乏氧诱导因子-1α基因沉默对肺癌细胞乏氧应答相关基因表达的影响  

Identification of Genes Involved in Adaptive Response to Hypoxia and Their Expression Attenuations by Gene Silencing of HIF-1α in A549 Lung Cancer Cells

作　　者：宋现让[1] 宋宝[1] 魏玲[1] 柳永蕾[1] 王兴武[1] 

机构地区：[1]山东省肿瘤医院基础研究中心,济南250117

出　　处：《中国生物化学与分子生物学报》2007年第10期864-869,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金资助(No.30570547)~~

摘　　要：乏氧诱导因子-1α(HIF-1α)是肿瘤细胞适应乏氧微环境的关键调控因子,具有作为治疗靶基因的潜力,以克服乏氧诱导的治疗抗拒等效应.下调其表达可能影响肿瘤细胞内一系列乏氧应答相关基因的表达.本研究采用已构建的HIF-1αRNAi慢病毒载体转导肺腺癌A549细胞,经杀稻瘟素(blasticidin)筛选建立HIF-1α基因稳定沉默的A549细胞株.应用cDNA微阵列技术检测并比较HIF-1α基因沉默A549细胞株和其亲本细胞株在常氧和乏氧状态下的基因表达谱改变.应用定量RT-PCR方法验证部分cDNA芯片差异表达基因的表达改变.HIF-1α基因稳定沉默细胞株A549/HIF-1α(-),在常氧和乏氧条件下HIF-1α mRNA水平分别较A549细胞下降89.2%和88.1%,HIF-1α蛋白水平分别下降97.2%和88.4%.在乏氧条件下,cDNA微阵列检测的1280个基因中,52个基因表达上调,15个基因表达下调.HIF-1α基因沉默显著影响其中27个基因的乏氧诱导效应.定量RT-PCR验证其中ENO2、BCL-2、CXCR4和MMP11的表达水平,与cDNA芯片结果相符合.结果提示,HIF-1α基因沉默能够在一定程度上阻断肺癌细胞的乏氧应答,在克服乏氧导致的肺癌治疗抗拒方面具有潜力.Hypoxia inducible factor-1α （HIF-1α ）is a key regulating factor in the adaptive response of cancer cells to hypoxia and may be a potential therapeutic target to neutrolize the impacts induced by hypoxia, such as increased aggressiveness and therapeutic resistance. A HIF-1 a silenced stable A549 cell line A549/HIF-1α（-） was constructed by transducing A549 ceils with HIF-1α specific RNAi lentiviral vectors and screened by blasticidin, cDNA microarray analyses with 1 280 tumor-related genes were performed in A549/HIF-1α（-） and A549 under normoxic and hypoxic conditions. Differentially expressed genes were selectively verified using quantitative RT-PCR. When incubated in 19% O2 or 0.5% O2 ,the level of HIF-1α mRNA in A549/HIF-1α （-） was decreased by 89.2% and 88.1% ,respectively, compared to A549, and the HIF-1α protein level was decreased by 97.2% and 88.4%. eDNA microarray analyses have shown that hypoxia incubation up-regulated 52 genes and down-regulated 15 genes in all 1 280 tested genes. The silencing of HIF-1α had blocked the changes of 27 genes induced by hypoxia, and among them, ENO2, BCL-2, CXCR4 and MMP11 were subsequently verified by quantitative RT-PCR. The results indicated that the silencing of HIF- 1 α could disrupt the adaptive response to hypoxia in lung cancer cells and might be used as a potential targeted intervention to overcoming hypoxia-induced therapeutic resistance.

关 键 词：乏氧诱导因子-1Α RNA干扰 A549细胞 乏氧应答 

分 类 号：R734.2[医药卫生—肿瘤]