MiRNA调控COPD大鼠肺泡巨噬细胞TLR2受体表达的作用机制研究

COPD patients are more susceptible to Aspergillus and mechanisms underling these phenomena are poorly understood. Alveolar macrophages play an important role in preventing Aspergillus invasion. Through pathogen recognition receptors such as TLR2, alveolar macrophages are able to recognize conidia and then clear them. MiRNAs can regulate immune reaction process by regulating the expression of TLRs and activation of related signal pathways. Research has found that exposure to cigarette smoke can lead to alterations in miRNAs of lung. Our previous study showed that TLR2 protein expression is failed to increase with the stimulation of Aspergillus conidia in COPD rats. The phagocytic function of alveolar macrophages is impaired in COPD rats and the release of inflammatory factors is also not as high as normal rats. We speculate that miRNA can regulate the expression of TLR2 and then affect the AM defense function to Aspergillus in COPD rats. In the present study, MiRNA array will be used to determine the miRNA profile alternation of alveolar macrophage in COPD rats. Bioinformational approaches will be used to identify miRNAs that might target TLR2. We will clarify the mechanism of miRNA regulating the expression of TLR2 with the methods of point mutations, increase or inhibit the expression of target miRNA(s). The aim of the study is to explore the molecular mechanism of inferior defense capability of alveolar macrophage to Aspergillus caused by cigarette smoke.

慢性阻塞性肺病（COPD）患者曲霉易感性增高，具体机制尚未阐明。肺泡巨噬细胞（AM）通过TLR2等模式识别受体识别并清除曲霉孢子，在曲霉防御中发挥重要作用；miRNA能通过调节TLRs的表达及信号通路的活化调控免疫效应过程，烟雾暴露影响肺组织中多种miRNAs的表达；另本项目组前期研究发现，COPD大鼠AM在孢子刺激后表面TLR2受体不能正常表达上调，吞噬曲霉孢子及释放炎症因子的能力下降。据此推测：某个/些miRNA(s)可能通过调控AM表面TLR2受体表达，影响COPD大鼠AM对曲霉的防御功能。本课题拟利用miRNA芯片技术测定COPD与正常大鼠AM中差异表达的miRNAs，通过生物信息学分析筛选可能参与TLR2受体表达调控的目标miRNA(s)，用定位点突变、增加或抑制miRNA表达等方法阐明miRNA对TLR2受体表达调控的影响机制，探索吸烟影响AM曲霉防御功能的分子机理。

慢性阻塞性肺疾病患者发生侵袭性肺曲霉病的风险在免疫正常宿主中居首位，但具体机制不清。本课题组前期研究证实烟雾暴露导致肺泡巨噬细胞(AM)表面模式识别受体TLR2不能正常表达上调，组推测某个/些miRNA可能通过调控AM表面TLR2受体表达，影响COPD大鼠AM对曲霉的防御功能。. 本研究分为动物实验与细胞实验两部分。首先通过动物实验筛选出调控COPD大鼠AMTLR2表达的miRNA。本研究利用miRNA微阵列基因芯片分析测定COPD大鼠与正常大鼠AM中差异表达的miRNA有13种（Fold change ≧2.0；P <0.05)。其中11种miRNA在COPD组表达上调,2种miRNA下调,然后对芯片结果中的差异显著且信号强度高的miRNAs予以qRT-PCR验证，结果与芯片一致。利用miRNA靶基因预测软件分析筛选出rho-miR-344b-1-3p可能靶标TLR2mRNA。在细胞实验中我们采用荧光素酶双报告基因检测系统证实过表达的miR-344b-1-3p可显著降低TLR2-3’UTR的报告基因活性(P=0.000481637)，而miR-344b-1-3p inhibitor可显著升高TLR2-3’UTR报告基因活性（P=0.000428327）。初步确认miR-344b-1-3p通过结合到TLR2 3'UTR上对TLR2基因表达进行调控。进一步研究miR-344b-1-3p对AMTLR2表达及信号通路、分泌炎症因子的影响。结果发现经Pam3CSK4刺激1小时后，CSE预处理的NR8383细胞+转染大鼠 miR-344b-1-3p inhibitor慢病毒组其TLR2mRNA及细胞膜TLR2表达较对照组明显增高，而且与TLR2相关的多条信号通路配体蛋白表达及炎症因子释放量明显增高。. 本研究首次证实miR-344b-1-3p调控大鼠AM TLR2的表达，继而影响了TLR2相关的多条信号通路的配体蛋白表达及炎症因子的释放，这是本项目的关键科学问题。本研究的科学意义在于首次发现miR-344b-1-3p通过调控TLR2表达，影响肺泡巨噬细胞固有免疫功能，而COPD患者肺泡巨噬细胞固有免疫功能的损害可能是其曲霉易感性增高的重要原因。明确肺泡巨噬细胞固有免疫功能损的分子机制，有助于寻找新的治疗靶点，为预防和治疗COPD患者呼吸系统感染提供新思路。