TLR2通路microRNA结合位点SNP与哮喘易感关系研究

Asthma is one of the most common chronic respiratory diseases. It is a complex genetic disease resulting from interactions between multiple genes and environmental factors. MicroRNAs are novel regulators of gene expression that act via hybridization to the 3′-untranslated region (UTR) of target mRNA molecules, leading to the degradation of mRNA or repression of translation. Thus although the field is still in an early stage of development, it has great potential to reveal a new level of epigenetic regulation. Moving forward, it is important to pursue studies consisting of larger patient populations with accompanying high accuracy and excellent quality of clinical data. We supposed that microRNA and its binding site polymorphisms in toll-like receptor 2 signal pathway may contribute to the risk of asthma. To test the hypothesis, we will carry out a case-control study in two different Chinese Han populations (the test set and the validation set) to find susceptive polymorphisms based on our previous study. Then functional consequences were examined by using luciferase reporter assay to determine the association of miRNA and binding site SNPs and risk of asthma. It is possible that miRNA binding site SNPs may bring a fundamental change to our understanding of the pathophysiology of asthma. This may then lead to the development of novel personalized therapeutic strategies in asthma.

哮喘系受遗传因素和环境因素的双重影响的慢性气道炎症性疾病。研究发现，microRNA作为基因表达新调控者，主要通过与靶基因的3'UTR结合而作用。microRNA靶基因结合位点多态性与哮喘遗传易感性的研究还为一相对空白的领域。本项目在已建立哮喘样本资源库和前期研究的基础上，拟针对TLR2信号通路相关microRNA靶基因相关位点，开展关联研究。课题组将（1）完成两期独立的病例对照研究（总例数约1200例），筛选真正的microRNA靶基因相关位点；（2）通过荧光素酶重组载体法，结合生物信息学方法进行验证及功能分析，研究microRNAs和SNPs对目标基因表达影响，功能性论证风险多态致哮喘的分子机制及以及个体化治疗的可行性。

支气管哮喘系多基因参与的具有遗传易感性的慢性气道炎症性疾病，是当今世界最常见的慢性气道炎症性疾病之一，其发病率逐年增高，已成为严重的公共卫生问题。本课题组首先研究我国人群TLR2通路基因多态性（SNPs）在哮喘发病机制中的作用。研究发现了5个有意义的阳性位点，其中AKT2基因的2个SNP（rs33933140及rs2304186）基因型频率分布在哮喘组与对照组之间有明显差异。利用生物软件预测分析发现AKT2基因rs2304186 G碱基突变与hsa-miR-625结合而发挥生物学效应。根据软件预测miRNA-625与 AKT2 3’-UTR互补配对，成功构建了野生型与突变型荧光素酶报告基因质粒，利用双荧光报告基因检测系统检测其基因活性，证实miRNA-625 能够与AKT2 3’-UTR 互补配对结合，转录后抑制AKT2 基因的表达。在此基础上课题组进一步进行了人支气管上皮细胞（16HBE）功能验证，通过体外培养的16HBE细胞, LPS诱导细胞活化，分别上调和下调miR-625-5P，验证了miR-625-5P通过靶向AKT2的3’-UTR区，从而下调AKT2的蛋白表达水平。miR-625-5P可下调AKT2和IkBα的磷酸化，抑制NF-kB信号通路的活化。最后，探讨虫草素对哮喘炎症防治的作用机理，通过虫草素对体外培养支气管上皮细胞进行处理，发现虫草素能下调AKT2信号通路，从而达到抑制支气管上皮细胞炎症的作用。本研究筛选出了影响哮喘发病及其相关表型的基因以及预测哮喘的新型指标，对进一步评价和识别遗传与环境因素在哮喘中的交互作用，阐明哮喘发生的分子遗传学机制具有重要的理论意义，有望为将来哮喘的防治提供新的思路和策略。