Abstract
Skeletal tissue originates from mesenchymal stem cells (MSCs) with differentiation potential into the osteoblast lineage regulated by essential transcriptional and post-transcriptional mechanisms. Recently, miRNAs and histone modifications have been identified as novel key regulators of osteogenic differentiation of MSCs. Here, we identified miR-99a and its target lysine (K)-specific demethylase 6B (KDM6B) gene as novel modulators of osteogenic differentiation of bone mesenchymal stem cells (BMSCs). Microarray profiling and further validation by quantitative real-time RT-PCR revealed that miR-99a was up-regulated during osteoblastic differentiation of BMSCs, and decreased in differentiated osteoblasts. Transfection of miR-99a mimics inhibited osteoblastic commitment and differentiation of BMSCs, whereas inhibition of miR-99a by inhibitors enhances these processes. KDM6B was determined as one of important targets of miR-99a, which was further confirmed by luciferase assay of 3'-UTR of KDM6B. Moreover, HOX gene level decreased after transfection of miR-99a mimics in BMSCs, which indicated that KDM6B is a bona fide target of miR-99a. Furthermore, in a model of in vivo bone regeneration, osteoblast-specific gain- and loss-of-function experiments performed using cranial bone defects revealed that miR-99a mimics-transfected BMSCs reduced bone formation, and conversely, miR-99a inhibitors-transfected BMSCs increased in vivo bone formation. Tissue-specific inhibition of miR-99a may be a potential novel therapeutic approach for enhancing BMSCs-based bone formation and regeneration.