RNA干扰沉默缺氧诱导因子-1α对人乳腺癌细胞功能的影响

Effect of Silencing Hypoxia-inducible Factor-1α by RNA Interference on Human Breast Carcinoma Cell Line

目的研究靶向针对缺氧诱导因子-1α(HIF-1α)的短发夹RNA(shRNA)对人乳腺癌细胞系MCF-7细胞功能的影响。方法氯化钴模拟低氧环境。采用RNA干扰技术,构建针对HIF-1α的shRNA真核表达载体,转染MCF-7细胞。RT-PCR检测RNA干扰后MCF-7中血管内皮生长因子(VEGF)表达的变化。实时定量PCR和Westernblot技术分别检测HIF-1αmRNA和蛋白质水平。Sub-G1测定、AnnexinⅤ荧光标记和DNAladder检测细胞凋亡。流式细胞仪检测细胞周期的变化。结果缺氧后,MCF-7中的HIF-1α表达增加(P<0.01),而凋亡率较常氧降低(P<0.05)。shRNA转染MCF-7后,HIF-1α的表达下调91.63%(P<0.01)。阿糖胞苷诱导或无凋亡诱导剂时,干扰组凋亡率比未转染组分别增高1.75倍(P<0.01)和61.31倍(P<0.01)。与未转染组相比,干扰组中的VEGF下调66.8%(P<0.05)。干扰组细胞周期进程也较未转染组减缓。结论HIF-1α在MCF-7中发挥抗细胞凋亡的作用。本室构建的针对HIF-1α的shRNA能够促进MCF-7凋亡,下调VEGF表达,推迟细胞周期。

Objective To investigate the effect of short hairpin RNA （shRNA） targeting hypoxiainducible factor-1α（HIF-1α） on the human breast carcinoma MCF-7 cell line. Methods The hypoxia environment was achieved by treating ceils with cobalt chloride. The shRNA eukaryotic expression vector targeting HIF-1α was constructed, and transfected into MCF-7 ceils through lipofectamine 2000. Semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR） was used to study the expression of vascular endothelial growth factor （VEGF）. The mRNA and protein level of HIF-1α were detected by real-time PCR and Western blot. Sub-G1 apoptotic population analysis, Annexin V/PI binding assay, and DNA ladder analysis were applied to investigate the cell apoptosis. The cell cycle was detected by flow cytometry. Results The mRNA and protein level of HIF-1α increased after exposure of MCF-7 ceils to hypoxia （P 〈0. O1 ）. However, apoptosis was lower in hypoxia compared with normoxia （ P 〈0. 05 ）. The HIF-1 level of MCF-7 transfected with HIF-1α shRNA decreased approximately 91.63% （P 〈 0. 01 ）. When the cells were treated with or without apoptosis inducer Ara-C, the apoptosis of MCF-7 ceils transfected with HIF-1α shRNA increased by 1.75 times （ P 〈 0. 01 ） and 61.31 times （P 〈0.01 ） , respectively. The expression of VEGF in MCF-7 ceils transfected with HIF-1α shRNA decreased 66. 8% compared with untransfected cells （P 〈0.05 ）. Cell cycle progression was inhibited when the MCF-7 cells were transfected with HIF-1α shRNA. Conclusions HIF-1α plays an anti-apoptotic role in human breast carcinoma MCF-7 cell line. The shRNA we designed targeting HIF-1α in MCF-7 can promote cell apoptosis, inhibit the expression of VEGF, and delay cell cycle progression.