miR-184靶向AGO2调控AKT/mTOR信号通路对膀胱癌细胞增殖、迁移及侵袭能力的影响

目的研究miR-184在膀胱癌发生、发展过程中的作用及其相关机制。方法收集2015年1月至2017年1月南阳市中心医院87例膀胱癌患者的手术切除标本,采用qRT-PCR检测膀胱癌及癌旁组织中miR-184的表达;通过生物信息学方法预测miR-184的靶基因并采用双荧光素酶实验及免疫荧光进行验证。在人膀胱癌细胞系T24中转染miR-184过表达质粒,MTT、Transwell侵袭和划痕实验检测细胞增殖、侵袭和迁移能力,Western blot检测AKT/mTOR通路蛋白的表达。结果miR-184在膀胱癌组织中的相对表达水平1.238±0.026明显低于癌旁组织2.601±0.054,差异有统计学意义(t=22.57,P<0.01);且其表达水平与膀胱癌患者的TNM分期(t=-5.61,P<0.001)、淋巴结转移(t=-2.35,P=0.011)及组织学分级存在明显相关(t=2.171,P=0.033)。生物信息学预测及双荧光素酶报告基因实验证实miR-184能直接靶向结合AGO2基因3′-UTR。体外实验结果表明,转染miR-184模拟物后,膀胱癌细胞T24中AGO2 mRNA和蛋白的表达水平明显下调,细胞增殖、迁移及侵袭能力降低,p-mTOR、p-AKT蛋白表达降低(P<0.05)。结论miR-184在膀胱癌组织中低表达,上调miR-184表达可抑制细胞的增殖、侵袭和迁移能力,其作用机制可能与AKT/mTOR信号通路的调节有关。

Up regulation of miR-184 inhibitsthe proliferation,invasion and migration of bladder cancer cells by targeting AGO2 via AKT/mTOR signaling pathway

Objective To investigate the role of mir-184 in the development of bladder cancer.Methods The expression of miR-184 in bladder cancer and adjacent tissues was detected by qRT-PCR in 87 cases of bladder cancer resected from Jan.2015 to Jan.2017 in Nanyang Central Hospital of Henan Province.Predicting the target gene of miR-184 by bioinformatics and verifying by double luciferase and immunofluorescence.miR-184 overexpression plasmid was transfected into human bladder cancer cell line T24.MTT,Transwell invasion and wound healing assay were used to detect cell proliferation,invasion and migration.Western blot was used to detect the expression of AKT/mTOR pathway protein.Results The relative expression level of miR-184 in bladder cancer tissues(1.238±0.026)was significantly lower than that in adjacent tissues(2.601±0.054),and the difference was statistically significant(t=22.57,P<0.01),and the expression level was significantly correlated with TNM stage,lymph node metastasis and histological grade of bladder cancer patients(t=-5.61,P<0.001;t=-2.35,P=0.011;t=2.171,P=0.033).Bioinformatics prediction and double luciferase reporter gene experiments confirmed that miR-184 could directly target AGO2 gene 3′-UTR.The results of in vitro experiments showed that the expression of AGO2 mRNA and protein in T24 cells decreased significantly,the proliferation,migration and invasion ability decreased,and the expression of p-mTOR and p-AKT protein decreased(P<0.05).Conclusion miR-184 is low expression in bladder cancer.Up regulation of mir-184 can inhibit cell proliferation,invasion and migration.The mechanism may be related to the regulation of AKT/mTOR signaling pathway.