Small EVs miRNA profiles and their expressions in EVs-depleted plasma as an optimal combination panel for...

Treatment-resistant schizophrenia (TRS) remains a major clinical challenge, with limited progress in identifying reliable biomarkers for targeted intervention. MicroRNAs (miRNAs) encapsulated in small extracellular vesicles (sEVs) have emerged as candidates for long-range intercellular communication due to their stability and cell specificity. Whiles sEV-derived miRNAs (sEV-miRNAs) profiling elucidates vesicle-mediated signaling, parallel assessment in sEV-associated miRNAs in EVs-depleted (dEV) plasma is critical to determine their packaging selectivity and potential roles in systemic regulation. In this proof-of-concept study, we hypothesized that specific miRNAs are selectively enriched in sEVs, and compared sEV-miRNA expression profiles between TRS and non-TRS (NTRS). We then conducted a side-by-side comparison of selective sEV-associated miRNAs expression in dEV plasma. In the screening set (N = 16), three differentially expressed sEV-miRNAs (miR-184, miR-3131, and miR-3135b) were identified and advanced for validation in sEVs and dEV compartments. In the validation set (N = 95), miR-184 and miR-3131 were elevated in sEVs from TRS patients, yet displayed reduced expression in dEV plasma, suggesting compartment-specific roles and their origin in signal transduction. An optimal marker panel combining sEV-miR-184 and dEV-miR-3131 achieved robust performance in distinguishing TRS from NTRS, yielding an area under the curve (AUC) = 0.986; accuracy = 0.926, with sustained performance in 5-fold cross-validation (AUC = 0.920; accuracy = 0.865). Functional enrichment implicated these miRNAs in synaptic signaling and neuroinflammatory pathways. Collectively, these findings highlight altered post-transcriptional regulation in TRS and the potential of sEV-miRNAs in advancing treatment strategies in precision psychiatry.