MiR-200a and miR-200b restrain inflammation by targeting ORMDL3 to regulate the ERK/MMP-9 pathway in asthma

Asthma is one of the most frequent and serious diseases worldwide. Inflammation has been reported to correlate with airway remodeling, which is critical for the progression of asthma. Better understanding of novel molecules modulating asthma and the underlying mechanism will benefit explorations of new treatments. <b>Method:</b> To explore the role of miR-200a and miR-200b in asthma, miR-200a mimics/inhibitor and miR-200b mimics/inhibitor were employed in A549 cells, respectively. Expression levels of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1β, were measured by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). A dual luciferase reporter assay was performed to identify whether miR-200a/200b directly bound to Orosomucoid 1-like 3 (ORMDL3). ERK, p-ERK and MMP-9, involved in downstream pathways of ORMDL3, were detected using qRT-PCR and western blotting. <b>Results:</b> MiR-200a/200b silencing significantly increased the expression of inflammatory cytokines, including TNF-α, IL-4, IL-5, IL-13 and IL-1β, in A549 cells. ORMDL3 was the target gene of miR-200a/200b, with high expression levels in miR-200a inhibitor and miR-200b inhibitor groups. MiR-200a and miR-200b played synergistic roles in the regulation of the inflammatory effect in A549 cells. Expression levels of p-ERK and MMP-9 were significantly increased in miR-200a inhibitor and miR-200b inhibitor groups and were rescued by ERK inhibitor and MMP-9 inhibitor, respectively. <b>Conclusion:</b> These findings suggest that miR-200a and miR-200b are required to regulate asthma inflammation. Reduction in miR-200a/200b promotes the development of asthma inflammation by targeting ORMDL3 to activate the ERK/MMP-9 pathway. Therefore, elevating miR-200a and miR-200b and decreasing ORMDL3 might be potential strategies for inhibition of the asthma process.

哮喘是全球范围内最常见且严重的疾病之一。已有研究表明，炎症与气道重塑密切相关，而气道重塑对哮喘的病情进展至关重要。深入解析调控哮喘的新型分子及其潜在作用机制，将有助于探索全新的哮喘治疗方案。<b>方法：</b>为探究miR-200a与miR-200b在哮喘中的作用，本研究分别采用miR-200a模拟物（mimics）/抑制剂（inhibitor）以及miR-200b模拟物/抑制剂处理A549细胞。采用实时定量聚合酶链反应（quantitative real time polymerase chain reaction, qRT-PCR）与酶联免疫吸附试验（enzyme-linked immunosorbent assay, ELISA）检测肿瘤坏死因子-α（TNF-α）、白细胞介素-4（IL-4）、白细胞介素-5（IL-5）、白细胞介素-13（IL-13）及白细胞介素-1β（IL-1β）等炎性细胞因子的表达水平。采用双荧光素酶报告基因实验验证miR-200a/200b是否可直接结合类黏蛋白1样3（Orosomucoid 1-like 3, ORMDL3）。采用qRT-PCR与蛋白质印迹法（western blotting）检测ORMDL3下游通路相关的细胞外调节蛋白激酶（ERK）、磷酸化细胞外调节蛋白激酶（p-ERK）及基质金属蛋白酶-9（MMP-9）的表达水平。<b>结果：</b>沉默miR-200a/200b可显著上调A549细胞中TNF-α、IL-4、IL-5、IL-13及IL-1β等炎性细胞因子的表达水平。ORMDL3是miR-200a/200b的靶基因，在miR-200a抑制剂组与miR-200b抑制剂组中其表达水平显著升高。miR-200a与miR-200b在调控A549细胞炎性反应中具有协同作用。miR-200a抑制剂组与miR-200b抑制剂组中p-ERK及MMP-9的表达水平显著升高，且可分别被ERK抑制剂与MMP-9抑制剂逆转。<b>结论：</b>本研究结果表明，miR-200a与miR-200b可调控哮喘炎性反应。miR-200a/200b表达下调可通过靶向ORMDL3激活ERK/MMP-9通路，进而促进哮喘炎性反应的发生发展。因此，上调miR-200a与miR-200b的表达、下调ORMDL3的水平，可能成为抑制哮喘病程进展的潜在治疗策略。