Hai-Li Huang1, Ya-Peng Shi2, Hui-Juan He1, Ya-Hong Wang1, Ting Chen1, La-Wei Yang1, Teng Yang1, Jie Chen3, Jun Cao4, Wei-Min Yao2, Gang Liu1. 1. Clinical Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China. 2. Department of Respiratory Medicine, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China. 3. Department of Cardiothoracic Surgery, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China. 4. Pathological Diagnosis and Research Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, China.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: miR-4673 modulates PTX-induced apoptosis, MMP loss and ROS generation by targeting OGG1.
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION:miR-4673 modulates PTX-induced apoptosis, MMP loss and ROS generation by targeting OGG1.