Establishment of a glioma cell line stably transfected with survivin specific targeting RNA interference vector

Objective To construct the specific RNA interference vector targeting human survivin gene and to establish a human glioma cell line stably transfected with the vector.Methods The specific RNA interference(RNAi) fragments targeting SVV and firefly luciferase gene were designed and synthesized,which were cloned into pSilencer 3.1-H1neo plasmid vector,and the specific RNA interference vector pSilencer 3.1-SVV targeting SVV gene and non-specfic sequence vector pSilencer 3.1-FLF were constructed.The pSilencer 3.1-SVV vector,pSilencer 3.1-FLF and blank pSilencer 3.1-H1neo vector were transfected respectively into U251 cells by lipofectamine 2000,and then the transfected cells were selected by G418.Expression of mRNA and protein of survivin were respectively investigated by real-time reverse transcription polymerase chain reaction,Western blot and immunocytochemistry methods.Results The specific RNA interference vector pSilencer3.1-SVV targeting survivin gene was constructed successfully.The stable transfectants U251-S,U251-F and U251-P were obtained.Expression of mRNA and protein of survivin were inhibited significantly in U251-S cells,whereas survivin gene expression levels were hardly changed in U251-F and U251-P cells.Conclusion Survivin gene expression can be suppressed markedly by specific RNA interference vector in U251 cells,and the current results have established the experimental foundation for the further study on the biological functions and its mechanisms of survivin in U251 cells.