Silencing of survivin in glioma cell line U251 by short hairpin RNA expression vector pGenesilsurvivin targeting survivin

Objective To construct a short hairpin RNA(shRNA)expression vector encoding 3 shRNAs targeting survivin to down-regulate the expression of survivin gene in glioma cell line U251. Methods Three siRNA sequences of 19 nucleotides were derived from the full-length coding region of survivin gene and then 2 complementary oligonucleotides with 9 bases for loop sequence were designed for shRNA DNA templates.The shRNA templates were cloned into siRNA expression vector pG1,pG2 and pG3 respectively.The full sequences containing U6 promoter and other 2 shRNA DNA templates at the downstream were digested with corresponding restrict enzymes from recombinant pG2 and pG3 respectively and then cloned into recombinant pGenesil-1 to generate the shRNA expression vector pGenesil-survivin encoding 3 shRNAs targeting survivin.The recombinant pGenesil-survivin was transfected into U251 by Metafectene according to the protocol.Reverse transcriptase-polymerase chain reaction(RT-PCR)and Western Blotting were performed to analyze the interfering effect at the RNA level and protein level respectively.Results The recombinant pGenesil-survivin contained 3 siRNA targeting survivin as confirmed by DNA sequencing and survivin expression was significantly down-regulated by siRNA as validated by RT-PCR and Western Bloting.Conclusion RNA interfering(RNAi)mediated by the shRNA expression vector pGenesil-survivin could significantly down-regulate the expression of survivin in glioma cell line U251.