hPDL-MSC Extracellular Vesicles-delivered miRNAs Regulates Osteogenesis via miRISC-mediated Targetome Silencing

Periodontal homeostasis is maintained by a delicate balance between tissue regeneration and degradation, orchestrated through complex regulatory networks of cellular and molecular signaling. miRNAs act as pivotal post-transcriptional regulators that fine-tune these signaling cascades. Despite increasing recognition of their regulatory potential, the specific miRNAs governing stem cell maintenance and differentiation in the periodontium remain insufficiently defined. In this study, we identified hsa-181c-5p and hsa-let-7c-3p within the extracellular vesicles secreted from human periodontal ligament mesenchymal stem cells (hPDL-MSCs) as a candidate regulator contributing to PDL-MSC homeostasis. hsa-let-7c-3p promotes the proliferation and migration while suppressing osteogenic differentiation of hPDL-MSCs. In the meanwhile, miR-181c-5p was consistently upregulated in hMSCs during osteogenesis, and its gain- and loss-of-function produced concordant effects. Integrative analyses combining AGO2-based immunoprecipitation sequencing (RIP-Seq), RNA-Seq, dual-luciferase reporter, and western blot identified miRISC-mediated targetome of miRNAs. Collectively, our study unveiled novel miRNA-mRNA networks through which hPDL-MSC–derived EVs affecting osteogenic differentiation and proliferation, providing new mechanistic insight into miRNA-mediated stem cell regulation and periodontal regeneration.