| Objective: To identify the differentially expressed circulating exosomal microRNAs in a certain subtype of ischemic stroke,and to explore their roles in the pathophysiological mechanism.Methods: This study was mainly composed of three parts: clinical cohort analysis,in vivo experiments,and in vitro mechanism exploration.Firstly,plasma exosomal microRNA expression profiles of patients with three main subtypes of ischemic stroke as well as healthy controls included in the retrospective cohort study were detected by high-throughput sequencing.Characteristically differential microRNAs were subsequently screened out using bioinformatics analysis,followed by diagnostic efficacy evaluation,target genes prediction and pathway enrichment analysis.Secondly,mouse model of atherosclerosis was established by feeding APOE-/-mice with a western-diet,whose plasma exosomes were extracted by ultracentrifugation.After measuring the exosomal characteristic microRNA expression,the extracted exosomes were then injected into the mice after transient middle cerebral artery occlusion（MCAO）.Immunofluorescence staining were applied to find the potential target cells of atherosclerotic-associated exosomes,and exosomal effects on the neurological function were evaluated by behavioral tests,Nissl’s staining and immunofluorescence staining.Finally,co-culture system consisting of macrophages/foam cells and potential target cells with oxygen and glucose deprivation（OGD）and transfection system of microRNA mimics/inhibitors were constructed in vitro and the effects of atherosclerotic-associated exosomes and characteristic microRNAs on target cells were explored using quantitative real-time reverse transcription polymerase chain reaction（q RTPCR）.Results:（1）The expression level of Novel-miRNA-3 in plasma exosomes of patients with large artery atherosclerotic stroke was characteristically higher when compared with the other three groups（P < 0.05）.Novel-miRNA-3 possessed certain diagnostic efficacy（AUC >0.8）and might be involved in several biological processed,such as neurogenesis and inflammation response;（2）Atherosclerosis-associated exosomes with higher expression of Novel-miRNA-3（P < 0.01）may lead to worse behavioral performance,larger infarct volume and overactivation of microglia in mice.（3）The expression level of Novel-miRNA-3 in exosomes secreted by foam cells derived from macrophages was higher（P < 0.01）.Both exosomes secreted by foam cells and Novel-miRNA-3 itself could increase the expression of pro-inflammatory genes and decrease the expression of anti-inflammatory genes in microglia,which could be partly reversed by transfection of Novel-miRNA-3inhibitor.Conclusions:（1）Novel-miRNA-3 was identified as a novel microRNA whose expression level characteristically increased in plasma exosomes of patients with large artery atherosclerotic stroke;（2）Atherosclerosis-associated exosomes with higher expression of Novel-miRNA-3 in mice could over-activate microglia of the peripheral region of cerebral infarction,thereby aggravating neurological damage;（3）Foam cells derived from macrophages could secrete exosomes with higher expression of Novel-miRNA-3 making microglia more prone to pro-inflammatory phenotype.Intervention of Novel-miRNA-3expression could reverse its excessive polarization to a certain extent. |
