Wang Jing, Li Hong-Yan, Wang Hu-Shan, Su Zhen-Bo
Department of Respiratory Medicine, China-Japan Union Hospital of Jilin University, Changchun, China.
Department of Infections, China-Japan Union Hospital of Jilin University, Changchun, China.
Cell Physiol Biochem. 2018;51(2):692-710. doi: 10.1159/000495327. Epub 2018 Nov 21.
BACKGROUND/AIMS: Chronic respiratory conditions continue to plague millions of people worldwide. We aimed to elucidate the detailed mechanisms of microRNA-485 (miR-485) in airway smooth muscle cell (ASMC) proliferation and apoptosis in chronic asthmatic mice.
A mouse model of chronic asthma was established. Ovalbumin was used to induce chronic asthma in the mice. The levels of transforming growth factor β (TGF-β), interleukin (IL)-4, IL-5, IL-13 and IL-17 in bronchoalveolar lavage fluid in mice were measured by enzyme-linked immunoassays (ELISAs). ASMCs were transfected with miR-485 mimic, miR-485 inhibitor and siRNA-Smurf2. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analyses were applied to detect the mRNA and protein levels of Smurf2, α-SMA, TGF-β1 and decapentaplegic homolog (Smads). The MTT assay was utilized for cell proliferation, while flow cytometry was conducted to assess cell cycle distribution and apoptosis.
Lower expression of miR-485 and higher expression levels of TGF-β1, IL-4, IL-5, IL-13 and IL-17 were detected in mice with chronic asthma. Smurf2 was identified as the target gene of miR-485. Upregulation of miR-485 mimic and downregulation of Smurf2 decreased expression levels of Smurf2, α-SMA, TGF-β1 and Smad3, inhibited cell proliferation and increased apoptosis, while contrary results were observed in ASMCs transfected with miR-485 inhibitor.
Overexpressed miR-485 inhibits cell proliferation and promotes apoptosis of ASMCs through the Smurf2-mediated TGF-β/Smads signaling pathway in mice with chronic asthma.
背景/目的：慢性呼吸道疾病继续困扰着全球数百万人。我们旨在阐明微小RNA-485（miR-485）在慢性哮喘小鼠气道平滑肌细胞（ASMC）增殖和凋亡中的详细机制。
建立慢性哮喘小鼠模型。用卵清蛋白诱导小鼠慢性哮喘。通过酶联免疫吸附测定（ELISA）测量小鼠支气管肺泡灌洗液中转化生长因子β（TGF-β）、白细胞介素（IL）-4、IL-5、IL-13和IL-17的水平。用miR-485模拟物、miR-485抑制剂和siRNA-Smurf2转染ASMC。应用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质印迹分析检测Smurf2、α-平滑肌肌动蛋白（α-SMA）、TGF-β1和果蝇蛋白（Smads）的mRNA和蛋白质水平。采用MTT法检测细胞增殖，同时进行流式细胞术评估细胞周期分布和凋亡。
在慢性哮喘小鼠中检测到miR-485表达降低，TGF-β1、IL-4、IL-5、IL-13和IL-17表达水平升高。Smurf2被鉴定为miR-485的靶基因。miR-485模拟物上调和Smurf2下调降低了Smurf2、α-SMA、TGF-β1和Smad3的表达水平，抑制细胞增殖并增加凋亡，而在转染miR-485抑制剂的ASMC中观察到相反的结果。
在慢性哮喘小鼠中，过表达的miR-485通过Smurf2介导的TGF-β/Smads信号通路抑制ASMC增殖并促进其凋亡。
