microRNA-449a靶向Notch1抑制膀胱癌细胞J82的迁移

【摘要】〓目的〓研究microRNA-449a(miR-449a)对膀胱癌细胞J82迁移能力的影响以及对靶基因Notch1表达的影响。方法〓通过mimics转染膀胱癌细胞株J82使其过表达miR-449a,利用Transwell实验、细胞划痕实验观察细胞迁移能力的变化;采用实时定量PCR和蛋白印迹检测细胞Notch1表达水平的变化,并通过荧光素酶实验验证miR-449a与Notch1基因的直接调控关系。结果〓与对照组相比,转染miR-449a组的J82细胞的迁移能力减弱。过表达miR-449a后,J82细胞Notch1的mRNA表达无明显变化(P=0.5739),但Notch1蛋白表达下调(P=0.0135)。荧光素酶报告基因实验显示,miR-449a能明显抑制Notch1-3’UTR的荧光素酶活性(P=0.0016)。结论〓过表达miR-449a可能通过靶向降低Notch1基因的蛋白表达,抑制膀胱癌细胞的迁移。

Inhibitory effects of microRNA-449a on migratory ability of bladder tumor cell line J82 by targeting Notch1

Objective To explore the influence of microRNA-449a （miR-449a） on migratory ability of bladder cancer cell hne J82 and evaluate the correlation between miR-449a and Notch1. Methods After transfection of microRNA mimics into J82 cells, cell migratory ability was assessed by transwell chamber assay and wound healing assay. Expression of Notch1 was evaluated by quantitative realtime polymerase chain reaction and western blotting. Luciferase assay was used to confirm the relationship between miR-449a and Notch1. Results Transwell chamber assays and wound healing assays revealed that transfection of miR-449a suppressed the cell migration of J82 cells. Ectopic overexpression of miR- 449a caused significant decrease of protein levels of Notch1 （P=0.0135）, while the mRNA levels of Notch1 showed little change （P=0.5739）. In addition, luciferase assays showed that miR-449a significantly reduced the normalized Notch1 3＇UTR luciferase activity （P=0.0016）. Conclusion Overexpressed miR- 449a can inhibit migration of bladder cancer cells by antagonizing Notch1, thus indicating its tumor suppressive function in bladder cancer.