Department of Intensive Care Unit, The Lihuili Affiliated Hospital, Ningbo University, Ningbo, Zhejiang, China.
Department of Ultrasound, Yinzhou No. 2 Hospital, Ningbo, Zhejiang, China.
Cell Biochem Biophys. 2022 Jun;80(2):457-466. doi: 10.1007/s12013-022-01072-6. Epub 2022 Mar 12.
Asthma is a chronic pulmonary inflammatory disease. MicroRNA (miR)-629-3p expression is reported to be up-regulated in the sputum of asthma patients. Nonetheless, miR-629-3p's role and mechanism in asthma remain largely unknown. This study is aimed at exploring miR-629-3p's role in regulating the injury and inflammation of bronchial epithelial cells.
Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to detect the expression levels of miR-629-3p and forkhead box a2 (FOXA2) mRNA in 16HBE cells treated with interleukin-13 (IL-13). 16HBE cell viability was evaluated using the cell counting kit-8 (CCK-8) assay, and cell apoptosis was analyzed by a flow cytometer. The levels of C-C motif chemokine ligand 11 (CCL11), C-C motif chemokine ligand 26 (CCL26), C-C motif ligand 2 (CCL-2)/mono-cyte chemotactic protein-1 (MCP-1), interleukin-1 beta (IL-1b), and interleukin 6 (IL-6) in 16HBE cell supernatant were detected through enzyme-linked immunosorbent assay (ELISA). The downstream target genes of miR-629-3p were predicted through bioinformatics. Besides, the targeted relationship between miR-629-3p and FOXA2 mRNA 3'-UTR was verified by dual-luciferase reporter gene assay. Western blot was utilized to determine the regulatory effects of miR-629-3p on the expression of FOXA2 protein in 16HBE cells.
MiR-629-3p expression was significantly enhanced in IL-13-stimulated 16HBE cells while the FOXA2 mRNA and protein levels were significantly down-regulated. The transfection of miR-629-3p mimics inhibited 16HBE cells' viability, and promoted the apoptosis and the secretion of chemokines CCL11, CCL26, CCL-2/MCP-1, IL-1b, and IL-6 of 16HBE cells, whereas inhibiting miR-629-3p had the opposite effects. Moreover, FOXA2 was identified as a downstream miR-629-3p target, and its overexpression reversed the effects of the miR-629-3p on 16HBE cells.
MiR-629-3p promotes IL-13-induced 16HBE cells' injury and inflammation by targeting FOXA2.
哮喘是一种慢性肺部炎症性疾病。据报道，微小 RNA（miR）-629-3p 在哮喘患者的痰中表达上调。然而，miR-629-3p 在哮喘中的作用和机制在很大程度上仍然未知。本研究旨在探讨 miR-629-3p 在调节支气管上皮细胞损伤和炎症中的作用。
采用定量实时聚合酶链反应（qRT-PCR）检测白细胞介素 13（IL-13）处理的 16HBE 细胞中 miR-629-3p 和叉头框蛋白 A2（FOXA2）mRNA 的表达水平。采用细胞计数试剂盒-8（CCK-8）法检测 16HBE 细胞活力，采用流式细胞仪分析细胞凋亡。采用酶联免疫吸附试验（ELISA）检测 16HBE 细胞上清液中 C-C 基序趋化因子配体 11（CCL11）、C-C 基序趋化因子配体 26（CCL26）、C-C 基序趋化因子配体 2（CCL-2）/单核细胞趋化蛋白-1（MCP-1）、白细胞介素 1β（IL-1β）和白细胞介素 6（IL-6）的水平。通过生物信息学预测 miR-629-3p 的下游靶基因。此外，通过双荧光素酶报告基因检测验证 miR-629-3p 与 FOXA2 mRNA 3'-UTR 的靶向关系。Western blot 用于检测 miR-629-3p 对 16HBE 细胞中 FOXA2 蛋白表达的调控作用。
IL-13 刺激的 16HBE 细胞中 miR-629-3p 表达明显增强，而 FOXA2 mRNA 和蛋白水平明显下调。miR-629-3p 模拟物转染抑制 16HBE 细胞活力，并促进 16HBE 细胞趋化因子 CCL11、CCL26、CCL-2/MCP-1、IL-1β和 IL-6 的分泌，而抑制 miR-629-3p 则有相反的作用。此外，FOXA2 被鉴定为 miR-629-3p 的下游靶基因，其过表达逆转了 miR-629-3p 对 16HBE 细胞的作用。
miR-629-3p 通过靶向 FOXA2 促进 IL-13 诱导的 16HBE 细胞损伤和炎症。
