贵州医科大学学报

2020, v.45;No.237(06) 660-667

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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

闫智永;张爽;唐桥斐;
YAN Zhiyong;ZHANG Shuang;TANG Qiaopei;Department of Otolaryngological ,the Second Affiliated Hospital of Shenyang Medical College;

摘要(Abstract):

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-149-5 p 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-149-5 p agonist control group( intranasal infusion of miR-149-5 p agomir negative control) and the miR-149-5 p agonist group( intranasal infusion of miR-149-5 p 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-149-5 p 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 receptor γt( 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-149-5 p and Notch1 3'-UTR was predicted using miRDB site. Luciferase reporter vector with 3'-UTR containing wild-type or mutant miR-149-5 p response elements from Notch1 was constructed and transfected into 293 T cells.The NC + Notch1( MUT) group,miR-149-5 p agomir + Notch1( MUT) group,NC + Notch1( WT)group and miR-149-5 p 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-149-5 p 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-149-5 p was significantly increased,the serum IgE and IL-6 levels decreased,the IL-10 levels increased,the expression of Foxp3 protein increased,and the expression of Notch1 and RORγt protein decreased( P <0. 05 or P < 0. 01). The miRDB site predicted the existence of a complementary site between miR-149-5 p and Notch1 3 '-UTR. The results of double luciferase experiment showed that the relative activity of luciferase in miR-149-5 p agomir + Notch1( WT) group was lower than that in NC + Notch1( WT) group( P <0. 05). Conclusion: miR-149-5 p may regulate the expression of Foxp3 and RORγt proteins by targeting Notch1,affecting the Treg/Th17 cell balance,and thereby improving nasal allergy symptoms in AR mice.

关键词(KeyWords): 变应性鼻炎;miR-149-5p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
allergic rhinitis(AR);miR-149-5p gene;neurogenic locus notch homolog protein 1(Notch1);T helper cell 17(Th17);regulatory T cells(Treg);fork-head box p3(Foxp3);tretinoic acid-related orphan nuclear receptor γt(RORγt)

Abstract:

Keywords:

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

作者(Author): 闫智永;张爽;唐桥斐;
YAN Zhiyong;ZHANG Shuang;TANG Qiaopei;Department of Otolaryngological ,the Second Affiliated Hospital of Shenyang Medical College;

Email:

DOI: 10.19367/j.cnki.2096-8388.2020.06.008

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