The Study On Suppression Of Mcl-1 Via RNA Interference Sensitizing Human Hepatocellular Carcinoma HepG2 Cells Towards Chemotherapy

PART ONE Construction of Recombinant Plasmids Contain shRNA Targeting Mcl-1 Gene and Their Effects on Cellular Growth and Apoptosis of Hepatoma Cell Line HepG2Objective To construct recombinant plasmids containing short hairpin RNA(shRNA) that targets the myeloid cell leukemia-1(Mcl-1) gene,to assay the expression of Mcl-1 and their effects on cellular proliferation and apoptosis of HepG2 cells transfected with recombinant plasmids.Methods Three shRNAs were designed according to the coding sequence of Mcl-1 gene, and cloned into the downstream of H1 promoter of psiRNA-hH1neo.Then the recombinant plasmids were transfected into HepG2 cells with lipofectamine2000.The Mcl-1 mRNA was measured with RT-PCR and Mcl-1 protein with Western Blot.The best effective silencing plasmid was screened,the cellular proliferation capacity was detected with MTT assay and the apoptotic state was analysed with flow cytometry.Results Three recombinant plasmids were successfully constructed and confirmed through DNA sequencing,then designated as pMclsi-1,pMclsi-2 and pMclsi-3 respectively.The expression of Mcl-1 mRNA and protein in HepG2 cells obviously decreased when transfecting with the plasmids,and the pMclsi-1 had the best silencing effect.,with the inhibitory efficiency of 93.7%at mRNA level and 86.4%at protein level.MTT assay indicated pMclsi-1 can inhibit the cellular proliferation, with the cell viability decreased from 70.7%24h after transfection to 53.67%48h after transfection,which obviously higher than control group(p＜0.05);and the apoptosis rate with flow cytometry increased from 6.5%24h after transfection to 15.6%48h after transfection,which obviously higher than control siRNA(3.9%,p＜0.05).Conclusion The constructed recombinant plasmids containing Mcl-1 shRNA can specifically block the Mcl-1 expression,and Mcl-1 gene may promote HepG2 cells' proliferation and inhibit their apoptosis. PART TWO The Study on Suppression of Mcl-1 via RNA Interference Sensitizing Human Hepatocellular Carcinoma Cells towards ChemotherapyObjective To study the effect on shRNA targeting Mcl-1 sensitizing human hepatocellular carcinoma cells towards chemotherapeutics mitomycin(MMC) or PI3K/Akt inhibitor LY294002.Methods The eukaryotic expression vector of psiRNA-Hhneo-Mcl-1 was transfected into the HepG2 cells by lipofectamin2000.The Mcl-1 protein by PI3K/Akt inhibitor LY294002 was measured with Western Blot.The cellular proliferation capacity and the apoptosis state with chemotherapeutics MMC,LY294002 or combined with this recombinant plasmid were detected with MTT assay and flow cytometry respectively.Results The expression of Mcl-1 protein in HepG2 cells obviously decreased when added LY294002.MTT assay indicated pMclsi-1 combined with MMC or LY294002 can significantly inhibit the cellular proliferation,obviously higher than MMC,LY294002 or transfected pMclsi-1 alone;and the apoptosis rate with flow cytometry obviously increased compared to MMC,LY294002 or transfected pMclsi-1 alone.Conclusion LY294002 can inhibit Mcl-1 protein expression,which indicate the expression of Mcl-1 in HepG2 cells may be correlate to PI3K/Akt path.Suppression of Mcl-1 via RNA interference can sensitize human hepatocellular carcinoma cells towards chemotherapeutics MMC or PI3K/Akt inhibitor LY294002. PART THREE Study on the Expression of Mcl-1 in Human Hepatocellular Carcinoma and its Relation with p53Objective:To investigate the expression of Mcl-1 in human hepatocellular carcinoma cells (HCC) and tissues.To identify the relationship between Mcl-1 and p53.Methods:HCC specimens of 30 patients were examined by immunohistochemistry for Mcl-1 and p53 expression.RT-PCR was used to detect the expression of Mcl-1 gene in various hepatocellular carcinoma cell lines HepG2,Hep3B,SMMC7721 and hepatic cell line L02.Western Blot was used to detect the expression of Mcl-1 and p53 protein in HepG2,Hep3B and L02 cell lines.Finally,changes of Mcl-1 or p53 expression in various HCC cell lines were examined when transfected with Mcl-1 siRNA,the Mcl-1 expression plasmid or the wide-type p53 expression plasmid.Results:Mcl-1 protein was enhanced in HCC tissues compared to adjacent non-tumor tissue,p53 protein was also enhanced in HCC tissues and there was a significant correlation with Mcl-1.In addition,Mcl-1 was prominently expressed in HepG2 and Hep3B cells, weakly expressed in SMMC7721 cells,and no significantly expressed in L02 cells.The expression of p53 was not influenced by Mcl-1 siRNA in HepG2 cells or transfected with the Mcl-1 expression plasmid in L02 cells.Furthermore,the expression of Mcl-1 was also not significantly changed when transfected with the wild-type p53 expression plasmid in Hep3B cells.Conclusions:Mcl-1 was overexpressed in HCC tissues,and its expression correlate to p53 expression.However,Mcl-1/p53 interaction may not through regulation of expression of Mcl-1 or p53,but through regulation of their function.