短发夹RNA靶向抑制suvivin基因对人胶质瘤U251细胞凋亡和细胞周期的影响

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

目的构建表达靶向抑制survivin基因的表达短发夹结构(shRNA)的RNA干扰载体,导入人胶质瘤细胞U251中,研究靶向抑制survivin基因对U251细胞的凋亡诱导以及细胞周期阻滞作用.方法在survivin全长序列中选取设计3条含19个核苷酸(19nt)靶序列两条反向重复序列,中间以9个核苷酸的茎环序列,两端分别加上对应的酶切位点,分步酶切连接,构建出含有3条shRNA模板且能独立编码shRNA的重组干扰载体pGenesil-1/survivin;采用Metafectene转染试剂将干扰质粒导入到胶质瘤细胞U251;采用荧光定量PCR以及Westernbloting分别从mRNA和蛋白水平检测干扰效果;采用Annexin-V/PI双色标记的流式细胞仪法检测siRNA诱导的细胞凋亡,PI染色检测细胞周期阻滞.结果实时荧光定量PCR以及Westernblotting检测显示,survivin基因的mRNA转录水平以及蛋白水平的表达均得到显著抑制;流式细胞仪检测分析显示,survivin基因表达被抑制后,U251细胞凋亡率明显增高,细胞周期出现明显的G2/M阻滞.结论靶向survivin基因的重组siRNA干扰载体pGenesi-l/survivin介导的RNAi显著靶向抑制了survivin基因在人胶质瘤细胞U251中的表达,并明显地诱导了U251细胞发生凋亡和G2/M期细胞周期阻滞.

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 survivin-targeting small interfering RNA （siRNA）. 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 pGenesil-1 to generate survivin-targeting siRNA expression vector pGenesil-1/survivin encoding 3 shRNAs targeting survivin by digestion and ligation step by step. pGenesil/survivin was transfected into U251 by Metafectene. Real-time quantitive PCR and Western blotting were performed to validate the interfering efficacy of survivin at mRNA level and protein level respectively. Annexin-V/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 downregulated as validated by real-time quantitive RT-PCR 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 pGenesi-l/survivin could significantly down-regulate the expression of survivin and induce remarkable apoptosis and G2/M cell cycle arrest in glioma cell line U251.