Zhou Yao, Yang Qianyuan, Xu Hong, Zhang Jiamin, Deng Huan, Gao Haiyan, Yang Jin, Zhao Deyu, Liu Feng
Department of Respiratory Medicine, Nanjing Children's Hospital Affiliated to Nanjing Medical University, Nanjing, Jiangsu, China 210008.
Department of Pediatrics, Jiangsu Huai'an Maternity and Children's Hospital, Huai'an, Jiangsu, China 223002.
PLoS One. 2016 Feb 22;11(2):e0148821. doi: 10.1371/journal.pone.0148821. eCollection 2016.
Mast cells play a central role in asthma. Moreover, serum miRNA-221-3p (miR-221) has been shown to be markedly increased in children with asthma. In the current study, we aimed to examine miR-221 expression in an asthma model and elucidate the mechanisms regulating interleukin (IL)-4 secretion in mast cells. Using polymerase chain reaction, we found that miR-221 was upregulated in a murine asthma model and in P815 mast cells after lipopolysaccharide (LPS) stimulation. Moreover, miR-221 upregulated IL-4 secretion from P815 cells, as shown by enzyme-linked immunosorbent assays. Bioinformatics analysis, luciferase reporter gene assays, and western blotting showed that phosphatase and tensin homolog (PTEN) was a target of miR-221 and could block IL-4 secretion stimulated by miR-221. The phosphorylation of p38 (protein) and activity of nuclear factor-kappaB (NF-κB) were increased after overexpression of miR-221, as shown by electrophoretic mobility shift assays. Finally, treatment with specific inhibitors could block IL-4 secretion. In conclusion, miR-221, which was overexpressed in a murine asthma model, stimulated IL-4 secretion in mast cells through a pathway involving PTEN, p38, and NF-κB.
肥大细胞在哮喘中起核心作用。此外，血清微小RNA-221-3p（miR-221）已被证明在哮喘儿童中显著增加。在本研究中，我们旨在检测哮喘模型中miR-221的表达，并阐明调节肥大细胞中白细胞介素（IL）-4分泌的机制。通过聚合酶链反应，我们发现miR-221在小鼠哮喘模型和脂多糖（LPS）刺激后的P815肥大细胞中上调。此外，酶联免疫吸附测定显示，miR-221上调了P815细胞中IL-4的分泌。生物信息学分析、荧光素酶报告基因测定和蛋白质印迹表明，磷酸酶和张力蛋白同源物（PTEN）是miR-221的靶标，并且可以阻断miR-221刺激的IL-4分泌。电泳迁移率变动分析显示，miR-221过表达后，p38（蛋白）的磷酸化和核因子-κB（NF-κB）的活性增加。最后，用特异性抑制剂处理可阻断IL-4分泌。总之，在小鼠哮喘模型中过表达的miR-221通过涉及PTEN、p38和NF-κB的途径刺激肥大细胞中IL-4的分泌。
