Liu Ying, Miao Yi, Gao Xin, Wang Yuan-Yuan, Wang Huan, Zheng Ya-Wen, Zhao Zhi-Yuan
Cell Physiol Biochem. 2018;50(6):2365-2389. doi: 10.1159/000495097. Epub 2018 Nov 13.
BACKGROUND/AIMS: The etiology of asthma, which is a complicated disorder with various symptoms, including wheezing, coughing, and airflow obstruction, remains poorly understood. In addition, the effects of microRNAs (miRs) have not been explored. This study explored the effect of microRNA-200a (miR-200a) on airway smooth muscle cells (ASMCs) and airway remodeling in asthmatic mice. Furthermore, we speculated that miR-200a achieves its effect by targeting FOXC1 via the PI3K/AKT signaling pathway based on differentially expressed gene screening, target miR predictions and a bioinformatics analysis.
Eighty mice were assigned to a saline group or an ovalbumin (OVA) group, and the OVA group was transfected with a series of inhibitors, activators, and siRNAs to test the established mouse model. Airway reactivity and the ratio of eosinophils (EOSs) to leukocytes were detected. An ELISA was adopted to measure the levels of interleukin (IL)-4, IL-6, IL-8, tumor necrosis factor (TNF)-α, and immunoglobulin E (IgE). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to determine the expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1, TGF-β1 and p-AKT in ASMCs. Finally, CCK-8 assays were performed to detect cell proliferation and flow cytometry to detect apoptosis and cell cycle entry.
The bioinformatics analysis indicated that miR-200a mediated the PI3K/AKT signaling pathway by targeting FOXC1. In addition, mouse models of asthma were established. An elevated expression of miR-200a, a decreased mRNA and protein expression of FOXC1, PI3K, AKT, NF-κB, cyclin D1 and TGF-β1, a decreased expression of p-AKT, suppressed cell proliferation, accelerated apoptosis, and an increased number of cells at the G0/G1 phase were observed following the upregulation of miR-200a and downregulation of FOXC1.
The overexpression of miR-200a may downregulate FOXC1, thereby inhibiting the activation of the PI3K/AKT signaling pathway and ultimately suppressing ASMC proliferation and airway remodeling in asthmatic mice. This evidence supports the potential that miR-200a represents a new approach to treating asthma.
背景/目的：哮喘是一种复杂的疾病，有喘息、咳嗽和气流阻塞等多种症状，其病因仍知之甚少。此外，微小RNA（miR）的作用尚未得到探索。本研究探讨了微小RNA-200a（miR-200a）对哮喘小鼠气道平滑肌细胞（ASMCs）和气道重塑的影响。此外，基于差异表达基因筛选、靶miR预测和生物信息学分析，我们推测miR-200a通过PI3K/AKT信号通路靶向FOXC1发挥其作用。
将80只小鼠分为生理盐水组或卵清蛋白（OVA）组，对OVA组小鼠转染一系列抑制剂、激活剂和小干扰RNA（siRNA）以验证所建立的小鼠模型。检测气道反应性以及嗜酸性粒细胞（EOSs）与白细胞的比例。采用酶联免疫吸附测定（ELISA）法检测白细胞介素（IL）-4、IL-6、IL-8、肿瘤坏死因子（TNF）-α和免疫球蛋白E（IgE）的水平。运用逆转录定量聚合酶链反应（RT-qPCR）和蛋白质免疫印迹法检测ASMCs中FOXC1、PI3K、AKT、核因子κB（NF-κB）、细胞周期蛋白D1、转化生长因子（TGF）-β1和磷酸化AKT（p-AKT）的表达。最后，进行细胞计数试剂盒-8（CCK-8）检测以检测细胞增殖，采用流式细胞术检测细胞凋亡和细胞周期进程。
生物信息学分析表明，miR-200a通过靶向FOXC1介导PI3K/AKT信号通路。此外，成功建立了哮喘小鼠模型。上调miR-200a并下调FOXC1后，观察到miR-200a表达升高，FOXC1、PI3K、AKT、NF-κB、细胞周期蛋白D1和TGF-β1的mRNA和蛋白表达降低，p-AKT表达降低，细胞增殖受到抑制，细胞凋亡加速，处于G0/G1期的细胞数量增加。
miR-200a的过表达可能下调FOXC1，从而抑制PI3K/AKT信号通路的激活，最终抑制哮喘小鼠ASMCs的增殖和气道重塑。这一证据支持了miR-200a可能成为治疗哮喘新方法的潜力。
