The effect of Smac/DIABLO associating with Survivin shRNA on the cell growth and apoptosis of Lovo / 中国癌症杂志

ABSTRACT

Background and purpose:The Smac/DIABLO(the second mitochondrial derived activator of caspase/direct IAP binding protein with low pI)is a new kind of mitochondria protein,it inhibits IAPs(inhibitors of apoptosis protein),including Survivin,and activates caspase-9 and caspase-3,accordingly promotes apoptosis.In this study,we investigated the effect of over-expressing mitochondrial protein-Smac/DIABLO while silencing inhibitor of apoptosis protein-Survivin on the cell growth,cell cycle and apoptosis of human colon cancer cells through cotransfecting both genes.Methods:Constructed survivin-shRNA-EGFP plasmid was transected with Smac-pcDNA3.1 plasmid into Lovo cells at either half each dose or total dose,respectively.Western blot was used to survey the protein level of Smac and Survivin;Hoechst 33258 staining was used to detect the karyomorphological diversity of apoptotic cells and evaluate apoptotic ratio roughly;PI staining and flow cytometry analysis were used to examine cell cycle;caspase-3 Detection Kits was used to detect the activity of caspase-3.Results:The expression of Smac protein increased but Survivin protein decreased 48 hr after transfection.Karyomorphological diversify of apoptotic cells were obviously observed by hoechst 33258 staining.The apoptosis rate in Smav+Survivin shRNA group was(18.5?1.7)%,which was higher than that in Smac group(9.6?1.8)% and Survivin shRNA group(15.0?0.3)%,and all of three groups were significantly higher than the control groups;The cells of G0/G1 phase increased to(51.0?6.2)% in Smac group,and the cells of S stage increased to(53.3?1.3)% in Survivin shRNA group(P