miR-25对三阴性乳腺癌细胞增殖和凋亡的影响及潜在机制

The effects of miR- 25 on proliferation and apoptosis of triple negative breast cancer cells and the potential mechanism

目的:研究miR-25对人三阴性乳腺癌(triple negative breast cancer,TNBC)细胞增殖和凋亡的影响,并初步探讨其可能机制。方法:在人TNBC细胞株MDA-MB-231中转染miR-25抑制性核苷酸(AS-miR-25)或对照核苷酸(NC oligo),通过MTT法和流式细胞术分别检测细胞的增殖和凋亡;采用实时定量PCR(qRT-PCR)和Western blot测量凋亡调节因子1(modulator of apoptosis 1,MOAP1)蛋白质和mRNA表达水平变化,采用双荧光素报告系统检测miR-25对MOAP1的直接调节。采用AS-miR-25和MOAP1 siRNA共转染MDA-MB-231细胞,MTT和流式细胞术检测细胞增殖、凋亡的变化。结果:下调miR-25后,MDAMB-231细胞的增殖能力较对照明显降低(P<0.05),凋亡显著增加(P<0.05),MOAP1 mRNA和蛋白质表达水平显著增高(P<0.05)。双荧光素报告实验显示MOAP1受miR-25的直接调节。共转染AS-miR-25和MOAP1 siRNA可显著缓解下调miR-25引起的增殖抑制和诱导凋亡。结论:miR-25可通过下调MOAP1表达水平调节TNBC细胞增殖和凋亡,提示miR-25在TNBC恶性生物学进程中可能发挥重要促进作用。

Objective： To investigate the roles of miR -2 5 in regulating proliferation and apoptosis of triple nega-tive breast cancer （TNBC） cells and the underlying mechanisms. Methods ：MDA - MB -2 3 1 cells were transfected with miR - 25 inhibitory oligo （ AS - miR - 25 ） or non - targeting oligo （NC oligo）. MTT assay and flow cytometry were applied to measure cell proliferation and apoptosis. Real - time PCR and Western blot were used to measure lev-els of modulator of apoptosis 1 （ MOAP1 ）. A dual - luciferase reporter assay was carried out to establish the direct regulation of MOAP - 1 by miR -25. To investigate the role of MOAP1 in functions of miR -25 ,we co - transfected MDA - MB -231 cells with AS - miR -25 and MOAP1 siRNA and then measure cell proliferation and apoptosis. Re-sults ： Compared to control cells,MDA - MB -231 cells depleted with miR -25 showed significantly decreased cell proliferation （P 〈0. 05） and increased cell apoptosis ra te （P 〈 0. 05 ） . MOAP1 mRNA（ P 〈0. 05） and protein （P 〈0.05） levels were significantly elevated. In addition,the dual - luciferase reporter assay indicated that MOAP1 was a direct target of miR -25 in TNBC. Co - transfection of AS - miR -25 and MOAP1 siRNA significantly alleviated the inhibitory effects caused by depletion of miR -25. Conclusion ： miR -2 5 promoted MDA - MB -2 3 1 cell proliferation and inhibited apoptosis partially through down - regulating MOAP1 expression level, indicating that miR -25 might- playa promoting role in formation and development of TNBC.