IRF-3介导RSV调控哮喘易感基因ORMDL3表达的分子机制研究

Orosomucoid 1-like protein 3 (ORMDL3) is a potential asthma candidate gene and is involved in the pathogenesis of asthma. The expression level of the ORMDL3 gene was upregulated in asthma and recurrent wheeze patients. Respiratory syncytial virus (RSV) infection was regarded as an important dangerous factor in the development and exacerbation of asthma and recurrent wheeze. But the molecular mechanisms involved in ORMDL3 regulation have not been fully elucidated. Our preliminary studies showed that poly (I:C) upregulated the expression levels of ORMDL3 gene. The transcription factor binding site of IRF-3 in the promoter region of ORMDL3 played an important role in the upregulation of ORMDL3 by poly (I:C). So we hypothesized that the regulatory effects of poly (I:C) and RSV infection to the expression of ORMDL3 were mediated by IRF-3. This study will research the relationship among the expression levels of IRF-3, ORMDL3 and airway inflammation in RSV-infected mouse asthma model in vivo. It will also demonstrate the regulatory effects of IRF-3, poly (I:C) and RSV infection to the promoter activities, the expression levels of RNA and protein of ORMDL3, and the binding of IRF-3 with the promoter by many experimental techniques of molecular biology. After using the method of knockdown of IRF-3 by siRNA, we will observe the changes of the promoter activities, the binding of IRF-3 with the promoter and the expression levels of ORMDL3 after the stimulation by poly (I:C) and RSV infection. This study may reveal a new mechanism among RSV, IRF-3, ORMDL3 and asthma and may provide experimental basis for the prevention of virus-inducted asthma and development of new targets for the therapy of asthma.

ORMDL3 是哮喘易感基因，与哮喘发病相关，哮喘和反复喘息患儿该基因表达增高，呼吸道合胞病毒 (RSV) 可诱发和加重哮喘和反复喘息，但机制不详。我们前期研究表明RNA病毒类似物poly (I:C) 可上调 ORMDL3 基因表达，该基因启动子区 IRF-3 位点在其中起重要作用，因此假设 poly (I:C) 和 RSV 可通过启动子区 IRF-3 位点调控 ORMDL3 的表达。本课题拟通过多种分子生物学实验技术和动物实验研究 RSV 对哮喘小鼠 IRF-3、ORMDL3 表达及气道炎症的影响；在细胞水平论证 poly (I:C) 和 RSV 对 ORMDL3 启动子、RNA和蛋白表达的调节作用及 IRF-3 在其中的介导作用。本研究将有助于阐明 RSV、IRF-3、ORMDL3 和哮喘之间的联系，为防治病毒诱发哮喘及研发哮喘药物新靶点提供理论和实验依据。

ORMDL3是哮喘易感基因，与哮喘发病相关。哮喘和反复喘息患儿该基因表达增高，呼吸道合胞病毒可诱发和加重哮喘及反复喘息，但机制不详。本课题的目的是研究RSV感染和poly (I:C) 是否通过ORMDL3启动子区的IRF-3结合位点调控ORMDL3的表达，进而参与哮喘和反复喘息的发病。方法：通过过表达或敲低转录因子、启动子萤光素酶活性分析、定量RT-PCR和Western blot等技术研究IRF-3对ORMDL3基因启动子、RNA和蛋白表达的影响，凝胶迁移试验 (electrophoretic mobility shift assay, EMSA) 和染色质免疫沉淀 （chromatin immunoprecipitation, ChIP） 法论证IRF-3与 ORMDL3 启动子的结合，观察poly (I:C)、RSV感染对ORMDL3的RNA和蛋白表达、启动子活性和IRF-3与启动子结合的影响，siRNA抑制IRF-3表达后，观察poly (I:C)、RSV感染对ORMDL3表达、启动子活性和IRF-3与 ORMDL3 启动子结合作用的影响。结果：1、在细胞中，RSV感染和poly (I:C) 能上调ORMDL3的RNA和蛋白表达，以及ORMDL3的启动子活性。2、ORMDL3启动子中的IRF-3结合位点在介导RSV感染和poly (I:C) 的上调ORMDL3的RNA和蛋白表达，以及ORMDL3的启动子活性中发挥重要作用。3、ORMDL3在哮喘小鼠肺组织中的表达较正常小鼠明显升高。反复气喘儿童外周血Cbl-b 的表达较正常儿童低，而ORMDL3的表达高于正常儿童，两者之间存在负相关。Cbl-b可通过STAT6调控哮喘易感基因ORMDL3的启动子活性和mRNA的表达。4、3岁以下反复喘息儿童外周血ORMDL3基因表达水平较正常对照组儿童增高，在特应性体质患儿更为明显。结论：RSV感染和poly (I:C) 是通过ORMDL3启动子区的IRF-3结合位点调控ORMDL3的表达。本研究可望设计一些药物，这些药物针对该通路中的ORMDL3，或其上下游的其他分子，包括IRF-3、内质网应激、鞘脂类合成或T淋巴细胞分化中的关键分子，来治疗RSV感染及预防RSV感染诱发和加剧哮喘和反复喘息的发生，并可为研发哮喘药物新靶点提供理论和实验依据。