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目的研究缺氧诱导因子-1在人乳腺癌细胞系MCF-7细胞中对凋亡的作用。方法氯化钴(CoCl2)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。Real-time PCR和Western-blot技术分别检测HIF-1αmRNA和蛋白质水平。Sub-G1测定,AnnexinⅤ荧光标记和DNA ladder检测细胞凋亡。结果缺氧后MCF-7细胞HIF-1α的蛋白质水平表达增加,但缺氧后细胞的凋亡率比正常氧水平时低。实时定量PCR和Western blot结果证实转染短发夹RNA后,MCF-7细胞中HIF-1α基因表达被成功抑制。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明显增高。结论这项研究证明HIF-1在MCF-7中发挥抗细胞凋亡的作用,为针对HIF-1α的shRNA有效地用于乳腺癌的治疗提供新的思路。
Objective To investigate the effect of hypoxia inducible factor-1 (apoptosis) on human breast cancer cell line MCF-7. Methods Cobalt chloride (CoCl2) simulates hypoxia environment. The RNA interference technique was used to construct shRNA eukaryotic expression vector targeting HIF-1α and transfected into MCF-7 cells. Real-time PCR and Western-blot were used to detect HIF-1αmRNA and protein levels respectively. Sub-G1 assay, Annexin Ⅴ fluorescent labeling and DNA ladder detection of apoptosis. Results The protein expression of HIF-1α in MCF-7 cells was increased after hypoxia, but the apoptosis rate of HIF-1α cells in hypoxia was lower than that in normal oxygen. Real-time PCR and Western blot confirmed that HIF-1α gene expression in MCF-7 cells was successfully inhibited after transfection with short hairpin RNA. When induced by cytarabine or without induction of apoptosis, the apoptosis rate in the interference group was significantly higher than that in the untransfected group. Conclusions This study demonstrates that HIF-1 plays an anti-apoptotic role in MCF-7 and provides a new idea for the efficient targeting of HIF-1α shRNA for the treatment of breast cancer.