【摘要】 目的观察缺氧诱导因子1α(HIF-1α)基因对乳腺癌MCF-7细胞增殖的影响,并探讨其可能的机制。方法构建并筛选HIF-1α基因的RNAi表达质粒,以脂质体LipofectamineTM2000介导转染MCF-7细胞。转染后48h,采用实时定量RT-PCR技术检测转染细胞中HIF-1αmRNA的转录水平,筛选有效的HIF-1αRNAi质粒;CCK-8法比较转染前后乳腺癌细胞增殖变化,实时定量RT-PCR检测顺滑蛋白(SMO)mRNA表达。结果成功构建含HIF-1α短发夹状RNA(shRNA)1～4的RNAi表达质粒,其中HIF-1αshRNA-4干扰抑制效率为74%,干扰效果最强(P均<0.05)。HIF-1αshRNA-4干扰48、72、96 h后,MCF-7细胞的生长抑制率明显升高(P均<0.05)。HIF-1αshRNA-4转染后MCF-7细胞SMO mRNA的相对表达量为0.56±0.06,低于转染前的1.07±0.16(P<0.05)。结论 HIF-1α表达可促进乳腺癌细胞增殖,可能与其参与调控SMO的表达有关。更多还原

【Abstract】 Objective To observe the effect of hypoxia-inducible factor-1α( HIF-1α) on the proliferation of breast cancer MCF-7 cells and to investigate the mechanism. Methods RNAi expression plasmids of HIF-1α were constructed and selected,and the breast cancer MCF-7 cells were transfected by the mediation of LipofectamineTM 2000. After 48 h,the transcription level of HIF-1α mRNA in transfected cells was detected by real-time RT-PCR to select HIF-1α RNAi plasmid with high interfering efficiency. CCK-8 kit was used to observe the changes in the proliferation of breast cancer cells before and after transfection,and real-time RT-PCR was used to detect the expression of Smathened protein(SMO) mRNA. Results pGPU6 / GFP / Neo / HIF-1α-shRNA1-4 plasmids were successfully constructed,the interference rate of HIF-1α shRNA-4 was 74% and had the highest interference efficiency(all P < 0. 05). HIF-1α-shRNA-4 significantly inhibited the proliferation of MCF-7 cells after interference for 48 h,72 h and 96 h( all P < 0. 05). The expression level of SMO mRNA decreased after HIF-1α shRNA-4 transfection in MCF-7 cells(0. 56 ± 0. 06 vs. 1. 07 ± 0. 16)( P < 0. 05). Conclusion The expression of HIF-1α can promote the proliferation of breast cancer cells,which might be closely associated with the regulation of SMO expression.更多还原