Role of microRNA-17-92 and PDLIM5 Signaling in Pulmonary Arterial Hypertension

 描述（由申请人提供）：微小RNA（miRNA）是小的非编码内源性RNA分子，被认为参与肺动脉高压（PAH）的发病机制，但其确切作用尚不清楚。我们发现，平滑肌细胞（SMC）特异性敲除miR-17~92可以减轻缺氧诱导的肺动脉高压（PH），而miR-17~92的重建可以恢复PH，表明miR-17~92在PH的发病机制中发挥重要作用。我们发现miR-17 ~92直接靶向脯氨酰羟化酶2（PHD 2）和PDZ和LIM结构域5（PDLIM 5）蛋白。抑制miR-17~92诱导PHD 2表达并抑制HIF活性，诱导PDLIM 5表达并降低TGF-β 1/Smad信号传导和SMC标志物表达，SMC特异性敲除PHD 2和PDLIM 5增强缺氧诱导的肺动脉重构，而PDLIM 5过表达抑制缺氧诱导的肺动脉重构。这些结果表明miR-17~ 92通过调节PHD 2和PDLIM 5的表达来调节PH。在体外培养的PASMC和体内培养的小鼠肺组织中，慢性缺氧导致miR-17~92的表达呈双相变化：早期表达增加，随后表达减少。我们发现从PAH患者分离的PASMC中miR-17~92的表达降低，并且这种miR-17~92的降低解释了IPAH-PASMC的去分化表型。我们推测miR-17~92的上调可能是人类和实验性肺动脉高压发病机制中共同的起始事件，而miR-17~92表达的晚期降低可能是抑制肺动脉高压进一步发展的适应性机制。我们的假设是miR-17~92通过以下途径启动肺动脉高压的发病：1）直接抑制PHD 2，激活HIF通路; 2）直接抑制PDLIM 5以激活TGF-β 1/Smad 2/3通路。miR-17~92在慢性缺氧中表达的双相性是一个新的发现，我们将研究其意义和相关机制。具体目标1、我们将研究miR-17~92和PHD 2调节缺氧诱导的PH的分子机制。我们将研究PHD 2是否是miR-17~92的一个新的直接靶点，以及miR-17~92和PHD 2调节HIF活性和PH的分子机制。具体目标2是确定miR-17~92和PHD 2调节HIF活性和PH的分子机制。我们将研究PDLIM 5是否是miR-17~92的一个新的直接靶点，以及PDLIM 5负调控TGF-β 1/Smad信号通路和抑制PH进展的分子机制。在特异性目的3中，我们将探讨miR-17~92双相表达的分子机制及其在PH进展中的意义。我们将研究HIF和E2 F1在慢性缺氧早期上调miR-17~92中的作用，p53在慢性缺氧晚期抑制miR-17~92中的作用，以及在缺氧晚期敲除miR-17~92、PHD 2和PDLIM 5是否减轻或加重小鼠PH。

 DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that are thought to be involved in the pathogenesis of pulmonary arterial hypertension (PAH) though their exact roles are not known. We found that smooth muscle cell (SMC)-specific knockout of miR-17~92 in mice attenuated hypoxia-induced pulmonary hypertension (PH) and reconstitution of miR-17~92 restored it, indicating an important role for miR-17~92 in pathogenesis of PH. We identified that miR-17~92 directly targets prolyl hydroxylase 2 (PHD2) and PDZ and LIM domain 5 (PDLIM5) proteins. Suppression of miR-17~92 induced PHD2 expression and inhibited HIF activity, induced PDLIM5 expression and decreased TGF-ß/Smad signaling and expression of SMC markers, all of which attenuated PH. SMC-specific knockout of PHD2 and PDLIM5 enhanced hypoxia-induced pulmonary artery remodeling whereas overexpression of PDLIM5 inhibited hypoxia-induced PH. These results indicate that miR-17~92 modulates PH by regulating the expression of PHD2 and PDLIM5. In PASMC in-vitro and mouse lungs in-vivo, chronic hypoxia resulted in a biphasic expression of miR-17~92: an early increase followed by a decrease in expression. We found that miR-17~92 expression was reduced in PASMC isolated from PAH patients and that this reduction in miR-17~92 accounted for the de-differentiated phenotype of the IPAH-PASMC. We speculate that up regulation of miR-17~92 may be a common initial event in the pathogenesis of both human and experimental PH and that the late decrease in miR-17~92 expression may be an adaptive mechanism to inhibit further progression of PH. Our hypothesis is that miR-17~92 initiates the pathogenesis of PAH by: 1) directly suppressing PHD2 to activate the HIF pathway; 2) directly suppressing PDLIM5 to activate the TGF-ß/Smad2/3 pathway. The biphasic nature of miR-17~92 expression in chronic hypoxia is a novel finding and we will investigate its significance and the mechanisms involved. In Specific Aim 1, we will determine the molecular mechanisms by which miR-17~92 and PHD2 regulate hypoxia- induced PH. We will investigate whether PHD2 is a novel direct target of miR-17~92 and the molecular mechanisms by which miR-17~92 and PHD2 regulate HIF activity and PH. Specific Aim 2 is to determine the molecular mechanisms by which miR-17~92 and PDLIM5 regulate hypoxia-induced vascular remodeling and PH. We will investigate whether PDLIM5 is a novel direct target of miR-17~92 and the molecular mechanisms by which PDLIM5 negatively regulates TGF-ß/Smad signaling and inhibits the progression of PH. In Specific Aim 3, we will determine the molecular mechanisms underlining the biphasic expression of miR-17~92 and its implication in PH progression. We will investigate the roles of HIF and E2F1 in up regulation of miR-17~92 in the early phase of chronic hypoxia, the role of p53 in inhibition of miR-17~92 in the late phase of chronic hypoxia, and whether knockout of miR-17~92, PHD2, and PDLIM5 in the late stage of hypoxia diminishes or accentuates PH in mice.