目的: 探讨miR-181a过表达通过调控调节性T细胞（regulatory T cells,Tregs）参与变应性鼻炎（allergic rhinitis,AR）的机制。方法: 选择20名AR患儿和20例年龄相近,无过敏史的儿童为研究对象。Pearson相关分析外周血Tregs数量与miR-181a表达之间关系。从AR患儿外周血中分离纯化Tregs。将miR-181a模拟物和抑制剂转染到Tregs中。用流式细胞仪和ELISA法评价Tregs的功能。将小鼠分为7组,每组10只：对照组、AR组、AR+阴性对照组、AR+miR-181a模拟组、AR+miR-181a抑制剂组、AR+miR-181a模拟+重组骨桥蛋白（osteopontin,OPN）组、AR+miR-181a模拟+OPN的慢病毒shRNA组。用卵清蛋白建立AR小鼠模型,然后通过功能增益和功能损失法确定miR-181a在AR中的作用。结果: 与对照组相比,AR患儿外周血Tregs数量减少,并且miR-181a表达显著降低。Pearson相关分析显示外周血Tregs数量与miR-181a表达呈显著正相关（R2=0.335,P<0.001）。miR-181a模拟物促进了Tregs的增殖,并上调了IL-10和TGF-β的mRNA表达。荧光素酶报告试验表明,miR-181a直接靶向OPN的3'UTR。在动物实验中,与AR和AR+miR-NC组相比,AR+miR-181a模拟组炎症减轻,AR+miR-181a抑制剂组炎症更严重。重组OPN蛋白显著逆转了miR-181a模拟物的抗炎作用。此外,AR组小鼠Tregs明显减少,miR-181a模拟物显著增加了AR小鼠的Treg细胞,而重组OPN蛋白显著逆转了miR-181a模拟物诱导的Tregs增多。结论: miR-181a的上调显著降低了OPN的表达,进而减少了嗜酸性粒细胞和增强Tregs功能,减轻了AR发病过程中气道炎症的发生。
Abstract
Objective To explore the mechanism of miR-181a overexpression in regulating Treg cells to participate in allergic rhinitis (AR). Methods 20 children with AR and 20 children with similar age and without allergic history were selected as the research objects. Pearson correlation analysis was used to analyze the relationship between the number of Treg and the expression of miR-181a. The Tregs were purified from PBMCs in AR children. The miR-181a mimics and inhibitors were transfected into Tregs. The function of Tregs were evaluated by flow cytometry and ELISA assay. Mice were divided into 7 groups, with 10 mice in each group: control group, AR group, AR+ negative control group, AR+miR-181a simulation group, AR+miR-181a inhibitor group, AR+miR-181a simulation+recombinant OPN protein group, and AR+miR-181a simulation +OPN lentivirus shRNA group. AR models were established using ovalbumin, after which the functional role of miR-181a in AR was determined using gain- and loss-of-function approaches. Results Compared with the control group, the number of Treg and the expression of miR-181a in the PBMCs of patients with AR were significantly decreased. Pearson correlation analysis showed that the number of Treg in PBMCs was positively correlated with the expression of miR-181a (R2=0.335, P<0.001). MiR-181a mimics promoted the proliferation of Tregs and upregulated the mRNA expression of IL-10 and TGF-β. Luciferase assay showed that miR-181a directly targeted the 3＇UTR of OPN. In animal experiments, compared with AR and AR + miR-NC group, the inflammation in AR + miR-181a simulation group was reduced, and the inflammation in AR + miR-181a inhibitor group was more serious. The recombinant OPN protein significantly reversed the anti-inflammatory effect of miR-181a mimic. In addition, Treg cells in AR group decreased significantly, and miR-181a mimic significantly increased the number of Treg cells in AR mice, while the recombinant OPN protein significantly reversed the increase of Treg cells induced by miR-181a mimic. Conclusion Upregulation of miR-181a significantly decreases the expression of OPN, and subsequently decreases eosinophils and enhances Treg function, by which it contributes to reduce the airway inflammation in the pathogenesis of AR.
关键词
miR-181a /
变应性鼻炎 /
小鼠 /
调节性T细胞 /
骨桥蛋白
Key words
miR-181a /
allergic rhinitis /
mouse /
regulatory T cells /
osteopontin
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基金
陕西省自然科学基金“MiR-181a过表达参与调控Treg细胞参与变应性鼻炎的机制研究”（2019-z-624）
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