缺氧诱导因子-1α对A549细胞株中生存素表达及其生物学特性的影响

Effect of hypoxia inducible factor-1α on the biological and the transcription of survivin in A549 cells

目的观察非小细胞肺癌细胞株A549中缺氧诱导因子-1α(HIF-1α)对生存素转录的调控作用及对A549细胞增殖、凋亡及迁移的影响。方法用凝胶电泳迁移率实验(EMSA)检测生存素启动子序列与核蛋白结合的情况。EMSA实验分为阴性对照反应组、结合反应组、探针冷竞争反应组、突变探针的冷竞争反应组和特异性抗体反应组。应用脂质体将载体pcDNATM6.2-GW/EmGFP miRNA HIF-1α质粒和阴性(错义链)、内参(β-肌动蛋白)微小RNA(miRNA)质粒转染A549细胞。转染分为未处理组(正常A549细胞)、HIF-1α沉默组(转染HIF-1αmiRNA质粒)、阴性对照组和内参组。杀稻瘟菌素筛选阳性克隆扩大培养;用逆转录-聚合酶链反应(RT-PCR)和蛋白免疫印迹法筛选出对A549细胞干扰效果最佳的miRNA HIF-1α干扰载体;常氧、缺氧条件下检测各组中HIF-1α、生存素在mRNA和蛋白水平的变化;用细胞计数法、流式细胞术和转移小室迁移试验检测各组细胞增殖、凋亡及迁移细胞数。结果 (1)γ-32P标记的生存素启动子序列-26^-9 bp与A549细胞核蛋白作用,该条带可与HIF-1α抗体结合产生特异性抗体结合条带;(2)常氧下HIF-1α沉默组的HIF-1α和生存素mRNA分别为0.313±0.051和0.060±0.008,明显低于未处理组的1.042±0.036和1.071±0.016(F=436.433,0.002,均P<0.05);HIF-1α和生存素的蛋白含量分别为0.559±0.051和0.051±0.003,未处理组分别是1.452±0.146和0.194±0.007,两组比较差异有统计学意义(F值为53.525和331.646,均P<0.05)。缺氧条件下HIF-1α沉默组中HIF-1α、生存素的mRNA和蛋白分别为0.604±0.040、0.394±0.018、0.997±0.026、0.172±0.006,明显受到抑制(mRNA的t值分别为7.809和-28.936,均P<0.01;蛋白的t值分别为13.523和-14.202,均P<0.01);常氧条件下,转染miRNA后A549细胞增殖(24 h为0.273±0.008)无明显变化,但迁移细胞数(30±6)比未处理组(121±17)减少;缺氧条件下,转染miRNA后,A549细胞生长速度(24 h为0.273±0.008)减慢,细胞体外的迁移细胞数(75±4)减少(均P<0.05)。结论 A549细胞中生存素核心启动子上存在HIF-1α结合位点;miRNA沉默A549细胞HIF-1α表达后,可有效下调生存素基因表达,常氧条件下仅抑制细胞迁移,缺氧条件下不仅促进细胞凋亡,还可抑制细胞增殖和迁移。

Objective To evaluate the effect of hypoxia inducible factor-1α（HIF-1α） on the transcriptional regulation of survivin and the proliferation, apoptosis and migration of A549 cells. Methods The binding of nuclear protein to the survivin promoter sequences was detected using electrophoretic mobility shift assay（EMSA）. It was divided into 5 groups in EMSA： negative control reaction, binding reaction, probe cold competing reaction, mutant probe cold competing reactionSuper-shift reaction. The plasmid pcDNATM6.2-GW/EmGFP miRNA HIF-1α was stably transfected into adenocarcinoma of lung A549 cell by LipofectamineTM 2000. Cells were divided into 4 groups in trsnsfection： untreated group, HIF-1α miRNA group, negative control group, β-actin group.The expression of HIF-1α and survivin in A549 cells were detected by RT-PCR and Western blot, to screen the best silencing vector A549/HIF-1α-miRNA. The transfected cells were exposed to normoxia and hypoxia, cell proliferation, apotosis and migration were measured by CCK8, flow cytometry（FCM） and transwell chambers methods. Results DNA-neucleoprotein bands were observed when A549 nuclear extracts incubating with the γ-32P labeled 18-bp probe（nucleotides-26to-9） of survivin promoter in EMSA assay. The specific bands were competed away by the cold 18-bp probe, but not the mutated cold probe in competition assay. The bands could bind the antibody of HIF-1α, exhibited in super-shift reaction. Under the normal condition, in the HIF-1α miRNA group, the mRNA of HIF-1α and survivin were 0.313±0.051 and 0.060±0.008, respectively, which were significantly decreased compared with those of the control group（1.042±0.036 and 1.071±0.016, respectively）（F=436.433, 0.002, all P0.0.5）. HIF-1α and survivin protin in the HIF-1α miRNA group（0.559±0.051 and 0.051±0.003） were significantly lower than those in control group（F=53.525 and 331.646, all P0.05）.Under hypoxia, HIF-1α, survivin mRNA and protein in the transfection of HIF-1α miRNA, were significantly restraining（0.604±0.040, 0.394±0.018, 0.997±0.026 and 0.172±0.006）, mRNA： t=7.809,-28.936, P0.01; protein： t=13.523,-14.202, P0.01. Under the normal condition, transfection of A549 cells with HIF-1α miRNA resulted in no changes in the proliferation, and apoptos in the of A549 cells, however, the migration of the cells（30±6） was substantially suppressed by HIF-1α silence compared with the control group（121±17）. Under hypoxia, transfection of cell with HIF-1α miRNA significantly inhibited cell proliferation（0.273±0.008）（F=8.048, P0.01）. The apoptotic ratio of transfected cells under hypoxia was also higher than that of control（F=26.359, P0.01）. The cell migration was also suppressed by HIF-1α silence under hypoxia（F=21.924, P0.05）. Conclusions There might be a binding site（nucleotides-26 to-9 bp） for HIF-1α in survivin promoter sequence of A549 cells. The recombinant plasmid HIF-1α miRNA effectively inhibits the expression of HIF-1α as well as survivin in A549 cells. HIF-1α silence can inhibit the cell migration under normoxia, and can＇t only promote cell apoptosis, but inhibit cell proliferation migration under hypoxia.