miR-20b调控肺泡巨噬细胞产生VEGF的机制及其在哮喘发病中作用的研究

Vascular endothelial growth factor (VEGF), the most critical angiogenesis-promoting factor, plays an important role in chronic airway inflammation and airway remodeling in asthma. Our previous study showed that alveolar macrophage (AM) is the main source of VEGF in the airway of mice with chronic asthma. Further, the expression of miR-20b is decreased in AM and miR-20b negatively regulates the expression of VEGF in AM. These findings suggest that miR-20b plays an important role in the regulation of VEGF production in AM and miR-20b might be involved in the pathogenesis underlying asthma. In the current study, we will determine whether or not miR-20b targets VEGF mRNA and directly regulates the expression of VEGF at the post-transcriptional level, or if miR-20b regulates the expression of VEGF indirectly by targeting intracellular signaling molecules, such as JAK1, STAT3, HIF1, VHL, and PTEN. Such knowledge will help us demonstrate the mechanism by which miR-20b regulates the production of VEGF in AM. Then, we will determine the effect of miR-20b on airway inflammation, pulmonary vascular permeability, and airway remodeling in mice with chronic asthma to elucidate the role of miR-20b in the pathogenesis of asthma. Finally, the upstream transcription factors of the miR-20b gene were predicted and identified in order to clarify the mechanism of transcriptional regulation of miR-20b gene expression. This research will help to clarify the molecular mechanisms involved in asthma and provide a theoretical and experimental basis for the discovery of new targets for the clinical treatment of asthma.

VEGF是最关键的血管生成促进因子，在哮喘的慢性气道炎症及气道重塑中起到了重要作用。我们前期的研究表明：慢性哮喘小鼠肺泡巨噬细胞（AM）是气道中VEGF的主要来源，其miR-20b的表达降低；miR-20b负性调节AM VEGF的表达。提示miR-20b在调控AM产生VEGF的机制中起重要作用，也可能参与哮喘的发病过程。本课题拟首先观察AM中miR-20b是否靶向VEGF，在转录后水平直接调节VEGF的表达，或者通过靶向与VEGF表达有关的胞内信号分子而间接调节VEGF的产生，以揭示miR-20b调控AM产生VEGF的机制；然后观察miR-20b对慢性哮喘模型小鼠气道炎症及气道重塑的影响，揭示其在哮喘发病中的作用；最后进行miR-20b基因上游转录因子的预测与鉴定，以阐明miR-20b表达的转录调控机制。本研究将有助于阐明哮喘发病的分子机制，为临床哮喘治疗发现新靶点提供理论和实验依据。

支气管哮喘（简称哮喘）是呼吸系统的常见病、多发病之一。VEGF 是最关键的血管生成促进因子，在哮喘的慢性气道炎症及气道重塑中起到了重要作用。既往的研究表明：慢性哮喘小鼠肺泡巨噬细胞（AM）是气道中VEGF 的主要来源，其miR-20b 的表达降低；miR-20b 负性调节AM VEGF 的表达。但miR-20b 是否参与哮喘的发病，及调控VEGF产生的机制尚未完全明确。本研究中，我们通过构建含VEGF 3'-UTR或与VEGF 表达有关的胞内信号分子3'-UTR的双荧光素酶报告载体，检测miR-20b对荧光素酶活性的影响；观察miR-20b鼻滴对慢性哮喘小鼠气道炎症的影响；并对miR-20b 基因上游转录因子进行了预测与鉴定。结果表明，miR-20b可以直接靶向VEGF 3'-UTR而负性调节其表达，或miR-20b可以间接靶向STAT3 3'-UTR而调控VEGF表达。慢性哮喘小鼠经miR-20b鼻滴后气道炎症减轻。miR-20b鼻滴诱导哮喘小鼠肺组织中Gr1+CD11b+MDSCs数目增多，但对骨髓、外周血和脾脏中MDSC数目并无明显影响。提示miR-20b可能通过诱导扩增的机制引起哮喘小鼠肺内MDSC增多。进一步研究发现：miR-20b鼻滴诱导哮喘小鼠肺组织中CD11b+Ly6ClowLy6G+型MDSCs数目增多，但对CD11b+Ly6C+Ly6G-型MDSCs的比例无明显影响。miR-20b鼻滴抑制了哮喘小鼠BALF中Th2型细胞因子的产生，但对Th1型细胞因子的产生并无明显影响；miR-20b的鼻滴同时增加了哮喘小鼠BALF中CCL2的含量及BALF和血清中TGF-β的含量。由于MDSC 可以TGF-β1依赖性方式抑制哮喘小鼠的气道炎症。因此miR-20b可能通过MDSC达成抑制哮喘小鼠气道炎症。转录因子Sp-1负向调控静止巨噬细胞中miR-20b的表达；并且其逆转了TNF-α诱导RAW264.7 细胞miR-20b表达降低。有趣的是，转录因子CREB却正向调控了miR-20b的表达。这说明miR-20b基因启动子区既有正向也有负向调控的转录因子。本研究将有助于阐明哮喘发病的分子机制，也为临床哮喘治疗发现新靶点提供理论和实验依据。