目的:过敏性鼻炎(AR)是一种常见的吸入性过敏性炎症性疾病,发病率较高。环状RNA(circularRNAs,circRNAs)已被揭示参与AR的发病机制。本研究探讨了cirMIRLET7BHG(hsa_circ_0008668)在AR进展中的生物学功能。
方法:采用卵清蛋白(OVA)暴露的人鼻上皮细胞系(HNepC)和小鼠作为AR的体外和体内模型。免疫荧光染色用于确定上皮紧密连接蛋白的表达。通过RT-qPCR和Western印迹评估靶分子水平。通过RNA-FISH和免疫荧光观察到cirMIRLET7BHG和IGF2BP1的定位。通过跨上皮电阻和异硫氰酸荧光素-葡聚糖(FD4)通透性确定上皮屏障的损伤。血清IgE浓度,sIgE,IFN-γ,通过ELISA检测IL-4和IL-5。细胞凋亡,病理变化,通过TUNEL评估鼻粘膜组织中的嗜酸性粒细胞浸润,H&E,和天狼星红染色,分别。通过RNA下拉法分析了分子机制,RIP,和MeRIP测定。
结果:在AR患者和实验模型中发现cirmMIRLET7BHG的表达增加。cirMIRLET7BHG的下调通过减轻上皮厚度来减轻OVA诱导的过敏症状,嗜酸性粒细胞浸润,凋亡,和小鼠的炎症反应。随后,circIRLET7BHG缺乏通过降低上皮通透性来预防OVA诱导的上皮屏障功能障碍,抑制紧密连接蛋白。机械上,甲基转移酶样3(METTL3)通过m6A甲基化增强cirmMIRLET7BHG表达,通过与IGF2BP1相互作用增强ADAM10mRNA的稳定性。
结论:METTL3介导的m6A修饰增加了circirmMIRLET7BHG表达,因此通过与IGF2BP1相互作用提高了ADAM10mRNA的稳定性,从而通过诱导上皮屏障功能障碍促进AR。
OBJECTIVE: Allergic rhinitis (AR) remains a frequent aspiratory allergic inflammatory disorder with a high incidence. Circular RNAs (circRNAs) have been revealed to participate in the pathogenesis of AR. This study investigated the biological function of circMIRLET7BHG (hsa_circ_0008668) in AR progression.
METHODS: Ovalbumin (OVA)-exposed human nasal epithelial cell line (HNEpC) and mice were adopted as the in vitro and in vivo models of AR. Immunofluorescence staining was used to determine epithelial tight junction protein expression. Target molecule levels were assessed by RT-qPCR and Western blotting. Localization of circMIRLET7BHG and IGF2BP1 was observed by RNA-FISH and immunofluorescence. Epithelial barrier damage was determined by transepithelial electrical resistance and fluorescein isothiocyanate-dextran (FD4) permeability. Serum concentrations of IgE, sIgE, IFN-γ, IL-4, and IL-5 were detected by ELISA. Apoptosis, pathological changes, and eosinophil infiltration in nasal mucosa tissues were evaluated by TUNEL, H&E, and Sirius red staining, respectively. Molecular mechanism was analyzed by RNA pull-down, RIP, and MeRIP assays.
RESULTS: An increased expression of circMIRLET7BHG was found in AR patients and experimental models. Down-regulation of circMIRLET7BHG attenuated OVA-induced allergic symptoms via relieving epithelial thicknesses, eosinophil infiltration, apoptosis, and inflammatory response in mice. Subsequently, circMIRLET7BHG deficiency prevented OVA-induced epithelial barrier dysfunction by reducing epithelial permeability, and inhibiting tight junction proteins. Mechanistically, methyltransferase-like 3 (METTL3) enhanced circMIRLET7BHG expression via m6A methylation, which enhanced ADAM10 mRNA stability via interaction with IGF2BP1.
CONCLUSIONS: METTL3-mediated m6A modification increased circMIRLET7BHG expression that consequently raised ADAM10 mRNA stability via interplay with IGF2BP1, thereby promoting AR by inducing epithelial barrier dysfunction.