microRNA-let-7在低氧诱导肺高压肺血管重构中的作用及机制研究

Pulmonary hypertension(PH) is characterized by hyperproliferation of pulmonary artery smooth muscle cells(PASMCs) within the vascular wall of resistant pulmonary arteries, leading to vascular lumen occlusion. Recent studies found that microRNAs may regulate multiple disease pathways in the pulmonary vasculature, but their importance in PH is just beginning to emerge. Further, researchers identified miRNAs associated with multiple pathogenic pathways (TGF-β, hypoxia and inflammation) central to PH which including let-7 by a network-based bioinformatics approach. However, the role of let-7 in pulmonary vascular remodeling in pulmonary hypertension remains unknown. Our pilot study showed that the expression of let-7b/g was downregulated in hypoxia-induced PH rats, and the let-7g mimic significantly inhibited proliferation of primary cultured PASMCs induced by hypoxia. We will be planning to explore the important potential role of let-7 family in mediating the pulmonary vascular remodeling in PH rats and proliferation of primary cultured PASMCs. Furthermore, we will investigate the underlying mechanisms responsible for the down-regulation of let-7 in pulmonary arteries in hypoxia-induced PH rats(focusing on positive feedback regulation between c-myc and let-7) and the downstream signaling that let-7 inhibits vascular remodeling(focusing on let-7/RAS/c-myc/bmi-1/p16). This study will contribute to the understanding of pathogenesis of PH, and provide new strategies to seek for the new drugs directing at let-7 target.

肺高压（pulmonary hypertension，PH）时肺动脉阻力增加的主要病因是肺血管重构导致肺动脉腔隙狭窄。近期有学者运用miRNAs网络药理学发现let-7家族与TGF-β、低氧及炎症诱导的PH关系密切。然而，let-7家族在PH中的作用还未见文献报道。我们的预实验发现，let-7家族中let-7b/g在低氧诱导的PH大鼠肺动脉中表达下调，let-7g mimic能显著抑制低氧诱导的原代大鼠肺动脉平滑肌细胞增殖，因此我们推测let-7家族参与低氧诱导PH肺血管重构。本项目拟通过在体动物与细胞实验相结合，探讨低氧PH发病过程中let-7下调的机制（HIF-1α/PKC/ERK1/2/c-myc/let-7正反馈调节）及let-7抑制肺血管重构的机制（let-7/RAS/c-myc/bmi-1/p16）。本项目将有助于阐明PH的发病机制，为寻找防治PH的新靶点奠定实验和理论基础。

肺动脉高压（PH）是动脉性肺动脉高压、左心脏病变肺动脉高压、肺疾病及血氧不足肺动脉高压、慢性栓塞或梗塞肺动脉高压以及混合性肺动脉高压的总称。主要病因是肺小动脉原发病变而导致肺动脉阻力增加，最终可导致患者右心衰竭而死亡。PH作为一种慢性血管性疾病，至今没有有效的治愈手段，患者的平均生存期仅有7年。PH病因涉及环境与遗传因素，其发病机制尚未完全阐明。业已证明，肺血管重构在PH发生发展的病理生理过程中起重要作用，发生牵涉到多种因子，如TGF-β、5-羟色胺、内皮素-1、NO等。新近有学者运用网络药理学发现了与TGF-β、低氧及炎症诱导的PH有关的miRNAs，其中包括let-7家族。然而let-7在PH肺血管重构中的作用及机制尚不清楚。.结果发现，低氧PH大鼠右心室收缩压和平均肺动脉压较正常组明显升高；肺动脉显著重构。PH大鼠肺动脉中let-7b和let-7g表达显著下调，原位杂交显示let-7g主要表达在肺血管。低氧处理PASMCs可引起细胞增殖，同时let-7g呈时间依赖性降低。let-7g过表达可显著取消低氧引起的促增殖作用。Let-7g抑制剂可直接促进PASMCs增殖。PASMCs过表达let-7g可显著抑制低氧诱导的靶基因c-Myc和下游蛋白bmi-1上调及p16下调，导致细胞停留在G0/G1期。抑制c-Myc也可显著抑制低氧诱导的bmi-1表达上调及p16表达下调。Let-7g的另一个靶基因LOX-1在PH时表达上调，let-7g过表达可显著抑制低氧诱导的LOX-1表达上调，而抑制LOX-1又可通过PKC/OCT-1反过来抑制低氧诱导的let-7g下调。.结果提示，let-7g在低氧PH中表达下调，具有抑制PASMCs增殖作用。Let-7g下调的上游机制涉及LOX-1/PKC/OCT-1通路。抑制PASMCs的机制涉及与c-Myc/Bmi-1/p16信号通路。