miR-146a介导STAT5B调控变应性鼻炎Treg细胞的作用机制

Previous studies have shown that STAT5B influenced the core immune response in allergic rhinitis by means of acting on the Treg cell through FOXP3. However, the regulatory mechanism of STAT5B involved in allergic rhinitis is still unclear. On the base of previous literature and studies, we found that the expression of miR146a in nasal mucosa of allergic rhinitis significantly decreased. Furthermore, the target gene prediction software tools indicate there are potentially specific binding sites between miR146a and STAT5B. Therefore,we hypothesize that the targeted regulation of miR146a to Treg cell by STAT5B is an important regulatory mechanism in allergic rhinitis. In order to verify the biological relationships between miR146a and STAT5B and reveal the mechanism how miR146a regulates STAT5B in allergic rhinitis, we propose three approaches at different levels, such as the validation in clinical tissue specimens, a variety of tests using in vitro cell lines and in vivo animal model, including immunohistochemistry, Western blot, and so on. This study is expected to explore the important issue of the gene regulation of Treg cells in allergic rhinitis from the perspective of miRNAs (e.g., miR146a). It will provide clinicians and researchers with new insights to further clarify the pathogenesis of allergic rhinitis and find new molecular targets for possible treatment and intervention in the future.

前期研究表明，STAT5B通过FOXP3作用于Treg细胞影响变应性鼻炎核心免疫反应。但在变应性鼻炎中机体调控STAT5B的机制尚不清楚。结合既往文献及前期研究基础我们发现，变应性鼻炎鼻黏膜上皮中miR146a表达显著降低，利用靶基因预测软件分析发现，miR146a与STAT5B存在潜在特异性结合位点。由此我们推测，miR146a通过STAT5B调控Treg细胞是变应性鼻炎中的重要调节机制。本研究拟从临床组织标本、细胞体外实验、动物模型三个层面通过免疫组化、蛋白印迹等多种试验方法，验证变应性鼻炎中miR146a与STAT5B的关联性，揭示miR146a调节STAT5B进一步影响变应性鼻炎的机制。本项目有望从miRNA的角度探索在基因水平调控变应性鼻炎Treg细胞的重要问题，有助于进一步阐明变应性鼻炎的发病机制，为将来建立可能的干预手段寻找到新的分子靶点。

变应性鼻炎（AR）是由特异性IgE介导的I型变态反应，是由2型细胞（TH2）相对增加介导的异常免疫反应引起的。调节性T细胞（Treg）可以直接抑制变应原特异性TH2细胞的活化。微小RNA（miRNA）通过抑制靶基因翻译以及靶向信使RNA（mRNA）降解来介导基因表达的转录后调控。miR-146a在Treg的功能维持和分化中起着重要作用，其机理仍知之甚少。STAT5b在人类淋巴细胞的发育和分化以及Treg的维持中起重要作用。因此我们推测，miR-146a可能通过调节STAT5b的表达和STAT5b的磷酸化而在免疫调节中发挥作用。我们采用RT-PCR和Western blot方法检测变应性鼻炎患者鼻黏膜组织中miR-146a和STAT5b的表达。通过感染慢病毒，构建miR-146a敲低细胞和miR-146a过表达的细胞株。通过RT-PCR和Western blot分析检测miR-146a和STAT5b的表达，通过流式细胞术检测Treg的细胞比例，用ELISA试剂盒测定细胞培养上清液中的IL-10和TGF-β的表达。我们发现AR组鼻黏膜中miR-146a和STAT5b的表达明显低于对照组（P<0.001，P=0.02），STAT5b在miR-146a过表达的细胞中升高（P<0.01），在miR-146a敲低的细胞中降低（P<0.01），STAT5b的基因表达与miR-146a呈正相关。miR-146a过表达组IL-10和TGF-β的表达高于对照组（P=0.44，P<0.01）。综上所述，我们证实了变应性鼻炎患者中miR-146a对STAT5b，p-STAT5b和Foxp3的表达具有积极的调节作用。miR-146a可以通过调节STAT5b的表达来影响Treg的分化，此外，miR-146a可以促进抗炎细胞因子的释放。本课题的研究结果为AR发病的分子机制提供重要理论基础，并可能筛选出对AR发病有意义的分子标记物并实施早期干预，为AR的早期预防及分子靶向治疗提供理论依据。