DataSheet1_Circulating Exosomal miRNAs as Novel Biomarkers Perform Superior Diagnostic Efficiency Compared With Plasma miRNAs for Large-Artery Atherosclerosis Stroke.PDF

Recently, exosomal miRNAs have been reported to be associated with some diseases, and these miRNAs can be used for diagnosis and treatment. However, diagnostic biomarkers of exosomal miRNAs for ischemic stroke have rarely been studied. In the present study, we aimed to identify exosomal miRNAs that are associated with large-artery atherosclerosis (LAA) stroke, the most common subtype of ischemic stroke; to further verify their diagnostic efficiency; and to obtain promising biomarkers. High-throughput sequencing was performed on samples from 10 subjects. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed on exosomes and plasma in the discovery phase (66 subjects in total) and the validation phase (520 subjects in total). We identified 5 candidate differentially expressed miRNAs (miR-369-3p, miR-493-3p, miR-379-5p, miR-1296-5p, and miR-1277-5p) in the discovery phase according to their biological functions, 4 of which (miR-369-3p, miR-493-3p, miR-379-5p, and miR-1296-5p) were confirmed in the validation phase. These four exosomal miRNAs could be used to distinguish LAA samples from small artery occlusion (SAO) samples, LAA samples from atherosclerosis (AS) samples, and LAA samples from control samples and were superior to plasma miRNAs. In addition, composite biomarkers achieved higher area under the curve (AUC) values than single biomarkers. According to our analysis, the expression levels of exosomal miR-493-3p and miR-1296-5p were negatively correlated with the National Institutes of Health Stroke Scale (NIHSS) score. The four identified exosomal miRNAs are promising biomarkers for the diagnosis of LAA stroke, and their diagnostic efficiency is superior to that of their counterparts in plasma.近年来，已有研究报道外泌体微RNA（exosomal miRNAs）与多种疾病存在关联，此类微RNA可用于疾病的诊断与治疗。然而，针对缺血性脑卒中的外泌体微RNA诊断生物标志物的相关研究仍较为匮乏。在本研究中，我们旨在筛选与缺血性脑卒中最常见亚型——大动脉粥样硬化性（large-artery atherosclerosis, LAA）脑卒中相关的外泌体微RNA，进一步验证其诊断效能，并筛选出具有应用前景的生物标志物。本研究首先对10名受试者的样本实施高通量测序。在发现阶段（共纳入66名受试者）与验证阶段（共纳入520名受试者）中，我们分别对外泌体及血浆样本开展实时荧光定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测。在发现阶段，我们基于生物学功能筛选出5个差异表达候选微RNA（miR-369-3p、miR-493-3p、miR-379-5p、miR-1296-5p及miR-1277-5p）；其中4个（miR-369-3p、miR-493-3p、miR-379-5p及miR-1296-5p）在验证阶段得到了证实。此4种外泌体微RNA可有效区分大动脉粥样硬化性脑卒中样本与小动脉闭塞（small artery occlusion, SAO）样本、大动脉粥样硬化性脑卒中样本与动脉粥样硬化（atherosclerosis, AS）样本，以及大动脉粥样硬化性脑卒中样本与对照样本，且其诊断效能优于血浆微RNA。此外，联合生物标志物的曲线下面积（area under the curve, AUC）值高于单一生物标志物。经分析，外泌体miR-493-3p与miR-1296-5p的表达水平与美国国立卫生研究院卒中量表（National Institutes of Health Stroke Scale, NIHSS）评分呈负相关。本研究筛选出的4种外泌体微RNA有望成为大动脉粥样硬化性脑卒中的诊断生物标志物，且其诊断效能优于血浆中的同类微RNA。