MicroRNA-34a通过Notch1对膀胱肿瘤细胞株T24增殖的影响

目的 探讨膀胱肿瘤细胞株中microRNA-34a(miR-34a)与Notch1的相互作用关系以及过表达miR-34a对T24细胞增殖的影响.方法 通过生物信息学软件预测miR-34a与Notch1的作用位点,并通过荧光素酶实验验证两者的直接调控关系.在膀胱癌细胞株T24过表达miR-34a,采用实时定量PCR和蛋白印迹检测Notch1表达水平的变化；分别通过新型四唑氮盐(MTS)实验和流式细胞术检测细胞增殖、凋亡以及细胞周期的变化.结果 T24细胞中荧光素酶报告基因实验显示,miR-34a在膀胱癌细胞中能与报告基因结合,使萤火虫荧光强度减弱(P=0.006).过表达miR-34a后,T24细胞内源性Notch1的mRNA水平和蛋白水平均明显下调；T24细胞生长明显受抑(P＜0.001),并呈现一定时间依赖性；凋亡率增加(P=0.003),G0-G1期细胞显著增多(P=0.002).结论 过表达miR-34a能通过降低靶基因Notch1的表达,抑制膀胱肿瘤细胞的增殖.

Inhibitory effects of microRNA-34a on proliferation of bladder tumor cell line T24 by targeting Notch1

Objective To explore the correlation between microRNA-34a (miR-34a) and Notch1, and evaluate the influence of miR-34a overexpression on proliferation of bladder cancer cell line T24. Methods Bioinformatics software were used for predicting the binding site of miR-34a and Notch1,and luciferase assay was performed for confirming the direct regulatory relationship between them.miR-34a plasmid was transfected into bladder cancer cell line T24.Notch1 expression was detected by quantitative real-time polymerase chain reaction and Western blotting.Cell proliferation was assayed by 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium(MTS) assay.Apoptosis and cell cycle were assessed by flow cytometry. Results Luciferase assays showed that miR-34a transfection significantly down-regulated the normalized Notch1 3’UTR luciferase activity(P=0.006).Transfection of miR-34a reduced the RNA and protein levels of Notch1.Cell proliferation assay revealed that miR-34a transfection suppressed the proliferation of T24 cells in a time-dependent manner(P<0.001).Ectopic expression of miR-34a caused significant increase of apoptotic cells(P=0.003) and induced cell cycle arrest at G0-G1 phase in T24 cells(P=0.002).Conclusion Overexpressed miRNA-34a can inhibit proliferation of bladder cancer cells by antagonizing Notch1, thus indicating its tumor-suppressive function in bladder cancer.