miR-30a-5p在人乳腺癌MCF-7细胞对他莫昔芬耐药性中的作用研究

Study on the Role of miR-30a-5p in Human Breast Cancer MCF-7 Cells Against Tamoxifen Resistance

关键词:  miR-30a-5p  自噬  乳腺癌细胞  他莫昔芬  耐药性

Key words:  miR-30a-5p  autophagy  breast cancer cell  tamoxifen  drug resistance

摘要:

目的 探究miR-30a-5p在人乳腺癌MCF-7细胞对他莫昔芬(tamoxifen,TAM)耐药性中的作用,并阐明相关作用机制。方法 通过短时间高浓度TAM刺激MCF-7细胞诱导耐药株,MTT法检测细胞耐药性的改变;qRT-PCR法检测MCF-7细胞及其耐药细胞株MCF-7/TAM中miR-30a-5p的表达情况;吖啶橙染色及Western blotting检测MCF-7/TAM细胞相对于MCF-7细胞自噬的变化;转染miR-30a-5p模拟物后,采用MTT法检测MCF-7/TAM细胞对TAM敏感性的变化,并通过吖啶橙染色和Western blotting观察miR-30a-5p对MCF-7/TAM细胞自噬的影响;采用生物信息学方法对miR-30a-5p进行靶基因预测,荧光素酶报告基因试验验证miR-30a-5p对ATG5的靶向调控作用;Western blotting检测miR-30a-5p对ATG5表达的影响。结果 TAM对MCF-7/TAM细胞的抑制作用明显弱于对MCF-7细胞;miR-30a-5p在MCF-7/TAM细胞中的表达水平明显低于在MCF-7细胞中;相对于MCF-7细胞,MCF-7/TAM细胞的自噬水平明显升高;过表达miR-30a-5p后,MCF-7/TAM细胞对TAM敏感性显著增加,自噬水平显著降低;Targetscan软件分析表明ATG5是miR-30a-5p的下游靶基因,通过荧光素酶报告基因试验进一步证明miR-30a-5p靶向调控ATG5;MCF-7/TAM细胞中上调miR-30a-5p能够抑制ATG5的表达。结论 miR-30a-5p靶向调控ATG5,并能够抑制细胞的自噬,进而增强乳腺癌细胞对TAM的敏感性。

Abstract:

OBJECTIVE To investigate the role of miR-30a-5p in human breast cancer MCF-7 cells against tamoxifen(TAM) resistance and to clarify the relevant mechanism. METHODS MTT assay was used to detect the changes of drug resistance in MCF-7 cells induced by short time and high concentration of TAM stimulation. The expression of miR-30a-5p in MCF-7 cells and its drug-resistant cell line MCF-7/TAM was detected by qRT-PCR. The changes of autophagy in MCF-7/TAM cells were detected by acridine orange staining and Western blotting. After transfection with miR-30a-5p mimic, the sensitivity of MCF-7/TAM cells to TAM was detected by MTT assay, and the effect of miR-30a-5p on autophagy of MCF-7/TAM cells was observed by acridine orange staining and Western blotting. Bioinformatics method was used to predict the target gene of miR-30a-5p, and luciferase reporter assay verified the targeted regulatory effect of miR-30a-5p on ATG5. The effect of miR-30a-5p on the expression of ATG5 were detected by Western blotting. RESULTS The inhibition of MCF-7/TAM cells to TAM was significantly decreased than that of MCF-7 cells, The expression of miR-30a-5p in MCF-7/TAM cells was significantly lower than that in MCF-7 cells. Compared with MCF-7 cells, the autophagy level of MCF-7/TAM cells was significantly increased. After overexpression of miR-30a-5p, the sensitivity of MCF-7/TAM cells to TAM was significantly increased, and the autophagy level was significantly reduced. Targetscan analysis showed that ATG5 was the downstream target gene of miR-30a-5p, and luciferase reporter gene experiment further proved that miR-30a-5p targeted regulation of ATG5, and up-regulated miR-30a-5p could inhibit the expression of ATG5. CONCLUSION miR-30a-5p targetedly regulates ATG5 and inhibits autophagy, thereby enhancing the sensitivity of breast cancer cells to TAM.