NKX2-1转录激活XR_596950双靶向调控PAH肺血管重构的分子机制

Seeking therapeutic targets is currently the most important task for the treatment of pulmonary hypertension (PAH). Pulmonary vascular remodeling (PVR) is one of the major pathological changes of PAH. Further exploration of the mechanisms that promote PVR is critical to discovering targets that could lead to prevention and treatment of PAH. As shown in our previous study that serotonin induces vascular remodeling, inflammation and that serotonin transporter (SERT) and serotonin 1B receptor (5-HT1BR) both regulate the proliferation of pulmonary artery smooth muscle cells (PASMCs). We also found miR-188-3p inhibits the proliferation of PASMCs regulated by SERT/5-HT1BR, lncRNA XR_596950 regulates PVR and probably via regulation of miR-188-3p expression by ceRNA; there are also NKX2-1 binding sites in the promoter region of XR_596950. This project intends to analyze the relationship between XR_596950 and PVR and the dual target of miR-188-3p on SERT/5-HT1BR-mediated PAH PVR; to study the mechanism of competitive combination of XR_596950 with miR-188-3p by ceRNA; and to reveal the mechanism of XR_596950 mediated via NKX2-1 transcriptional regulation of PVR.

寻求药物治疗靶点是肺动脉高压（PAH）亟待解决的问题。肺血管重构是PAH主要病理改变，进一步探索其作用机制对于PAH防治具有重要意义。课题组前期发现：5-羟色胺通过1B受体（5-HT1BR）和转运体（SERT）诱导血管重构、炎症等；SERT/5-HT1BR调控肺动脉平滑肌细胞（PASMCs）增殖；miR-188-3p抑制SERT/5-HT1BR调控PASMCs增殖；lncRNA XR_596950参与调控肺血管重构，并提示可能通过ceRNA方式调控miR-188-3p表达；XR_596950启动子区存在NKX2-1结合位点。本项目拟通过XR_596950与肺血管重构的关系为切入点，分析miR-188-3p双靶向SERT/5-HT1BR介导PAH肺血管重构作用；研究XR_596950竞争结合miR-188-3p的ceRNA机制；揭示XR_596950被NKX2-1转录激活调控肺血管重构机制。

肺动脉高压（PAH）肺血管重构主要表现为肺动脉平滑肌细胞（PASMCs）增殖。5-羟色胺（5-HT）在PAH患者血浆中显著升高。5-HT通过5-羟色胺转运体（SERT）内在化及5-羟色胺1B受体（5-HT1BR）转运诱导血管重构。有效抑制SERT、5-HT1BR可能成为逆转PAH患者血管重构的关键途径。我们应用miRNA及LncRNA芯片分析发现，大鼠PASMCs中5-HT 诱导XR_596950和NKX2-1表达增高，miRNA-188-3p降低。而XR_596950是否调控PASMCs增殖尚不清楚。因此，我们假设5-HT通过 NKX2-1/XR_596950/miR-188-3p/SERT/5-HT1BR调控PASMCs增殖。在PAH患者及3%O2或5-HT诱导人PASMCs中，NKX2-1，XR_596950，SERT和5-HT1BR表达升高，miR-188-3p降低。荧光素酶报告基因表明NKX2-1激活XR_596950启动子。在人PASMCs中，过表达NKX2-1，XR_596950，SERT，5-HT1BR和PCNA表达升高，miR-188-3p降低。而沉默NKX2-1，XR_596950，SERT，5-HT1BR和PCNA表达降低，miR-188-3p升高；过表达XR_596950，SERT，5-HT1BR和PCNA表达升高，miR-188-3p降低。沉默XR_596950，结果相反。同时过表达NKX2-1及沉默XR_596950发现，抑制XR_596950部分抵消过表达NKX2-1的效应。RNA Pull down表明miR-188-3p直接调控XR_596950。利用Mirwalk软件发现，miR-188-3p和SERT及5-HT1BR 3'UTR存在相似的应答元件。在人PASMCs转染SERT或5-HT1BR 3'UTR并过表达miR-188-3p发现，miR-188-3p调控SERT及5-HT1BR。过表达miR-188-3p，SERT，5-HT1BR，PCNA表达降低。抑制miR-188-3p升高SERT，5-HT1BR和PCNA水平。以上表明NKX2-1/lncRNA XR_596950/miR-188-3p/SERT/5-HT1BR调控5-HT或缺氧诱导的PASMCs增殖，抑制Lnc RNA XR_596950可能成为治疗PAH新靶点。