DataSheet1_Platelets-Derived miR-200a-3p Modulate the Expression of ET-1 and VEGFA in Endothelial Cells by Targeting MAPK14.DOCX

The interaction between platelets and vascular endothelial cells plays a pivotal role in the pathophysiology of acute ischemic stroke (AIS), especially in atherosclerosis formation. However, the underlying mechanism is not entirely clear. The aim of this study was to elucidate the role of platelets-derived miRNA in the development of atherosclerosis and AIS. We evaluated the miRNA expression profiles of serum microvesicles (MV) in five AIS patients and five healthy controls using RNA-seq, and then measured the levels of selected platelets derived miRNAs by qRT-PCR. miR-200a-3p expression in the serum MV and platelets had increased to 1.41 (p < 0.05) and 3.29 times (p < 0.001), respectively, in AIS patients compared with healthy controls, and was modified by severity of AIS. We transferred Cy5-miR-200a-3p into platelets, collected and identified platelets-derived MV (PMVs). Then, the gene expression of p38 MAPK/c-Jun pathway was analyzed using both miR-200a-3p gain- and loss-of-function experiments and PMVs coincubation with HUVEC. The results showed that activated platelets remotely modulated endothelins 1 (ET-1) and vascular endothelial growth factor A (VEGFA) levels in HUVEC through the release of miR-200a-3p-containing PMVs via targeting MAPK14. The results of ROC analyses showed that combination of platelet miR-200a-3p, serum ET-1 and VEGFA levels had an AUC of 0.817, a sensitivity of 70%, and a specificity of 89%. Our results presented new evidence that activated platelets could remotely modulate ET-1 and VEGFA expression in HUVEC via releasing miR-200a-3p-enriched PMVs, which provides a potential miRNA-based predictive biomarker and therapeutic strategy for atherosclerosis and AIS.

血小板与血管内皮细胞的相互作用在急性缺血性卒中（acute ischemic stroke, AIS）的病理生理过程中发挥关键作用，尤其在动脉粥样硬化形成进程中。然而其潜在机制尚未完全阐明。本研究旨在阐明血小板源性微小RNA（microRNA, miRNA）在动脉粥样硬化及急性缺血性卒中发生发展中的作用。我们通过RNA测序（RNA-seq）分析了5例急性缺血性卒中患者与5例健康对照者的血清微囊泡（microvesicles, MV）的miRNA表达谱，并通过实时定量聚合酶链反应（qRT-PCR）检测了选定的血小板源性miRNA水平。与健康对照相比，急性缺血性卒中患者血清微囊泡及血小板中的miR-200a-3p表达分别上调至1.41倍（p < 0.05）和3.29倍（p < 0.001），且其表达水平与急性缺血性卒中的严重程度相关。我们将Cy5标记的miR-200a-3p转染至血小板中，收集并鉴定了血小板源性微囊泡（platelets-derived MV, PMVs）。随后，分别通过miR-200a-3p的功能获得与功能缺失实验，以及血小板源性微囊泡与人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）共孵育实验，分析了p38丝裂原活化蛋白激酶（p38 MAPK）/c-Jun通路的基因表达情况。结果显示，活化的血小板通过释放携带miR-200a-3p的血小板源性微囊泡，靶向MAPK14，从而远程调控人脐静脉内皮细胞中内皮素1（endothelin 1, ET-1）及血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）的表达水平。受试者工作特征曲线（Receiver Operating Characteristic curve, ROC）分析结果显示，血小板miR-200a-3p、血清ET-1及VEGFA水平三者联合检测的曲线下面积（Area Under the Curve, AUC）为0.817，灵敏度为70%，特异度为89%。本研究结果提供了新的证据，表明活化的血小板可通过释放富含miR-200a-3p的血小板源性微囊泡，远程调控人脐静脉内皮细胞中ET-1与VEGFA的表达，这为动脉粥样硬化及急性缺血性卒中提供了一种基于miRNA的潜在预测生物标志物与治疗策略。