miR-149-5p对变应性鼻炎小鼠Notch1表达和Treg/Th17免疫平衡的影响  

Effect of miR-149-5p on Expression of Notch1 and Immune Balance of Treg/Th17 in Mice with Allergic Rhinitis

作　　者：闫智永[1] 张爽[1] 唐桥斐[1] YAN Zhiyong;ZHANG Shuang;TANG Qiaopei(Department of Otolaryngological,the Second Affiliated Hospital of Shenyang Medical College,Shenyang 110002,Liaoning,China)

机构地区：[1]沈阳医学院附属第二医院耳鼻喉科,辽宁沈阳110002

出　　处：《贵州医科大学学报》2020年第6期660-667,共8页Journal of Guizhou Medical University

基　　金：辽宁省科学技术计划项目(2018225020);沈阳医学院科技基金项目(20182038)。

摘　　要：目的:探讨miR-149-5p对变应性鼻炎(AR)小鼠神经源性位点缺口同系物蛋白1(Notch1)表达和调节性T细胞(Treg)/辅助性T细胞17(Th17)失衡的影响。方法:12只BALB/c小鼠,3只为正常组,余9只小鼠采用卵清蛋白(OVA)和氢氧化铝[Al(OH)3]建立AR模型,再随机均分为模型组(仅造模)、miR-149-5p激动剂对照组(鼻内滴注miR-149-5p agomir阴性对照)及miR-149-5p激动剂组(鼻内滴注miR-149-5p agomir);采用行为学评分考察各组各组小鼠过敏症状、苏木精/伊红染色(HE)分析各组小鼠鼻黏膜病理学变化、实时荧光定量PCR(q PCR)检测各组小鼠鼻黏膜组织miR-149-5p,采用酶联免疫吸附法(ELISA)分析各组小鼠血清免疫球蛋白E(Ig E)、白细胞介素-6(IL-6)、白细胞介素-10(IL-10)水平,采用蛋白印迹法检测各组小鼠鼻黏膜组织Notch1、维甲酸相关核孤儿受体γt(RORγt)及叉头状螺旋转录因子3(Foxp3)蛋白表达;利用miRDB网站预测miR-149-5p与Notch1 3’-UTR结合位点,构建Notch1 3’-UTR区野生型(WT)和突变型(MUT)荧光素酶报告载体,转染293T细胞,设置NC+Notch1(MUT)组、miR-149-5p agomir+Notch1(MUT)组、NC+Notch1(WT)组及miR-149-5p agomir+Notch1(WT)组,采用双荧光素酶实验分析并计算荧光素酶相对活性。结果:与模型组比较,miR-149-5p激动剂组小鼠行为学评分明显下降,鼻黏膜炎症反应减轻,上皮结构趋于完整,miR-149-5p的表达显著增加,血清中Ig E、IL-6水平下降,IL-10水平升高,Foxp3蛋白的表达增加,Notch1和RORγt蛋白的表达减少(P <0. 05或P <0. 01);miRDB网站预测miR-149-5p与Notch1 3’-UTR存在互补位点,双荧光素酶实验结果显示,miR-149-5p agomir+Notch1(WT)组的荧光素酶相对活性较NC+Notch1(WT)组降低(P <0. 05)。结论:miR-149-5p可能通过靶向Notch1调节Foxp3和RORγt蛋白表达,影响Treg/Th17细胞平衡,从而改善AR小鼠鼻部过敏症状。Objective:To investigate the effect of miR-149-5p on neurogenic locus notch homolog protein 1(Notch1)and regulatory cells(Treg)/T helper cell 17(Th17)imbalances of allergic rhinitis(AR)mice.Methods:12 BALB/c mice of which 3 mice were in the normal group,and the remaining 9 mice were treated with ovalbumin(OVA)and aluminum hydroxide[Al(OH)3]to establish the AR model.They were randomly divided into the model group(for modeling only),the miR-149-5p agonist control group(intranasal infusion of miR-149-5p agomir negative control)and the miR-149-5p agonist group(intranasal infusion of miR-149-5p agomir).The behavioral score was used to investigate the allergic symptoms of the mice in each group.Hematoxylin eosin staining(HE)was used to analyze the pathological changes of nasal mucosa in mice,Quantitative Real-time PCR(qPCR)was used to detect miR-149-5p in the nasal mucosal tissues of the mice.Serum levels of Immunoglobulin E(IgE),interleukin 6(IL-6)and interleukin 10(IL-10)were analyzed by enzyme linked immunosorbent assay(ELISA),and protein expression of Notch1,retinoic acid-related orphan nuclear receptorγt(RORγt)and fork-head box p3(Foxp3)in the nasal mucosal tissues of the mice were detected by Western blot(WB).The putative interaction site between miR-149-5p and Notch13'-UTR was predicted using miRDB site.Luciferase reporter vector with 3'-UTR containing wild-type or mutant miR-149-5p response elements from Notch1 was constructed and transfected into 293T cells.The NC+Notch1(MUT)group,miR-149-5p agomir+Notch1(MUT)group,NC+Notch1(WT)group and miR-149-5p agomir+Notch1(WT)group were set up.The relative luciferase activity was analyzed by double luciferase assay.Results:Compared with the model group,the behavior score of the miR-149-5p agonist group was significantly decreased,the inflammatory response of the nasal mucosa was reduced,the epithelial structure tended to be intact,the expression of miR-149-5p was significantly increased,the serum IgE and IL-6 levels decreased,the IL-10 levels increased,the expression o

关 键 词：变应性鼻炎 miR-149-5 p基因 神经源性位点缺口同系物蛋白1 辅助性T细胞17 调节性T细胞 叉头状螺旋转录因子3 维甲酸相关核孤儿受体γt 

分 类 号：R765.21[医药卫生—耳鼻咽喉科]