Role of short hairpin RNA targeting survivin in apoptosis and G2/M cell cycle arrest in glioma cell line U251

    XU Ruª²Xiang, TU Yanª²Yang, JIANG Xiaoª²Dan, FENG Jiangª²Nan

    Department of Neurosurgey, Guangdong Provincial Institute of Neuromedicine, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China, Gensil Bioª²technology Limited Company of Wuhan, Wuhan 430074, China

    ¡¾Abstract¡¿ AIM: To construct a recombinant short hairpin RNA (shRNA) expression vector targeting survivin and investigate apoptosis and cell cycle arrest in glioma cell line U251 induced by survivinª²targeting small interfering RNA (siRNA). METHODS: Three siRNA sequences of 19 nucleotides were derived from the fullª²length 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 pGenesilª²1 to generate survivinª²targeting siRNA expression vector pGenesilª²1/survivin encoding 3 shRNAs targeting survivin by digestion and ligation step by step. pGenesiª²l/survivin was transfected into U251 by Metafectene. Realª²time quantitive PCR and Western blotting were performed to validate the interfering efficacy of survivin at mRNA level and protein level respectively. Annexinª²V/PI double staining by flow cytometry analysis was used to evaluate apoptosis. PI staining was conducted to evaluate cell cycle arrest. RESULTS: The expression of survivin at both RNA level and protein level were significantly downª²regulated as validated by realª²time quantitive RTª²PCR and Western bloting. Flow cytometry analysis revealed glioma cell line U251 suffered a notable apoptosis and G2/M arrest after RNA interference mediated by siRNA targeting survivin. CONCLUSION: RNA interference (RNAi) mediated by the siRNA expression vector pGenesiª²l/survivin could significantly downª²regulate the expression of survivin and induce remarkable apoptosis and G2/M cell cycle arrest in glioma cell line U251. ......