Abstract
Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system with heterogenous clinical course, lacking non-invasive biomarkers for phenotype differentiation. This study aimed to explore circulating extracellular vesicle (EV)-derived microRNA (miRNA) signatures and related molecular profiles capable of distinguishing stable relapsing-remitting MS (RRMS) from secondary progressive MS (SPMS). Plasma samples were collected from stable RRMS (n = 30), SPMS (n = 30), and healthy controls (HC) (n = 30), followed by total EVs isolation and characterization using transmission electron microscopy, dynamic light scattering, and flow cytometry. RNA was extracted from EVs, and miRNA profiles were analyzed via RNA sequencing and RT-qPCR. Cytokines and neuronal/astroglial injury biomarkers were quantified using the BioPlex system and ELISA. Functional enrichment and network analyses of miRNA targets were performed, alongside logistic regression modeling to explore potential distinguishing features. Four EV-derived miRNAs (miR-760, miR-98-5p, miR-301a-3p, miR-223-3p) showed significant differences (p < 0.05) between stable RRMS and SPMS. An integrative model combining miRNAs with fibroblast growth factor (FGF) basic protein enabled accurate phenotypic differentiation (AUC = 0.942). miR-760 showed the strongest distinctive capacity for stable RRMS. Additionally, miR-98-5p was markedly up-regulated in both stable RRMS and SPMS compared to HC. Network analysis of miRNA targets suggested distinct immunoregulatory patterns across MS phenotypes. Plasma EV-derived miRNAs—particularly miR-760, and miR-98-5p—showed potential as molecular indicators associated with disease phenotype in MS. Integrating EV-miRNA profiling with protein markers support efforts toward more precise stratification of MS patients. Further studies in independent cohorts and functional validation are warranted before clinical translation.
Data availability
The raw and processed RNA sequencing data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE303912.To review GEO accession GSE303912:Go to (https:/www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE303912) Enter token oxufugusxnobngf into the box.
Abbreviations
- ALS:
-
Amyotrophic lateral sclerosis
- AUC:
-
Area under the curve
- BBB:
-
Blood-brain barrier
- BDNF:
-
Brain-derived neurotrophic factor
- BP:
-
Biological processes
- CC:
-
Cellular components
- cDNA:
-
Complementary DNA
- CI:
-
Confidence interval
- CIS:
-
Clinically isolated syndrome
- CNS:
-
Central nervous system
- CPDA:
-
Citrate phosphate dextrose adenine
- CRP:
-
C-reactive protein
- CSF:
-
Cerebrospinal fluid
- CV:
-
Coefficient of variation
- DLS:
-
Dynamic light scattering
- DMTs:
-
Disease-modifying therapies
- DSI:
-
Disease specificity index
- EAE:
-
Experimental autoimmune encephalomyelitis
- EDSS:
-
Expanded disability status scale
- ELISA:
-
Enzyme-linked immunosorbent assay
- ESR:
-
Erythrocyte sedimentation rate
- EV:
-
Extracellular vesicle
- FC:
-
Fold-change
- FDR:
-
False discovery rate
- FGF:
-
Fibroblast growth factor
- G-CSF:
-
Granulocyte colony-stimulating factor
- GDA:
-
Disease-gene association
- GFAP:
-
Glial fibrillary acidic protein
- GM-CSF:
-
Granulocyte macrophage colony-stimulating factor
- GO:
-
Gene ontology
- GPR:
-
G protein-coupled receptor
- HC:
-
Healthy control
- HL:
-
Hosmer-Lemeshow
- IFN:
-
Interferon
- IL:
-
Interleukin
- IP:
-
Interferon gamma-induced protein
- IQR:
-
Interquatrile range
- ISEV:
-
International society of extracellular vesicles
- KEGG:
-
Kyoto encyclopedia of genes and genomes
- MCP:
-
Monocyte chemoattractant protein
- MF:
-
Molecular functions
- MIP:
-
Macrophage inflammatory protein
- miRNA:
-
microRNA
- MOG:
-
Myelin oligodendrocyte glycoprotein
- MOGAD:
-
Myelin oligodendrocyte glycoprotein antibody-associated disease
- MRI:
-
Magnetic resonance imaging
- MS:
-
Multiple sclerosis
- NfL:
-
Neurofilament light chain
- NLRP3:
-
NBD-, LRR- and pyrin domain-containing protein 3
- NMOSD:
-
Neuromyelitis optica spectrum disorder
- OD:
-
Optical density
- PBMCs:
-
Peripheral blood mononuclear cells
- PBS:
-
Phosphate-buffered saline
- PDGF:
-
Platelet-derived growth factor
- PDI:
-
Polydispersity index
- PPMS:
-
Primary progressive multiple sclerosis
- RANTES:
-
Regulated on activation, normal T expressed and secreted
- ROC:
-
Receiver operating characteristic
- RORγt:
-
RAR-related orphan receptor gamma t
- RRMS:
-
Relapsing-remitting multiple sclerosis
- RT:
-
Room temperature
- RT-qPCR:
-
Quantitative real-time PCR
- SPMS:
-
Secondary progressive multiple sclerosis
- TEM:
-
Transmission electron microscopy
- Th:
-
T helper
- TNF:
-
Tumor necrosis factor
- Treg:
-
T regulatory
- VEGF:
-
Vascular endothelial growth factor
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Acknowledgements
We would like to thank the Cytometry Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Poland, for conducting the flow cytometry analyses.
Funding
The study was funded by the University of Lodz IDUB Excellence Initiative – Research University grant (No. 65/2021) and the National Science Centre grant (No. UMO-2018/31/B/NZ4/02688).
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Authors and Affiliations
Contributions
**Karina Wasilewska** : Methodology, Investigation, Data analysis and interpretation, Writing – original draft and editing, Visualization, Project administration. **Angela Dziedzic** : Methodology, Writing – review and editing, Supervision. **Shamundeeswari Anandan** : Writing – review and editing. **Elżbieta Miller** : Resources. **Łukasz Łaczmański** : Methodology, Data analysis and interpretation. **Radosław Zajdel** : Statistical analysis. **Sylwia Michlewska** : Methodology, Visualization. **Dorota Kujawa** : Investigation. **Marta Gancarek** : Data analysis and interpretation. **Justyna Raczkowska** : Investigation. **Lidia Włodarczyk** : Resources. **Patrycja Nowak** : Investigation. **Joanna Saluk** : Conceptualization, Funding acquisition, Methodology, Project administration, Writing – review and editing, Supervision.
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The authors declare no competing interests.
Ethics approval
The study was performed in accordance with the Declaration of Helsinki and approved by the University of Lodz Research Bioethics committee with resolution No. 3/KBBN- UŁ/IV/2018.
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Wasilewska, K., Dziedzic, A., Anandan, S. et al. Extracellular vesicle-derived miR-760 as a novel promising candidate biomarker differentiating stable RRMS from SPMS. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35189-y
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DOI: https://doi.org/10.1038/s41598-026-35189-y
