hsa-let-7c-3p Restrains the Proliferation-to-Osteogenesis Transition of Human Periodontal Ligament Mesenchymal Stem/Stromal Cells via NT5E

Small extracellular vesicles (sEVs) derived from mesenchymal stem/stromal cells (MSCs) contain microRNAs (miRNAs) that can modulate the cellular behavior of recipient cells. Human periodontal ligament mesenchymal stem/stromal cells (hPDL-MSCs) are a unique MSC population involved in periodontal tissue regeneration; however, the specific miRNAs that regulate their proliferative and differentiation-related properties remain incompletely understood. In this study, we profiled miRNAs enriched in sEVs secreted by highly proliferative human PDL-MSCs and identified hsa-let-7c-3p as a candidate regulator of hPDL-MSC behavior. Functional assays demonstrated that hsa-let-7c-3p enhanced proliferative and migratory behavior, reflected by an approximately 1.5-fold increase in metabolic activity and a 2-fold increase in migration, while suppressing osteogenic differentiation, with approximately 50% reductions in alkaline phosphatase activity and mineralization. To identify functionally relevant downstream targets, we intersected AGO2-RIP-seq-enriched transcripts, RNA-seq-defined genes altered by hsa-let-7c-3p overexpression, and in silico target predictions, identifying NT5E/CD73 as a candidate downstream target with experimental validation. Furthermore, NT5E knockdown partially phenocopied the effects of hsa-let-7c-3p overexpression on hPDL-MSC proliferation, migration, and osteogenic differentiation, supporting a functional link between hsa-let-7c-3p and NT5E/CD73. These findings highlight hsa-let-7c-3p as an underexplored sEV-associated 3p miRNA strand that restrains the proliferation-to-osteogenesis transition of hPDL-MSCs and identify NT5E/CD73 as a functionally relevant downstream target. Clinical trial number. Not applicable.