Li Bo, Xie Dalong, Zhang Hui
Department of Urinary surgery, Shengjing Hospital, China Medical University, Shenyang, Liaoning, China.
Department of Anatomy, College of Basic Medicine, China Medical University, Shenyang, Liaoning, China.
J Cancer. 2019 Jun 2;10(12):2628-2634. doi: 10.7150/jca.33117. eCollection 2019.
: Chemotherapy is a major therapeutic method for bladder urothelial carcinoma (BUC), which can effectively improve the prognosis of BUC patients, but the chemoresistance often leads to chemotherapy failure. This study will research the regulatory roles and molecular mechanism of miR-101-3p in BUC chemoresistance. : The quantitative real-time PCR was used to detect the expression of miRNA-101-3p and EZH2. The proliferation and chemoresistance were analyzed by CCK8 assay. Luciferase reporter assay was used to verify the combination between miR-101-3p and EZH2. Protein expression was detected by Western blotting. Flow cytometry was used to examine apoptosis rate. : The miR-101-3p expression was down-regulated in cisplatin (CDDP) resistant BUC cell line (T24/CDDP) and tissues, and was positively related to sensitivity of BUC to CDDP. In T24/CDDP cells, the up-regulation of miR-101-3p decreased the half maximal inhibitory concentration (IC50) to CDDP, depressed the expression of MRP1 protein, promote the CDDP-induced cytotoxicity, and advanced CDDP sensitivity. A series of in vitro experiments certified the EZH2 gene was a target gene of miR-101-3p, including luciferase reporter assay, western blotting and so on. Up-regulation of EZH2 largely reversed the regulatory effects of miR-101-3p enhancement on CDDP sensitivity in T24/CDDP cells. : The expression of miR-101-3p is positively related to CDDP sensitivity of BUC, miR-101-3p advances sensitivity of BUC to CDDP through targeted silencing EZH2.
化疗是膀胱尿路上皮癌(BUC)的主要治疗方法,它能有效改善BUC患者的预后,但化疗耐药性常导致化疗失败。本研究将探讨miR-101-3p在BUC化疗耐药中的调控作用及分子机制。
采用定量实时PCR检测miRNA-101-3p和EZH2的表达。通过CCK8法分析细胞增殖和化疗耐药性。采用荧光素酶报告基因实验验证miR-101-3p与EZH2之间的结合。通过蛋白质免疫印迹法检测蛋白表达。采用流式细胞术检测凋亡率。
miR-101-3p在顺铂(CDDP)耐药的BUC细胞系(T24/CDDP)和组织中表达下调,且与BUC对CDDP的敏感性呈正相关。在T24/CDDP细胞中,miR-101-3p的上调降低了对CDDP的半数抑制浓度(IC50),抑制了MRP1蛋白的表达,促进了CDDP诱导的细胞毒性,并提高了CDDP敏感性。一系列体外实验证实EZH2基因是miR-101-3p的靶基因,包括荧光素酶报告基因实验、蛋白质免疫印迹法等。EZH2的上调在很大程度上逆转了miR-101-3p增强对T24/CDDP细胞中CDDP敏感性的调控作用。
miR-101-3p的表达与BUC对CDDP的敏感性呈正相关,miR-101-3p通过靶向沉默EZH2提高BUC对CDDP的敏感性。