Specific inhibition of survivin gene expression by short hairpin RNA in U251 cells

Objective To construct the specific short hairpin RNA (shRNA) eukaryotic expression vector targeting human survivin gene, further to observe its effect on inhibiting survivin gene expression in human brain glioblastoma U251 cells.Methods According to survivin cDNA coding sequence, the specific RNA interference (RNAi) fragments targeting survivin gene were designed and synthesized, which were cloned into pWH1 plasmid vector, and the shRNA eukaryotic expression vector pWH1-SR targeting survivin gene was constructed. The pWH1-SR vector and blank pWH1 vector were transfected respectively into U251 cells by lipofectin medium, and the stable transfectants U251-SR and U251-P cells were established through selecting with G418. Expression of mRNA and protein of survivin was investigated by RT-PCR, Western blot and immunohistochemistry methods respectively in U251, U251-P and U251-SR cells.Results The specific shRNA eukaryotic expression vector pWH1-SR targeting survivin gene was constructed successfully, which was identified by restriction endonuclease digestion. The stable transfectants U251-SR and U251-P cells respectively containing pWH1-SR vector and blank pWH1 vector were obtained. Expression of mRNA and protein of survivin was inhibited significantly with inhibitory rates of 87% and 91% respectively in U251-SR cells, whereas survivin gene expression levels were hardly changed in U251-P cells.Conclusion Survivin gene expression can be suppressed markedly by specific shRNA in U251 cells, the current results establishing the experimental foundation for further studying the biological functions and its mechanisms of survivin in U251 cells.