Inhibition of Survivin gene expression in human osteosarcoma cell line MG63

OBJECTIVE:To explore the blocking effect of siRNA on the expression of Survivin in gene in osteosarcoma cell line MG63.METHODS:According to Survivin cDNA coding sequence,the specific RNA interference(RNAi)fragments targeting Survivin gene were designed and synthesized,which were cloned into pSilencer 3.0-H1 neo plasmid vector,and the shRNA eukaryotic expression vector siRNA Survivin targeting Survivin gene was constructed.After the vector was constructed,MG63 cells were transfected with negative control vector siRNA neg or RNAi vectors and selected by G418.Expression of mRNA and protein of Survivin in the stable transfected cells was investigated seperately by RT-PCR,Western blot,immunofluorescence microscopy,and flow cytometry.RESULTS:The specific siRNA eukaryotic expression vector PsvA and PsvB targeting Survivin gene were constructed successfully.The stable transfectants containing negative control vector siRNA neg,PsvA and PsvB were obtained.Expression of mRNA and protein of Survivin was inhibited significantly in MG63/PsvB cells.Whereas Survivin gene expression levels were hardly changed in the other groups.Otherwise,MG63/PsvB had significant decreases in cell number compared with other transfectants.Analysis of DNA content in PsvB transfectants revealed a 7-fold increase in the fraction of sub-G1 peak(apoptotic peak)as compared with vector control and PsvA transfectants,under the same experimental conditions,P0.01.CONCUSION:Survivin gene expression can be suppressed markedly by specific shRNA in MG63 cells,which establishes the experimental foundation for further studying the biological functions and its mechanisms of Survivin in MG63 cells.