Table1_Serum Extracellular Vesicle–Derived miR-124-3p as a Diagnostic and Predictive Marker for Early-Stage Acute Ischemic Stroke.DOCX

Background: A delay in the diagnosis of acute ischemic stroke (AIS) reduces the eligibility and outcome of patients for thrombolytic therapy. Therefore, early diagnosis and treatment of AIS are crucial. The present study evaluated the sensitivity and accuracy of serum extracellular vesicle (EV)-derived miR-124-3p in the diagnosis and prediction of AIS.Methods: An miRNA expression profile was downloaded from Gene Expression Omnibus (GEO) database and analyzed by R software. EVs were harvested from the serum of AIS patients using a total exosome isolation kit and characterized by Western blotting, a transmission electron microscope, and the nanoparticle tracking analysis. BV2 microglia were pre-stimulated with lipopolysaccharide (LPS), followed by miR-124-3p treatment for 24 h and subsequent analysis of viability, apoptosis, and migration (scratch assay), and Western blotting. The relative expression of the selected genes was assessed by qRT-PCR. The phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK in BV2 microglia cells was evaluated by Western blotting, while the luciferase reporter gene assay detected the correlation between key genes involved in the pro-inflammatory signaling pathways and miR-124-3p.Results:hsa-miR-124-3p was downregulated in AIS serum compared to the non-AIS serum (p < 0.05), and the gene expression of has-miR-124-3p in EVs was negatively correlated with serum pro-inflammatory cytokines and the NIHSS (p < 0.05). In addition, miR-124-3p promoted the viability and inhibited the apoptosis of LPS-induced BV2 microglia. Furthermore, miR-124-3p reduced the phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK, and promoted the migration in LPS-induced BV2 microglia (p < 0.05).Conclusion: Serum EV-derived miR-124-3p serves as a diagnostic and predictive marker for early-stage AIS.

背景：急性缺血性卒中（AIS）的早期诊断延迟会降低患者接受溶栓治疗的可及性和预后。因此，对AIS的早期诊断和治疗至关重要。本研究评估了血清细胞外囊泡（EV）来源的miR-124-3p在诊断和预测AIS敏感性及准确性的作用。方法：从基因表达综合数据库（GEO）下载miRNA表达谱，并使用R软件进行分析。采用总外泌体分离试剂盒从AIS患者的血清中提取EVs，并通过Western blotting、透射电子显微镜和纳米颗粒追踪分析对其进行表征。将BV2小胶质细胞预先用脂多糖（LPS）刺激，随后进行miR-124-3p处理24小时，随后分析其活力、凋亡和迁移（划痕实验），以及Western blotting。通过qRT-PCR评估选定基因的相对表达量。通过Western blotting评估BV2小胶质细胞中的Erk1/2、PI3K/Akt和p38MAPK的磷酸化，而荧光素酶报告基因检测检测到涉及促炎信号通路的关键基因与miR-124-3p之间的相关性。结果：与非AIS血清相比，hsa-miR-124-3p在AIS血清中的表达下调（p < 0.05），且EVs中has-miR-124-3p的表达与血清促炎细胞因子和NIHSS呈负相关（p < 0.05）。此外，miR-124-3p促进了LPS诱导的BV2小胶质细胞的存活并抑制了其凋亡。此外，miR-124-3p降低了LPS诱导的BV2小胶质细胞中Erk1/2、PI3K/Akt和p38MAPK的磷酸化，并促进了其迁移（p < 0.05）。结论：血清EV来源的miR-124-3p作为早期AIS诊断和预测的标志物。