缺氧对人乳腺癌MCF-7细胞miRNA-21表达的影响及与细胞增殖和凋亡的关系

Influence of Hypoxia on the Expression of miRNA-21 in Human Breast Cancer MCF-7 Cell and Its Relationship with Cell Proliferation and Apoptosis in Vitro

[目的]观察缺氧环境下人乳腺癌MCF-7细胞增殖及凋亡的情况,初步探讨缺氧对miRNA-21表达的影响及其与细胞增殖、凋亡的关系。[方法]CoCl2人工模拟缺氧环境,四甲基偶氮唑蓝比色法(MTT)检测缺氧环境下细胞增殖状况,流式细胞仪检测细胞的凋亡率,实时荧光聚合酶链反应(RT-PCR)测定不同缺氧程度下HIF-1α和miRNA-21表达水平的变化。[结果]与常氧对照组相比较,CoCl2处理组的细胞明显受到抑制,但100、200μmol/L CoCl2处理组的细胞在一定时间内仍增殖,抑制率随缺氧时间和药物浓度增加而上升。流式细胞仪检测结果显示,CoCl2处理组细胞凋亡率高于对照组(P<0.05)。RT-PCR检测显示,200、400μmol/L的CoCl2处理组培养24h后细胞的HIF-1α和miRNA-21表达高于对照组(P<0.05)。[结论]人乳腺癌MCF-7细胞可在一定缺氧程度和时间内生长,缺氧诱导细胞凋亡率增加,缺氧能上调HIF-1α、miRNA-21的表达,使之进一步耐受缺氧,其机制可能是上调的miRNA-21通过多个信号通路调控靶基因进而影响癌细胞的增殖及凋亡。

[Purpose ] To investigate the effects of hypoxia microenvironment on the proliferation and apoptosis of human breast cancer MCF-7 cell line,and preliminarily explore the effects of hypoxia on the cellular expression of microRNA-21 （miRNA-21） and the cell proliferation and apoptosis. [Methods]Hypoxia environment of breast cancer line MCF-7 was induced by COC12. MTT assay was used to monitor the situation of CoCl2-indUced hypoxia on proliferation of hu- man breast MCF-7 cell in vitro. The cell apoptotic ratio was detected by flow cytometry. Re- verse transcription polymerase chain reaction （RT-PCR） was used to examine the expression of HIF-1α mRNA in different hypoxia level,and the expression of miRNA-21 was assessed by stem-loop RT-PCR. [Results] Compared with the cells without COCl2 treatment,the cells with COCl2 exposure exhibited obvious growth inhibition which increased with COCl2 concentration and exposure time,but 100,200μmol/L COCl2 treatment group still had proliferation of cells in a certain period of time. According to the flow cytometry results,apoptosis rate in CoCl2treatment group was higher than that in control group. The mRNA expressions of HIF-1μ and miRNA-21 were higher in 200,400μ mol/L COCl2 treatment group than those in control group （P〈0.05）. [Conclusions] Human breast cancer MCF-7 cell are grown in a certain degree of hypoxia and time. The increase of cell apoptosis is induced by hypoxia. Hypoxia condition can up-regulate the expression of HIF-1α and miRNA-21 ,lead to hypoxia tolerance of MCF-7. The mechanism may be miRNA-21 up-regulated through multiple signaling pathways regulating target genes to affect cancer cell proliferation and apoptosis.