Depletion of MicroRNA-100-5p Promotes Osteogenesis Via Lysine(K)-Specific Demethylase 6B

Abstract

Senescence and osteogenic differentiation potential loss limited bone nonunion treatment effects of bone marrow-derived mesenchymal stem cells (BMSCs). MiR-100-5p/Lysine(K)-specific demethylase 6B (KDM6B) can inhibit osteogenesis, but their effects on bone union remain unclear. This study aims to investigate the effects of miR-100-5p/KDM6B on osteogenic differentiation and bone defects. Wild-type or microRNA 100 (miR-100) knockdown mice underwent critical-size defect (CSD) cranial surgery and collagen I/poly-γ-glutamic acid scaffold treatment. The crania was observed using microcomputed tomography, hematoxylin and eosin staining, Masson staining, alkaline phosphatase (ALP) staining, immunohistochemistry, and immunofluorescence. Primary-cultured BMSCs transfected with miR-100-5p mimic/inhibitor and KDM6B cDNA were evaluated for osteogenic differentiation using Alizarin Red staining, ALP activity detection, and Western blot analysis. Genetic transcription levels were detected using quantitative reverse transcription polymerase chain reaction. This study found that miR-100 depletion promotes defect healing in mouse calvaria, increases the proportion of new bone and osteoblasts in calvaria, and activates the expression of KDM6B and osteocalcin (OCN) proteins, promoting the transcription of bone morphogenetic protein-2, Runt-related transcription factor 2 (Runx2), OCN, and KDM6B, while methylation of lysine 27 on histone H3 (H3K27me3) decreased. Furthermore, miR-100-5p mimics suppressed osteogenic differentiation by inhibiting KDM6B with increased H3K27me3, ALP, Runx2, OCN, and osteopontin protein expression, while miR-100-5p inhibitors have opposite effects. Moreover, KDM6B can reverse miR-100-5p mimic effects. Notably, scaffolds carrying miR-100-5p mimics/inhibitors transfected BMSCs were placed in CSD mice and found that miR-100-5p inhibitors have a better effect on CSD healing and increase new bone without inflammatory cell infiltration. This study proved that miR-100-5p depletion promotes bone union and osteogenic differentiation of BMSCs via KDM6B/H3K27me3.

Keywords: bone marrow-derived mesenchymal stem cells; critical-size defect; lysine(K)-specific demethylase 6B; microRNAs.