环状RNA通过调控CD4+T细胞活化参与支气管哮喘发病机制的研究

Circular RNA (circRNA) is cyclic non-coding RNA that plays an important role in a variety of life activities such as cell proliferation and differentiation. Recent studies have shown that circRNA may participate in the cellular immune response, but its role in bronchial asthma inflammation has not been reported. CircRNA microarray was adopted to detect the circRNA in peripheral blood CD4+T cells from bronchial asthma patients and healthy volunteers. Compared with the healthy volunteers, a higher level of hsa_circ_0005519 was observed in peripheral blood CD4+ T cells from asthmatic patients. This study was to examine the expression of hsa_circ_0005519 by expanding the clinical samples, and analyze its correlation with the clinical symptoms of asthmatic patients. Besides, it was also to clarify whether it was involved in the pathogenesis as well as the mechanism of asthma. In the study, we used bioinformatics method to predict and construct the RBPs-circRNA-miRNA-mRNA/gene network, RNA-FISH and then designed gene overexpression and knockdown to perform the intervention experiment in vitro，to which clarify the function of the hsa_circ_0005519 participating in the upstream and downstream mechanisms of asthmatic inflammation by regulating CD4+ T cell-related pathway. The this study will help to confirm that circRNA may take part in the pathogenesis of asthma and further understand the molecular mechanism of airway inflammation and to find new potential bio-markers and targets of asthma, which is significant for the diagnosis and treatment methods.

环状RNA（circRNA）是环状的非编码RNA，在细胞的多种生命活动中发挥重要作用。研究表明circRNA可能参与免疫应答过程，但其在支气管哮喘（简称哮喘）免疫炎症中的作用尚未见报道。我们对哮喘患者和健康志愿者外周血CD4+T细胞提取RNA进行circRNA微阵列检测，发现在哮喘患者中 hsa_circ_0005519的表达较健康志愿者明显上调。本研究拟通过扩大临床样本检测 hsa_circ_0005519的表达，分析其与哮喘患者临床特征的相关性；通过生物信息学方法预测并构建相关调控网络以及RNA-FISH，然后进行基因过表达与敲减的细胞干预实验，研究 hsa_circ_0005519通过调控CD4+T细胞活化参与哮喘免疫炎症的上下游机制。本课题的实施将首次阐明circRNA在哮喘发病机制中的作用，进一步了解哮喘免疫炎症的分子机制，寻找新的哮喘生物学标志物和干预靶点，具有重要的科学意义。

环状RNA（circular RNA, circRNA）是环状的非编码RNA，在细胞的多种生命活动中发挥重要作用。研究表明circRNA可能参与免疫应答过程，但其在哮喘免疫炎症中的作用尚未见报道。本项目主要以hsa_circ_0005519和hsa_circ_0002594调控CD4+T细胞活化以及在哮喘诊断中的作用开展研究。通过circRNA检测芯片发现circRNA的表达谱在哮喘患者外周血CD4+ T细胞中发生了明显改变，其中急性发作期哮喘患者与健康志愿者相比有264个circRNA分子表达上调以及333个表达下降。在哮喘患者急性期CD4+ T细胞中has_circ_0005519的表达较健康志愿者明显上调，采用腺病毒载体敲低哮喘患者急性期CD4+T细胞中has_circ_0005519表达后，发现CD4+T细胞产生IL-13和IL-6等细胞因子明显降低。进一步研究发现has_circ_0005519是通过吸附has_let_7a_5p调控IL-13和IL-6的表达。进而本项目提出，在CD4+ T细胞中存在 “has_circ_0005519/has_let_7a_5p/IL-13&IL-6”参与哮喘的发病。通过对哮喘患者临床资料相关性分析发现，以正常对照组CD4+ T细胞中has_circ_0005519表达的75%位数作为界值，可将哮喘患者分为has_circ_0005519低和高2个亚型。这2个亚型之间，IL-13、IL-6、血嗜酸性粒细胞数目和呼出气一氧化氮（FeNO）水平均存在显著差异，这表明has_circ_0005519可作为哮喘分子表型分型新的指标，为哮喘的临床诊断和治疗提供更多的依据。.此外，本项目发现在哮喘患者外周血CD4+T 细胞中hsa_circ_0002594表达水平显著升高。通过对未使用ICS治疗的83位哮喘患者相关性分析发现，hsa_circ_0002594的表达在有哮喘家族史或皮肤点刺试验阳性的哮喘患者中显著升高，进一步研究发现hsa_circ_0002594水平与FeNO呈正相关，与乙酰甲胆碱PD20呈负相关。进一步研究发现与ICS治疗前相比，ICS治疗后哮喘患者CD4+T细胞中hsa_circ_0002594的水平明显降低。本项目首次在哮喘外周血CD4+T细胞中阐明circRNA的作用，并为寻找新的分子标志物提供实验依据。