In Silico Systems-Level Analysis of Extracellular Vesicle-Associated miRNAs Potentially Involved in Bone Regeneration

Background: Extracellular vesicle (EV)-associated microRNAs (miRNAs) have been implicated in intercellular signaling processes relevant to bone regeneration; however, the regulatory relationships through which selected EV-associated miRNAs may converge on biological processes relevant to bone repair remain incompletely understood. Objective: To investigate, using an in silico systems biology approach, the predicted regulatory architecture of five literature-selected EV-associated miRNAs potentially involved in biological processes relevant to bone regeneration. Methods: Five miRNAs (hsa-miR-146a-5p, hsa-miR-21-5p, hsa-miR-335-5p, hsa-miR-503-5p, and hsa-miR-129-5p) were selected a priori based on published evidence linking them to EV-associated signaling and/or osteogenesis-related biological processes. Candidate target genes were predicted using TargetScan Human 8.0 and prioritized according to Context++ score ranking, followed by predicted target-overlap analysis, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment pathway enrichment, and miRNA–messenger RNA (mRNA) network reconstruction using Cytoscape 3v.10. Results: The five miRNAs yielded 395 prioritized predicted miRNA–mRNA interactions corresponding to 387 unique predicted target genes, with most predicted targets associated with a single miRNA and a limited subset shared between pairs of miRNAs. Eight genes were identified as pairwise shared predicted targets, with no higher-order overlap involving three or more miRNAs. SMAD family member 7 (SMAD7) was identified as was identified as a pairwise shared predicted target of hsa-miR-21-5p and hsa-miR-503-5p and was selected for biological contextualization because of its involvement in transforming growth factor beta (TGF-β)/bone morphogenetic protein (BMP)-associated signaling. Functional enrichment identified statistical overrepresentation of signaling pathways regulating pluripotency of stem cells, as well as mitogen-activated protein kinase (MAPK), Hippo, TGF-β, Wnt, mechanistic target of rapamycin (mTOR), and forkhead box O (FoxO) signaling pathways. Conclusions: This exclusively computational analysis identified a predicted regulatory architecture characterized by predominantly miRNA-specific target relationships, limited pairwise target overlap, and pathway-level enrichment potentially relevant to bone-related biological processes. These findings are hypothesis-generating and do not establish miRNA abundance within a specific EV preparation, direct miRNA–mRNA regulation, pathway activation, or osteoregenerative activity. Experimental EV cargo characterization and functional validation in relevant cellular and in vivo models are required to determine the biological significance of the predicted relationships.