E2F1/microRNA-520c/LATS2/YAP信号通路参与低氧性肺动脉高压血管重构的机制研究

Pulmonary vascular remodeling is a key process during the development of pulmonary hypertension (PH), the underlying mechanism of which is closely associated with the gene expression and gene transcription regulation involved in the pulmonary vascular smooth muscle cell (PASMCs) pro-proliferation and anti-apoptosis phenotype switch. MicroRNA, a noncoding RNA molecule, has been demonstrated to be able to regulate the cellular function through modulation of target genes. On the basis of our microRNA chip data and bioinformatics analysis, we identified miR-520c as one of the differential expression gene and investigate the functional role of miR-520c both in vivo and vitro established PH models. By using the miR-520c mimics and inhibitors transfection as well as the luciferase assay, we intend to demonstrate that miR-520c promotes the PASMCs pro-proliferative phenotype by regulating the Hippo/YAP signaling via targeting LATS2. The underlying molecular mechanism through which miR-520c regulates the PASMCs disease phenotype transition is explored by using the LATS2 overexpression and RNA interference. The upstream mechanism of the miR-520 transcription was also investigated by using ChIP assay. The transcription factor E2F1 was involved in the regulation of hypoxia induce miR-520c/LATS2 signal pathway. Finally, we examined the effect of miR-520c inhibition on the vascular remodeling in PH rat models. Together, our research project may provide evidence that E2F1/miR-520c/LATS2 axis serve as a potential therapeutic targets for the treatment of PH.

肺动脉血管重构是肺动脉高压(PH)发病中的重要病理过程，其发生机制涉及肺动脉血管平滑肌细胞(PASMCs)的异常增殖和抗凋亡表型转换，但其具体机制仍需进一步阐明。申请者在前期研究中，对低氧性肺动脉高压患者血液标本进行microRNA差异表达筛选发现miR-520c表达明显增高。进一步研究证实miR-520c靶向调控LATS2/YAP通路参与肺动脉血管平滑肌细胞增殖的表型转换。本课题拟在此基础上，在肺动脉高压细胞和动物模型中深入研究miR-520c介导肺动脉血管重构中的作用方式及调控机理，阐明转录因子E2F1调控miR-520c转录的分子机制，并通过干预PH动物评价抑制miR-520c对逆转PH血管重构的作用，在临床病例标本中分析miR-520与低氧性肺动脉高压预后的相关性。上述研究将对阐明低氧性肺动脉高压肺血管重构的分子机制具有重要意义，可能为肺动脉高压的提供新的药物作用靶。

低氧性肺动脉高压是多数慢性呼吸系统疾病病程中的重要并发症之一，研究表明肺血管增生性重构导致肺血管阻力增加是肺动脉高压患者预后差的关键因素，但目前暂无逆转肺血管增生重构的有效药物。肺动脉血管重构是低氧相关肺动脉高压病理生理过程中的核心环节，其发生机制涉及肺动脉血管平滑肌细胞(PASMCs)的异常增殖和抗凋亡表型转换，但其具体机制仍需进一步阐明。申请者在前期研究中，对低氧性肺动脉高压动物组织标本以及低氧诱导PASMCs中发现miR-520c表达明显增高。进一步研究证实抑制miR-520c能逆转低氧诱导的PASMCs增殖并减轻低氧诱导的肺动脉血管重构。在此基础上，本课题在肺动脉高压动物和细胞模型中深入研究miR-520c介导低氧诱导肺动脉血管重构中的作用方式及调控机理。首先利用生物信息学分析预测 miR-520c的靶基因LATS2，并通过荧光素酶报告检测技术初步验证了LATS2是miR-520c的靶基因; 通过LATS2 siRNA转染PASMCs细胞进行功能回复实验，抑制LATS2表达可逆转miR-520c antagomir抑制低氧诱导hPASMCs细胞增殖，验证了miR-520c/LATS2/YAP信号通路介导低氧诱导hPASMCs增殖/抗凋亡细胞表型转换。通过miR-520c基因启动子区的生物信息学分析及染色质免疫共沉淀检测方法验证了转录因子E2F1与miR-520c启动子区域结合。进一步通过E2F1siRNA和/或 miR-520c agomir转染PASMCs细胞进行功能回复实验，验证了E2F1参与调控低氧诱导miR-520c表达上调及低氧诱导hPASMCs增殖。上述研究数据证明了E2F1/miR-520c/LATS2信号轴参与调控低氧诱导的肺动脉血管平滑肌细胞增殖，对阐明低氧性肺动脉高压肺血管重构的分子机制有重要意义，可能为肺动脉高压的提供新的药物作用靶点。