CircRNA_00286与miRNAs相互对话调控肺动脉高压右心室心肌细胞程序性死亡的机制研究

Pulmonary arterial hypertension is a kind of malignant disease. Right ventricular dysfunction is the key factor in determining the prognosis of pulmonary arterial hypertension. In recent years, the studies of non coded RNAs in right ventricular function are increasing, but the mechanism leading to the right ventricle dysfunction needs to be further explored. Our experiments found that circRNA_00286 was upregulated and its targeting miRNAs were downregulated in the right ventricular myocardium of monocrotaline induced pulmonary hypertension rat. Three targeting proteins PARP-1, AIF and MIF of miRNAs were significantly increased. According to previous studies indicating PARP-1/AIF/MIF mediated apoptosis and another programmed cell death style, parthanatos, the applicant speculate that thepregulation of circRNA_00286 expression and the downregulation of miRNAs could increase PARP-1, AIF and MIF expressions, promote myocardial cell apoptosis and parthanatos to participate in right ventricular myocardial remodeling in pulmonary arterial hypertension. The aim of this project is to explore the molecular mechanism of circRNA_00286 and miRNAs to mediated the procedural death of right ventricular cardiomyocytes via a series of experiments induced by gene editing and other technologies, which support that the regulation of targeting circRNA and miRNAs expressions is expected to be a new strategy for improving the right ventricular function of pulmonary arterial hypertension.

肺动脉高压是一类恶性疾病，右心室功能是决定其预后的关键因素。近年来非编码RNAs调节右心室功能的研究逐渐成为热点，但其如何导致右心室功能障碍的机制有待深入挖掘。我们前期预实验发现野百合碱诱导的肺动脉高压大鼠右心室circRNA_00286上调，能与其结合的三个miRNAs显著减少，上调PARP-1、AIF及MIF。结合文献报道PARP-1/AIF/MIF介导凋亡和另一种程序性死亡方式parthanatos发生，据此推测：circRNA_00286及目标miRNAs异常表达促进肺动脉高压右心室心肌细胞凋亡和parthanatos增加，导致右心室功能障碍。为此本项目拟采用基因修饰等技术经系列实验探讨circRNA_00286与miRNAs相互对话调控肺动脉高压心肌细胞死亡导致右心室功能障碍的分子机制，明确调节目标circRNA与miRNAs表达有望成为改善肺动脉高压右心室功能的新策略。

肺动脉高压(PAH)是一类以肺动脉重构为特征的进展性疾病，最终可能因右心衰竭而死亡，因此缓解PAH肺动脉重构和右心室重构是亟待解决的重要问题。申请人前期开展了6例特发性肺动脉高压 （Idiopathic PAH, IPAH）患者与6例健康对照者外周血单个核细胞全转录组测序的检测，结果提示两组之间存在大量差异性 circRNAs。随后申请人在60例 IPAH 患者和30例对照者中进行验证，结果发现 circRNA_00286、circGSAP、circPICALM 等表达显著降低，且 circGSAP 水平降低能独立预测PAH患者发病风险和死亡率增加，随后我们在IPAH和COPD-PH患者的血浆和肺组织中也分别进行了验证上述结果。体内实验发现circGSAP过表达提高了MCT-PH大鼠生存率，减轻了肺血管和右心室重构，上调了BMPR2水平。体外实验发现circGSAP过表达通过吸附miR-27a-3p抑或miR-942-5p制缺氧诱导的PMECs细胞周期G1/S转变、增殖、迁移和增加死亡率。BMPR2为鉴定为miR-27a-3p靶基因，SMAD4为miR-942-5p的靶基因。BMPR2沉默改善了miR-27a-3p抑制剂对PMECs增殖、迁移和死亡率的影响，miR-942-5bp抑制剂对PMECs影响也得到缓解被SMAD4 siRNA抑制。结果发现circGSAP下降越低PH患者预后越差。补充circGSAP通过与miR-27a-3p或miR-942-5p竞争性结合增加BMPR2/SMAD4来减轻PMECs的功能障碍，减轻MCT-PH大鼠的肺血管和右心室重构，为我们认识circRNA在PAH中的作用提供新思路，为寻找PAH的新药物靶点和基因治疗提供依据。

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