HIF-1α对乳腺癌MCF-7细胞增殖的影响及其机制探讨

Effect of HIF-1α on the proliferation of breast cancer MCF-7 cells and the mechanism

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。

Objective To observe the effect of hypoxia-inducible factor-1 α （HIF-1 α） 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-1 α mRNA in transfected cells was detected by real-time RT-PCR to select HIF-1 α 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-1 α shRNA-4 was 74％ and had the highest interference efficiency （all P ＜ 0.05）.HIF-1 α-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-1 α 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.