微小RNA-124-3p调控KIF26B对膀胱尿路上皮癌细胞顺铂耐药性的影响

Effect of miR-124-3p on relieving cisplatin resistance of bladder urothelial cancer cells by regulating KIF26B

目的探讨微小RNA(miR)-124-3p调控KIF26B对膀胱尿路上皮癌细胞的顺铂耐药性的影响。方法体外培养人膀胱移行细胞癌细胞(T24),使用顺铂(DDP)浓度递增的方法处理建立耐药T24细胞(T24/DDP),分为T24/DDP(NC)组、转染过表达miR-124-3p组(miR-124-3p模拟物)、转染低表达miR-124-3p组(miR-124-3p抑制剂)、共转染过表达miR-124-3p和过表达KIF26B组(miR-124-3p模拟物+oe-KIF26B)。采用细胞转染的方式将NC-模拟物、NC-抑制剂、miR-124-3p模拟物、miR-124-3p抑制剂、oe-KIF26B转染至T24/DDP细胞中;采用CCK-8检测细胞对顺铂的半数抑制浓度(IC_(50));采用蛋白印迹法检测多药耐药蛋白2(MRP2)以及细胞凋亡相关的蛋白;采用实时荧光定量检测miR-124-3p;采用双荧光素酶报告基因系统验证miR-124-3p和KIF26B的靶向关系;采用流式细胞仪检测细胞凋亡情况。结果与正常T24细胞相比,T24/DDP细胞IC_(50)值升高,细胞凋亡率降低,MRP2、X连锁凋亡抑制蛋白(XIAP)、Bcl-2、Bcl-XL表达上调,miR-124-3p表达水平降低。与NC-模拟物组相比,miR-124-3p模拟物组miR-124-3p表达水平显著升高,IC_(50)值降低,细胞凋亡率升高,MRP2、XIAP、Bcl-2、Bcl-XL表达下调。KIF26B的表达水平,过表达miR-124-3p抑制KIF26B的表达,低表达miR-124-3p促进KIF26B的表达,共转染miR-124-3p模拟物和oe-KIF26B后明显恢复单独转染miR-124-3p模拟物的作用。结论miR-124-3p通过调控KIF26B提升膀胱尿路上皮癌细胞对顺铂的敏感性。

Objective To explore the effect of miR-124-3p on relieving cisplatin resistance of bladder urothelial cancer cells.Methods Human bladder transitional cell carcinoma cells(T24)were cultured in vitro and treated with different concentrations of cisplatin to establish drug-resistant T24 cells(T24/DDP),which were divided into T24/DDP(NC)group,miR-124-3p transfected group and KIF26B transfected group.The NC-mimic,NC-inhibitor,miR-124-3p mimic,miR-124-3p inhibitor and overexpression(oe)-KIF26B were transfected into T24/DDP cells by cell transfection.CCK-8 was used to detect the IC_(50) of cisplatin.Western blot was used to detect multidrug resistance protein 2(MRP2)and apoptosis related proteins.The miR-124-3p was quantitatively detected by real-time fluorescence.The targeted regulation of miR-124-3p and KIF26B was verified by double luciferase reporter gene and Western blot.Cell apoptosis was detected by flow cytometry.Results Compared with normal T24 cells,the IC_(50) of T24/DDP cells increased,the apoptosis rate decreased,the expressions of MRP2,X-linked inhibitor of apoptosis protein(XIAP),Bcl-2 and Bcl-XL increased,and the expression level of miR-124-3p decreased.Compared with NC-mimic group,miR-124-3p expression level in miR-124-3p mimic group increased significantly,IC_(50) decreased,apoptosis rate increased,and expression of MRP2,XIAP,Bcl-2 and Bcl-XL decreased.Overexpression of miR-124-3p inhibited the expression of KIF26B,while low expression of miR-124-3p promoted the expression of KIF26B.After co-transfection of miR-124-3p mimic and oe-KIF26B,the effect of single transfection of miR-124-3p mimic was obviously restored.Conclusion miR-124-3p can enhance the sensitivity of bladder urothelial cancer cells to cisplatin by regulating KIF26B.