Huang Hai-Li, Shi Ya-Peng, He Hui-Juan, Wang Ya-Hong, Chen Ting, Yang La-Wei, Yang Teng, Chen Jie, Cao Jun, Yao Wei-Min, Liu Gang
Clinical Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China.
Department of Respiratory Medicine, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China.
Cell Physiol Biochem. 2017;42(3):889-900. doi: 10.1159/000478644. Epub 2017 Jun 26.
Our previous study identified a novel microRNA, miR-4673, which is upregulated in A549 cells exposed to paclitaxel (PTX). In this study, we investigated the role of miR-4673 in PTX-induced cytotoxicity.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, apoptosis assay, 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide (JC-1) staining and 2',7'-Dichlorofluorescein (DCFH) staining were used to evaluate cell viability, apoptosis, mitochondrial membrane potential (MMP) loss and reactive oxygen species (ROS) generation in A549 and H1299 cells. Bioinformatics analysis and Luciferase reporter assay were used to explore whether 8-oxoguanine-DNA glycosylase-1 (OGG1) is a target gene of miR-4673.
Enforced expression of miR-4673 decreased cell viability and increased PTX-induced apoptosis, MMP loss and reactive oxygen species (ROS) generation in A549 and H1299 cells. Bioinformatics analysis, which was used to identify potential target of miR-4673, revealed a binding site of miR-4673 in 3'UTR of OGG1. Luciferase reporters assays showed that miR-4673 specifically binds to 'CUGUUGA' in 3'UTR of OGG1. Enforced expression of miR-4673 decreased accumulation of OGG1. In addition, silencing OGG1 enhanced inhibitory effects of PTX on apoptosis, MMP loss and ROS generation, which is similar to effects of miR-4673. Moreover, enforced expression of OGG1 compromised promoting effects of miR-4673 on PTX-induced apoptosis, MMP loss and ROS generation.
miR-4673 modulates PTX-induced apoptosis, MMP loss and ROS generation by targeting OGG1.
我们之前的研究鉴定出一种新型微小RNA，即miR - 4673，其在暴露于紫杉醇（PTX）的A549细胞中表达上调。在本研究中，我们调查了miR - 4673在PTX诱导的细胞毒性中的作用。
采用3 -（4,5 - 二甲基噻唑 - 2 - 基）- 2,5 - 二苯基四氮唑溴盐（MTT）法、凋亡检测、5,5',6,6'-四氯 - 1,1',3,3'-四乙基 - 咪唑羰花青碘化物（JC - 1）染色和2',7'-二氯荧光素（DCFH）染色来评估A549和H1299细胞的细胞活力、凋亡、线粒体膜电位（MMP）丧失和活性氧（ROS）生成。利用生物信息学分析和荧光素酶报告基因检测来探究8 - 氧代鸟嘌呤 - DNA糖基化酶 - 1（OGG1）是否为miR - 4673的靶基因。
miR - 4673的过表达降低了A549和H1299细胞的活力，并增加了PTX诱导的凋亡、MMP丧失和ROS生成。用于鉴定miR - 4673潜在靶标的生物信息学分析揭示了miR - 4673在OGG1的3'非翻译区（UTR）中的一个结合位点。荧光素酶报告基因检测表明miR - 4673特异性结合OGG1的3'UTR中的“CUGUUGA”。miR - 4673的过表达降低了OGG1的积累。此外，沉默OGG1增强了PTX对凋亡、MMP丧失和ROS生成的抑制作用，这与miR - 4673的作用相似。而且，OGG1的过表达削弱了miR - 4673对PTX诱导的凋亡、MMP丧失和ROS生成的促进作用。
miR - 4673通过靶向OGG1调节PTX诱导的凋亡、MMP丧失和ROS生成。
