Integrated in silico and experimental analysis identifies microRNAs from extracellular vesicle-enriched fractions associated with multiple sclerosis

Abstract MicroRNAs (miRNAs) are key post-transcriptional regulators implicated in neuroinflammatory and neurodegenerative processes underlying multiple sclerosis (MS). This study aimed to identify candidate miRNAs associated with MS and investigate their involvement in disease-related molecular pathways. An integrated in silico workflow was used to identify MS-associated genes, predict regulatory miRNAs, and characterize enriched pathways. Candidate miRNAs were validated in serum EV-enriched fractions from treated and untreated MS patients and healthy controls using quantitative polymerase chain reaction (qPCR). In silico analysis prioritized four miRNAs (miR-204-5p, miR-17-5p, miR-21-5p, and miR-106a-5p) for validation. Their predicted target genes were enriched in pathways related to immune regulation and central nervous system function, including Th17 cell differentiation, MAPK signaling, cytokine signaling, and axon guidance. qPCR revealed significant differences in miR-21-5p and miR-106a-5p expression between MS patients and healthy controls, while miR-204-5p differed between untreated MS patients and healthy controls. Among the four miRNAs evaluated, miR-21-5p was significantly associated with MS status and showed moderate discrimination between MS patients and healthy controls (AUC = 0.7204). These findings identify serum EV-enriched miRNAs as candidate biomarkers of MS, although validation in larger and longitudinal cohorts is required.