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目的探讨microRNA-26b(miR-26b)的作用靶点及其对MCF-7细胞的抑制作用。方法通过荧光定量PCR方法及Western blot方法检测正常乳腺细胞系MCF-10A及乳腺癌细胞系MCF-7、MDA-MB-231及MDAMB-435的miR-26b及MCL-1的表达水平。MTT法检测miR-26b转染对MCF-7细胞活力的影响,Annexin V/PI染色法检测miR-26b转染对MCF-7细胞凋亡的影响。通过生物信息学分析预测miR-26b的靶基因,并将miR-26b转染入MCF-7细胞,检测miR-26b对MCL-1表达水平的影响。结果相比于MCF-10A细胞,乳腺癌细胞系MCF-7、MDA-MB-231及MDA-MB-435的miR-26b表达水平均显著下降,同时MCL-1表达水平均显著上升,提示miR-26在乳腺癌中其肿瘤抑制作用可能调节细胞MCL-1的表达。进一步研究发现MCL-1基因的3’-UTR区存在miR-26b的假定结合位点,且在乳腺癌细胞中转染miR-26b后,MCL-1的表达下降。转染miR-26b可抑制MCF-7细胞的活力并诱导其发生凋亡。结论 miR-26b下调MCF-7细胞MCL-1蛋白的表达并诱导其发生凋亡。
Objective To investigate the target of microRNA-26b (miR-26b) and its inhibitory effect on MCF-7 cells. Methods The expression levels of miR-26b and MCL-1 in normal breast cell line MCF-10A and breast cancer cell lines MCF-7, MDA-MB-231 and MDAMB-435 were detected by fluorescent quantitative PCR and Western blot. The effect of miR-26b transfection on the viability of MCF-7 cells was detected by MTT assay. The effect of miR-26b transfection on the apoptosis of MCF-7 cells was detected by Annexin V/PI staining. The miR-26b target gene was predicted by bioinformatics analysis, and miR-26b was transfected into MCF-7 cells to examine the effect of miR-26b on the expression level of MCL-1. Results Compared with MCF-10A cells, the expression levels of miR-26b in breast cancer cell lines MCF-7, MDA-MB-231, and MDA-MB-435 were significantly decreased, and the expression level of MCL-1 was significantly increased, suggesting that miRs -26 Tumor inhibition in breast cancer may regulate MCL-1 expression. Further studies revealed that the putative binding site of miR-26b exists in the 3’-UTR region of MCL-1 gene, and the expression of MCL-1 decreased after transfection of miR-26b in breast cancer cells. Transfection of miR-26b inhibited the viability of MCF-7 cells and induced apoptosis. Conclusion miR-26b down-regulates the expression of MCL-1 protein and induces apoptosis in MCF-7 cells.