摘要
目的探讨miR-99a对乳腺癌细胞侵袭及迁移的影响,并初步分析其影响乳腺癌细胞侵袭及迁移的可能分子机制。方法利用荧光实时定量PCR检测乳腺癌细胞系MDA-MB-231和MCF-7中miR-99a的表达。运用脂质体介导的转染方法分别将miR-99a模拟物(miR-99a mimics)、miR-99a抑制物(miR-99a inhibitors)以及相应对照miRNA转染MDA-MB-231和MCF-7细胞,通过Transwell侵袭实验检测细胞的侵袭力;采用Transwell迁移实验及划痕实验检测细胞的迁移能力;利用生物信息学方法预测miR-99a的靶基因,并对靶基因进行验证。结果(1)高转移潜能的MDA-MB-231细胞中miR-99a表达明显低于低转移潜能的MCF-7细胞,划痕实验中转染miR-99a mimics的与转染control mimics的MDA-MB-231细胞比较迁移能力显著减弱(P<0.05),而MCF-7细胞转染miR-99a inhibitors后迁移能力明显增强(P<0.01)。(2)Transwell的侵袭及迁移实验显示,转染miR-99a mimics后MDA-MB-231细胞的侵袭和迁移能力明显减弱(P<0.01);MCF-7细胞转染miR-99a inhibitors后迁移能力增强(P<0.01),而侵袭能力基本不变(P>0.05)。(3)生物信息学方法预测徼管相关蛋白(MTMR3)是miR-99a的靶点,实时定量PCR和3'UTR荧光素酶报告基因实验验证了该靶点。(4)干扰了MTMR3后MDA-MB-231细胞的迁移和侵袭能力明显减弱。结论(1)miR-99a对乳腺癌细胞的侵袭及迁移发挥负向调控作用。(2)miR-99a可能通过靶向于MTMR3发挥其对乳腺癌细胞迁移和侵袭的调控作用。
Objective To investigate the effect of miR-99a on invasion and migration potential of human breast cancer cells and its possible mechanism, Methods The expression level of miR-99a was detected in human breast cancer cell lines MDA-MB-231 and MCF-7 by qRT-PCR. Human breast cancer MDA-MB-231 cells were transfected with miR-99a mimics and control mimics by Lipofectamine. Cell invasion potential was evaluated by Transwell invasion assay; cell migration was detected by Transwell migration assay and wound healing assay. The possible target genes of miR-99a were forecasted by bioinformatics tools and the reliability of these genes was analyzed. Results Remarkable inhibition of cell migration was detected in MDA-MB-231 cells transfected with miR-99a mimics by wound healing and Transwell migration assay, compared to cells transfected with control mimics. MCF-7 cells transfected with miR-99a inhibitors showed increased migration, compared to cells transfected with control inhibitors. Transfection of miR-99a mimics into MDA-MB-231 cells led to a significant decrease in cell invasion as detected by Transwell invasion assay. Myotubularin related protein 3 （MTMR3） was forecasted by bioinformatics tools as a target of miR-99a, which was verified by RT-qPCR and 3＇UTR luciferase reporter assays. Knock down of MTMR3 in MDA-MB-231cells inhibited cell migration and invasion, Conclusion miR-99a negatively may regulate the invasion and migration potential of human breast cancer cells through targeting MTMR3.
出处
《浙江医学》
CAS
2016年第5期317-321,335,I0001,共7页
Zhejiang Medical Journal
基金
国家自然科学基金资助项目(81373115)
