MicroRNA-34a通过Notch1对膀胱肿瘤细胞株T24增殖的影响

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

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。

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（P0.001）.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.