miR-487b-3p impairs osteoblastogenesis by targeting Notch-regulated ankyrin-repeat protein (Nrarp).

MicroRNAs (miRNAs) have appeared as critical controllers of gene expression at post-transcriptional level either by degrading RNA transcripts or repressing translation. It is evident from the ever growing scientific literature that microRNAs play a significant role in osteoblast commitment and differentiation. Here, we report that over-expression of miR-487b-3p leads to inhibition of osteoblastic differentiation. Using in silico approaches, Nrarp was found to be the direct target of miR-487b-3p which was further validated by luciferase 3' UTR reporter assay. Nrarp inhibits Notch-1 signaling and promotes Wnt signaling by stabilization of LEF-1. Role of miR-487b-3p in regulating canonical Wnt and Notch signalling was determined by western blotting. Protein levels of Nrarp, RUNX-2, Lef1 and β catenin were reduced in osteoblasts cells transfected with miR-487b-3p whereas protein levels of Notch1, Hes1 and P- β catenin were up regulated when osteoblast cells were transfected with miR-487b-3p. These outcomes were reversed after treating cells with anti-miR-487b-3p. Further silencing of miR-487b-3p in neonatal Balb/c mice attenuated all the inhibitory actions of miR-487b-3p on osteoblast differentiation. Importantly, in vivo action of anti-miR-487b-3p to ovariectomized osteopenic BALB/c mice steered to significant enhancement in trabecular bone microarchitecture. Further the bio-mechanical properties of isolated femurs were enhanced in anti-miR-487b-3p treated mice. Overall, miR-487b-3p negatively regulates osteogenesis by suppressing Nrarp expression, which in turn, suppresses Runx-2 and Wnt signalling, both of which play a pivotal action in osteoblast differentiation.