微RNA-374c-5p通过靶向LIMK1增强人乳腺癌MDA-MB-231/DDP细胞对顺铂的敏感性

MicroRNA-374c-5p enhances sensitivity of human breast cancer MDA-MB-231/DDP cells to cisplatin by targeting LIMK1

目的:探讨人类(Homo sapiens,hsa)-微RNA(micro RNA,miR)-374c-5p对人乳腺癌MDA-MB-231/DDP细胞顺铂敏感性的影响及其作用机制。方法:采用实时荧光定量PCR法检测乳腺癌MCF-10A、MDA-MB-231和MDA-MB-231/DDP细胞中hsa-miR-374c-5p和Lim结构域激酶1(Lim domain kinase 1,LIMK1)的表达情况。将hsa-miR-374c-5p模拟物(mimics)和抑制剂(inhibitor)以及LIMK1 siRNA和过表达质粒分别转染乳腺癌MDA-MB-231/DDP细胞,采用实时荧光定量PCR法检测hsa-miR-374c-5p和LIMK1 mRNA表达变化,蛋白质印迹法检测LIMK1蛋白表达水平;分别采用CCK-8、FCM和平板克隆形成实验检测细胞增殖、凋亡和克隆形成能力的变化;荧光素酶报告基因实验验证hsa-miR-374c-5p与LIMK1的结合关系。结果:乳腺癌MDA-MB-231/DDP细胞中hsa-miR-374c-5p和LIMK1的表达趋势相反。过表达hsa-miR-374c-5p或敲低LIMK1表达均可抑制MDA-MB-231/DDP细胞的增殖活力(P值均<0.05)和克隆形成能力(P值均<0.05),而细胞凋亡率明显升高(P值均<0.05),对顺铂的敏感性明显增强(P值均<0.05);沉默hsa-miR-374c-5p后,情况相反(P值均<0.05);且在转染了hsa-miR-374c-5p mimics的细胞中过表达LIMK1可以逆转单一过表达hsa-miR-374c-5p引起的细胞表型改变(P值均<0.05)。hsa-miR-374c-5p可靶向调控LIMK1的表达。结论:Hsa-miR-374c-5p可以通过靶向调控LIMK1的表达,增强人乳腺癌MDA-MB-231/DDP细胞对顺铂的敏感性。

Objective:To investigate the effect Homo sapiens(hsa)-microRNA(miRNA,miR)-374c-5p on the sensitivity of human breast cancer MDA-MB-231/DDP cells to cisplatin and its mechanism Methods:The expressions of hsa-miR-374c-5p and Lim domain kinase 1(LIMK1)in breast cancer MCF-10 A,MDA-MB-231 and MDA-MB-231/DDP cells were detected by real-time fluorescent quantitative PCR.Then the hsa-miR-374c-5p mimics and inhibitor,LIMK1 siRNA and LIMK1 overexpressed plasmid were transfected into cisplatin-resistant human breast cancer MDA-MB-231/DDP cells,respectively.The relative expression levels of hsa-miR-374c-5p and LIMK1 mRNA were detected by real-time fluorescent quantitative PCR,the expression level of LIMK1 protein was detected by Western blotting,the cell viability was detected by CCK-8 method,the apoptosis was detected by flow cytometry,and the clone formation ability was tested by plate clone forming assay.Finally,the binding relationship between hsa-miR-374c-5p and LIMK1 was verified by luciferase reporter gene assay.Results:The expression levels of hsa-miR-374c-5p and LIMK1 were reversed in breast cancer MDA-MB-231/DDP cells.The overexpression of hsa-miR-374c-5p or the knockdown of LIMK1 decreased cell viability(both P<0.05)and clone forming ability(both P<0.05),increased apoptotic rate(both P<0.05),and increased the sensitivity of MDA-MB-231/DDP cells to cisplatin(both P<0.05);while the effects of hsa-miR-374c-5p knockdown on proliferation,clone formation,apoptosis and cisplatin-sensitivity of MDA-MB-231/DDP cells were reversed(all P<0.05);and the overexpression of LIMK1 reversed the phenotype changes of MDA-MB-231/DDP cells transfected with hsa-miR-374c-5p mimics(all P<0.05).Hsa-miR-374c-5p targeted LIMK1 mRNA directly and regulated the expression of LIMK1.Conclusion:Hsa-mi R-374 c-5 p enhances the sensitivity of human breast cancer MDAMB-231/DDP cells to cisplatin by regulating the expression of LIMK1.