靶向乏氧诱导因子-1α和生存素基因的RNA干扰腺病毒穿梭载体的构建  

Construction of RNAi adenoviral shuttle vector targeting both HIF-1α and survivin synchronously

作　　者：郭彩霞[1] 李艳博[2] 孙婷[3] 杨巍[4] 孙志伟[1] 龚守良[2] 

机构地区：[1]吉林大学公共卫生学院卫生毒理学教研室,吉林长春130021 [2]吉林大学公共卫生学院卫生部放射生物学重点实验室,吉林长春130021 [3]吉林大学基础医学院生物化学教研室,吉林长春130021 [4]苏州大学放射医学与公共卫生学院放射生物学教研室,江苏苏州215123

出　　处：《中国现代医学杂志》2009年第20期3041-3045,共5页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30600160)

摘　　要：目的构建能同时干扰乏氧诱导因子-1α(hypoxia induced factor1α,HIF-1α)和生存素(survivin)基因表达的RNA干扰(RNA interference,RNAi)腺病毒穿梭载体,并检测其对人肝癌SMMC-7721细胞HIF-1α基因和生存素基因表达的干扰作用。方法根据GeneBank数据库中HIF-1α和生存素基因的cD-NA序列设计干扰序列,构建干扰HIF-1α和生存素基因的重组单基因干扰质粒pGenesil-HIF-1α和pGene-sil-survivin。酶切、测序鉴定正确后,干扰质粒经脂质体介导转染SMMC-7721细胞,应用逆转录-聚合酶链式反应(RT-PCR)检测HIF-1α和生存素基因的mRNA表达水平。将两个单基因干扰质粒的干扰序列和U6启动子序列切下并连入腺病毒穿梭载体pshuttle,构建双基因干扰载体pshuttle-Genesil-survivin-HIF-1α,并检测其对HIF-1α和生存素基因mRNA表达的干扰作用。结果测序和酶切鉴定结果显示,pGenesil-HIF-1α和pGenesil-survivin重组质粒构建正确。SMMC-7721细胞分别被pGenesil-HIF-1α和pGenesil-survivin转染48h后,HIF-1α和生存素基因mRNA水平与正常和阴性对照组相比明显降低。双基因干扰载体pshut-tle-Genesil-survivin-HIF-1α酶切鉴定所得片段大小与预期相符,转染该质粒48h后,SMMC-7721细胞中HIF-1α和生存素基因mRNA的表达明显降低。结论成功构建能同时干扰HIF-1α和生存素基因的腺病毒穿梭载体,可有效地同时抑制SMMC-7721细胞内HIF-1α和生存素基因mRNA的表达。[Objective] To construct RNAi adenoviral shuttle vector targeting both HIF-1α and survivin synchronously and detect its silencing effect on HIF-1α and survivin genes in human hepatocarcinoma SMMC-7721 cell line. [Methods] Two pairs of RNA interfering sequence targeting HIF-1α or survivin gene were chosen and designed respectively according to their cDNA sequence showed in GeneBank. Then the RNAi plasmids targeting HIF-1α and survivin gene were constructed respectively, which were transfected to SMMC-7721 cells by the liposome-mediated pathway after identified by enzyme digestion and sequencing. The mRNA expressions of HIF-1α and survivin genes were detected by reverse transcriptase- polymerase chain reaction （RT-PCR）. U6 promoter and RNAi sequence in both RNAi plasmids were cut and ligated to the adenoviral shuttle vector pshuttle to generate pshuttle-Genesil- survivin-HIF-1α which was identified by enzyme digestion, and its effect on gene silence were detected. [ Results ] Enzyme identification and sequencing showed that the single-gene RNAi plasmids were constructed correctly.As compared to that in the normal and negative control groups, the mRNA expressions of HIF-1α and survivin in SMMC-7721 cells were decreased in 48 hours after transfectcd pGenesil-HIF-1α and pGenesil-survivin respectively. The fragment of the double-gene RNAi plasmid pshuttle-Genesil-survivin-HIF-1α acquired by enzyme digestion was coincident with the anticipated result, and HIF-1α and survivin mRNA levels in SMMC-7721 cells decreased significantly 48 hours after being transfected by pshutfle-Genesil-survivin-HIF-1α [ Conclusion] The RNAi adenoviral shuttle vector targeting both HIF-1α and survivin synchronously was constructed successfully, and it could inhibit mRNA expressions of HIF-1α and survivin genes obviously.

关 键 词：RNA干扰 腺病毒 乏氧诱导因子-1Α 生存素 

分 类 号：R730.54[医药卫生—肿瘤]