短发夹RNA靶向抑制suvivin基因在人胶质瘤细胞U251中的表达  

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

作　　者：涂艳阳[1] 徐如祥[1] 王向宇[1] 姜晓丹[1] 封江南 

机构地区：[1]南方医科大学珠江医院神经外科,广东省神经医学研究所,广州510282 [2]湖北省武汉市晶赛生物工程技术有限公司,430074

出　　处：《中华神经医学杂志》2006年第6期579-582,共4页Chinese Journal of Neuromedicine

基　　金：国家自然科学基金(30170961)

摘　　要：目的构建表达靶向抑制survivin基因的三个短发夹结构(shRNA)的RNA干扰载体,使其在胶质瘤细胞发挥干扰效应。方法在survivin全长序列中选取设计3条19个核苷酸靶序列,2条反向重复序列,间以9个核苷酸的茎环序列,加上对应酶切位点,形成3条shRNA的DNA模板,分别克隆到3个干扰载体pG1,pG2和pG3上：采用分布酶切连接的方法,分别将U6启动子以及下游的后2个shRNA模板酶切下来,依次连接到pG1对应酶切位点,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-survivin,测序鉴定：将干扰质粒导入到胶质瘤细胞U251,分别采用RT-PCR以及Western Bloting从mRNA和蛋白水平检测干扰后效果。结果重组的干扰载体含有3条正确的shRNA模板；RT-PCR以及Western Blotting检测显示,survivin的mRNA转录水平以及蛋白水平的表达均得到显著抑制。结论编码3条shRNA的干扰载体pGenesil-survivin介导的RNA干扰技术(RNAi)可以显著的靶向抑制survivin基因在人胶质瘤细胞U251中的表达。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 giioma cell line U251.

关 键 词：短发夹RNA SURVIVIN基因 神经胶质瘤 

分 类 号：R739.4[医药卫生—肿瘤]