miR-181a-5p靶向Kras对乳腺癌细胞MDA-MB-231增殖及凋亡的调控作用

miR-181a-5p regulates cell proliferation and apoptosis by targeting Kras in the breast cancer cell line MDA-MB-231

目的探讨miR-181a-5p靶向Kras对乳腺癌细胞MDA-MB-231增殖及凋亡的调控作用。方法选取乳腺癌细胞系MDA-MB-231和人乳腺细胞系HBL-100,分别采用RT-PCR、Western blotting检测miR-181a-5p及Kras蛋白的表达。选取MDA-MB-231细胞,分别转染miR-181a-5p mimics及阴性对照mimics,采用RT-PCR检测细胞中miR-181a-5p的表达,Western blotting检测Kras蛋白的表达,MTT法检测细胞增殖情况,流式细胞仪检测细胞周期及细胞凋亡情况。分别将野生型Kras 3'-UTR质粒(Kras-wt)和突变型Kras 3'-UTR质粒(Kras-Mut)与miR-181a-5p mimic,或阴性对照mimics共转染至MDA-MB-231细胞,采用双荧光素酶报告基因检测细胞荧光素酶的活性。结果与HBL-100细胞相比,MDAMB-231细胞中miR-181a-5p的表达明显降低(P<0.05),而Kras蛋白的表达明显升高(P<0.05)。与转染阴性对照mimics相比,转染miR-181a-5p mimic可使MDA-MB-231细胞中miR-181a-5p的表达明显上调(P<0.05),Kras蛋白的表达明显下调(P<0.05),并可使MDA-MB-231细胞增殖活性降低(P<0.05),凋亡细胞比例增加(P<0.05),G2/M期细胞增多,S期细胞减少(P<0.05)。与转染阴性对照mimics和(或)Kras-Mut相比,共转染miR-181a-5p mimic及Kras-wt后MDA-MB-231细胞中的荧光素酶活性明显降低(P<0.05)。结论在乳腺癌细胞MDA-MB-231中miR-181a-5p呈低表达,Kras呈高表达;过表达miR-181a-5p可通过靶向Kras抑制细胞增殖并诱导细胞凋亡。

Objective To explore the potential function of miR-181 a-5 p in regulating cell proliferation and apoptosis in the breast cancer cell line MDA-MB-231 by targeting Kras. Methods The expressions of miR-181 a-5 p and Kras in MDA-MB-231（a human breast cancer cell line） and HBL-100（a human breast cell line） were detected by RT-PCR and Western blotting, respectively. The MDA-MB-231 cells were transfected with miR-181 a-5 p mimics. Cells that were transfected with the negative control mimics were served as control. To verify the upregulation of miR-181 a-5 p using its mimics, the expression of miR-181 a-5 p was detected using RT-PCR and the expression of its target, Kras, was detected using Western blotting. Cell proliferation was measured by MTT assay. Cell apoptosis and cell cycle were detected with flow cytometer. To confirm that Kras is a real target of miR-181 a-5 p in MDA-MB-231 cells, wild-type Kras 3＇-UTR plasmid（Kras-wt） or mutant Kras 3＇-UTR plasmid（Kras-Mut） were co-transfected with miR-181 a-5 p mimic or control mimics. Dual luciferase reporter gene assay was conducted. Results Compared to HBL-100 cells, the expression of miR-181 a-5 p significantly lower（P〈0.05） in MDA-MB-231 cells, while the expression of its target, Kras, was significantly higher（P〈0.05）. Compared to cells transfected with negative control mimics, cells transfected with miR-181 a-5 p mimics showed an upregulation of miR-181 a-5 p（P〈0.05）, a reduction of its target Kras（P〈0.05）, a significant reduction in proliferative capacity（P〈0.05）, a higher rate of apoptosis（P〈0.05）, and a greater number of cells arrested in G2/M phase as well as a dramatic decline in the number of cells arrested in S phase（P〈0.05）. Compared to control cells, cells co-transfected with miR-181 a-5 p mimics and Kras-wt showed the lowest luciferase activity（P〈0.05）. Conclusions In MDA-MB-231 cells, miR-181 a-5 p was lowly expressed but its target Kras was highly expressed. Upregulated miR-181 a-5 p in MDA-MB-231 cell inhibited cell proliferation and induced apoptosis by targeting Kras.