Identification of microRNAs involved in osteoblast differentiation of murine embryonic stem cells

Elucidating the molecular events governing the osteogenic differentiation of mouse embryonic stem cells (ESCs) is of extreme importance for improving the treatment of bone-related diseases. Osteoblast differentiation is a key step in proper skeletal development and acquisition of bone mass; however, the physiological role of non-coding small RNAs, especially microRNAs (miRNAs), in osteoblast differentiation remains elusive. In this study miRNA profiling was used to identify miRNAs that are potentially involved in osteogenesis. In addition, functional characterization and target identification of these miRNAs were performed to further unravel the molecular mechanisms underlying osteogenesis. Among the more than 250 miRNAs examined, twenty-five miRNAs in particular were significantly expressed during osteogenic differentiation. Eleven miRNAs, namely miR-22, miR-127, miR-130a, miR-183, miR-291b-5p, miR-293, miR-300, miR-361, miR-467b, miR-665 and miR-690, were identified to be differentially expressed in undifferentiated ESCs versus ESCs differentiated into osteoblasts. Overexpressing and knocking down these miRNAs caused changes in cell survival, cell morphology, and osteogenic differentiation capacity as measured with calcium deposition, ALP activity and expression of osteogenic markers. Using three different miRNA target identification approaches, most targets were found to be associated with the Wnt signaling pathway. We conclude from this study that as the Wnt pathway has been shown to be one of the major pathways involved in osteogenic differentiation of ESCs, the current results suggest a role for the identified miRNAs in this process.