miR-129通过靶向调节CAMK2N1抑制乳腺癌细胞增殖、迁移和侵袭

miR-129 inhibits breast cancer cell proliferation,migration and invasion by targeting CAMK2N1

目的探讨microRNA-129(miR-129)在乳腺癌细胞增殖、迁移以及侵袭中的作用及其潜在的机制.方法运用实时荧光定量反转录PCR(real-time fluorescence quantitative reverse transcription PCR,qRT-PCR)检测三种乳腺癌细胞系(MCF-7,MDA-MB-231,T47D)以及一种正常乳腺上皮细胞系(MCF-10A)中miR-129的表达水平,并且通过体外CCK-8细胞活性实验,菌落形成实验研究miR-129在乳腺癌发生过程中的生物学功能,细胞划痕以及细胞迁移侵袭实验检测miR-129在乳腺癌中的迁移情况,通过Luciferase实验和Western blot分析证实了乳腺癌中miR-129下游作用靶标.结果与正常乳腺上皮细胞MCF-10A相比,肿瘤细胞系内miR-129表达显著降低(MCF-10A:1.21±0.3,MCF-7:0.52±0.2,MDA-MB-231:0.43±0.1,T47D:0.82±0.24;t值分别为4.02,4.23和3.87,P值均<0.05).上调miR-129能够明显抑制乳腺癌MCF-7细胞活性(t=4.14,P<0.05).低表达的miR-129能够明显促进MCF-7细胞增殖(miR-129 inhibitor:52±18;t=4.24,P<0.05),高表达miR-129能够明显抑制MCF-7细胞增殖(miR-129 mimic:156±12;t=4.13,P<0.05)并同时能够明显抑制MCF-7细胞的侵袭和迁移.此外,CAMK2N1则被证实是miR-129的直接作用靶点.结论 miR-129可以作为肿瘤抑制因子,并通过靶向CAMK2N1进而调节乳腺癌的疾病进程.

Objective To investigate the role and potential mechanism of microRNA-129(miR-129) in the progress of proliferation,apoptosis and migration in breast cancer. Methods The expression of miR-129 in three breast cancer cell lines (MCF-7,MDA-MB-231,T47D)and one normal breast epithelial cell line (MCF-10A)was detected by real-time fluorescence quantitative reverse transcription PCR(qRT-PCR). Cell colony formation and cell counting kit-8 (CCK-8)were used to characterize the role of miR-129 in the prolifera-tion of breast cancer. Wound healing and transwell were used to test the migration of miR-129 in breast cancer. A miR-129 target gene was identified with Western blot and luciferase activity assays. Results Compared with normal mammary epithelial cell MCF-10A,miR-129 was down-regulated in vitro cell lines (MCF-10A:1. 21 ± 0. 3,MCF-7:0. 52 ± 0. 2,MDA-MB-231:0. 43 ± 0. 1,T47D:0. 82 ± 0. 24;t values were 4. 02,4. 23 and 3. 87 respectively,all P values < 0. 05). Up-regulation of miR-129 can significantly inhibit the activity of breast canc-er MCF-7 cells(t = 4. 14,P < 0. 05). Over-expression of miR-129 could significantly inhibit the proliferation of MCF-7 cells (miR-129 inhibitor:52 ± 18;t = 4. 24,P < 0. 05). Otherwise,miR-129 inhibitor could promote the proliferation of MCF-7 cells(miR-129 mimic:156 ± 12;t = 4. 13,P < 0. 05)and also significantly inhibit the in-vasion and migration of MCF-7 cells. In addition,CAMK2N1 has been proved to be a direct target of miR-129. Conclusion The miR-129 can act as a tumor suppressor and regulate the progression of breast cancer by targe-ting CAMK2N1.