3'UTR Shortening in Pulmonary Vascular Disease

项目摘要 肺动脉高压(PH)是一种以平均肺动脉增加为特征的肺血管疾病 压力(MPAP)导致右室(RV)肥大和功能障碍，右心衰和 最终死亡。PH的病理过程以广泛的血管重构为特征，影响 肺动脉平滑肌细胞(PASMC)和右室壁。已将多个进程与 包括血管周围纤维增殖性沉积增加在内的肺动脉高压的发展 改变了细胞的代谢，增加了细胞的增殖和抗凋亡能力。近几年来的研究进展 RNA生物学领域的研究表明，交替的多聚腺苷(APA)会导致更短的3‘-非翻译区 (3‘非编码区)的mRNAs，可以避免mRNA的调节，导致这些转录本的过度表达。近期 研究表明，细胞应激后存在3‘非编码区。RNA一个25 kDa亚基的耗尽 结合蛋白裂解因子I(CFIM25)，已被证明导致APA和显著的3‘非编码区缩短。 来自我的实验室的具有挑衅性的初步数据显示CFIm25的耗尽和3‘非编码区缩短的证据 从肺动脉高压模型中分离出的肺动脉。这些观察结果与 分离的肺动脉平滑肌细胞3‘非编码区缩短的证据。然而，3‘UTR的影响 PH值的缩短仍不清楚。PASMCs中CFIm25基因敲除研究的通路分析 3‘端非编码区缩短与纤维增殖性沉积和细胞增殖相关的基因。已被占用 综上所述，我们的假设是，3‘非编码区缩短改变了许多影响基因表达的因素 PH的发展。目标1将解决CFIm25耗竭的时间变化如何导致血管增加 3‘端非编码区缩短重塑。我们还将评估CFIm25的表达水平，使用患者来源的 组织和PASMC来跟踪CFIm25的耗尽如何与PH的疾病严重程度相关。使用新的RNA- 在小鼠和人类组织中的SEQ方法我们的目标是确定在参与其中的通路中3‘非编码区长度的变化 对PH的发展有一定的促进作用。在目标2中，我们将评估CFIm25如何耗尽血管平滑肌细胞 加重了PH的发生发展。在这里，我们还将执行新的RNA-SEQ方法来识别缩短的 CFIm25耗尽后的3‘UTRs。AIM 3将测定患者的miRs-203和miR-509-3p水平 在PH和PH的实验模型中。我们将测试旨在提升 CFIM25的表达作为一种治疗PH的新方法。

Project Summary Pulmonary hypertension (PH) is a disorder of the lung vasculature defined by increased mean pulmonary arterial pressure (mPAP) leading to right ventricle (RV) hypertrophy and dysfunction, right-sided heart failure and ultimately death. The pathologic process in PH is characterized by extensive vascular remodeling affecting pulmonary artery smooth muscle cells (PASMC) and the RV wall. Several process have been associated with the development of pulmonary hypertension including increased perivascular fibro-proliferative deposition altered cellular metabolism and increased cell proliferation and resistance to apoptosis. Recent advances in the field of RNA biology have shown that alternative polyadenylation (APA) results in shorter 3'-untranslated regions (3'UTR) of mRNAs that can avoid mRNA regulation resulting in overexpression of these transcripts. Recent studies have shown that 3'UTR is present following cellular stress. Depletion of a 25kDa subunit of the RNA binding protein cleavage factor I (CFIm25), has been shown to result in APA and marked 3'UTR shortening. Provocative preliminary data from my lab demonstrate depletion of CFIm25 and evidence of 3'UTR shortening from isolated pulmonary arteries from models of pulmonary hypertension. These observations are in line with evidence of 3'UTR shortening in isolated pulmonary artery smooth muscle cells. However, the effects of 3'UTR shortening in PH remain unknown. Pathway analysis from CFIm25 knock-down studies in PASMCs revealed 3'UTR shortening of genes associated with fibro-proliferative deposition and cellular proliferation. Taken together, our hypothesis is that 3'UTR shortening alters gene expression of many factors influencing the development of PH. Aim 1 will address how temporal changes in CFIm25 depletion lead to increased vascular remodeling through 3'UTR shortening. We will also evaluate expression levels of CFIm25 using patient-derived tissues and PASMCs to track how depletion of CFIm25 correlates with disease severity in PH. Using novel RNA- seq approaches in murine and human tissue we aim to identify changes in 3'UTR length in pathways contributing to the development of PH. In Aim 2, we will evaluate how CFIm25 depletion in vascular smooth muscle cells worsens the development of PH. Here we will also perform novel RNA-seq approaches to identify shortened 3'UTRs following depletion of CFIm25. Aim 3 will determine the levels of miRs-203 and miR-509-3p in patients with PH and in experimental models of PH. We will test the therapeutic potential of strategies aimed at elevating CFIm25 expression as a novel treatment for PH.