG2 cell lines inhibition ratio (P>0.05);after48h, pZsGreen-c1-SIRT3HepG2cells grew slowly than the other two groups of cells(P<0.05), the cell group was the inhibition ratio was increased significantly (P<0.05); No significant difference was found between growth of blank control group (HepG2cell line) and experimental control group (pZsGreen-c1HepG2cell line)(P>0.05),as the same as the cell inhibition ratio (P>0.05).(3) The early apoptotsis and late apoptotsis stage cells of pZsGreen-c1-SIRT3HepG2cells group increased obviously than pZsGreen-c1HepG2and HepG2two cells lines with significant differences(P<0.01).(4) There was two no significant differences between each expression of SIRT3, Fas, Bax and P53of pZsGreen-cl HepG2and HepG2cell lines by RT-PCR and Western-blotting(P>0.01). The protein expression of pZsGreen-c1-SIRT3HepG2cell lines was significantly increased which had significant difference with their expression levels.(5) The MnSOD content which was detected by biochemical methods had no significant difference between pZsGreen-cl HepG2and HepG2cell lines(P>0.05),and the MnSOD content of pZsGreen-c1-SIRT3HepG2cell lines increased significantly than other groups(P<0.01).Conlusion(1) pZsGreen-c1-SIRT3inhibits the growth and induces apoptosis of HepG2;(2) SIRT3may increase the expression of P53and MnSOD which can increase the Bax and Fas to induce HepG2cell apoptosis. Total conclusion(1) SIRT3are highly expressed in normal liver and para-cancer tissues which is lowly or not expressed in hepatocellular carcinoma tissues, low expression or absence of SIRT3may play a role in the occurrence of hepatocellular carcinoma development process,(2) The pZsGreen-c1-SIRT3eukaryotic expression vector is built and identified,(3) pZsGreen-c1-SIRT3inhibits the growth and induces apoptosis of HepG2cell lines,(4) SIRT3may increase the expression of P53and MnSOD which can increase the Bax and Fas to induce apoptosis.