Effects of siRNA on the cell cycle of osteosarcoma and the survivin gene expression in vitro / 解放军医学杂志

ABSTRACT

Objective To explore the effects of siRNA on survivin gene expression in osteosarcoma cell line MG63 using siRNA eukaryotic expression vector,and on the cell cycle of osteosarcoma.Method The specific RNA interference(RNAi)fragments targeting survivin gene were synthesized,and they were cloned into pSilencer 3.0-H1 neo plasmid vector.After the vector was constructed,MG63 cells were transfected with negative control vector or RNAi vectors and selected by G418.Expression of protein of survivin in the stable transfected cells was determined by Western blot.These cells which were cultured on cover slips were observed with electron microscopy.Apoptosis analysis was performed with flow cytometry and Hoechst staining.Results Specific siRNA eukaryotic expression vector PsiS targeting survivin gene was constructed successfully.The stable transfectants containing negative control vector and PsiS were obtained.Protein expression of survivin was inhibited significantly in MG63/PsiS cells,whereas survivin gene expression levels were hardly changed in normal MG63 and negative control cells.There were much more pyknotic nuclei found in MG63/PsiS pool than those in MG63 and control.Analysis of DNA content in PsiS transfectants revealed a 7-fold increase in the fraction of sub-G1 peak(apoptotic peak)as compared with other transfectants under the same experimental conditions(P