MicroRNA-451 blockade promotes osteoblastic differentiation and skeletal anabolic effects by promoting YWHAZ-mediated RUNX2 protein stabilization.

Background: senile osteoporosis researchers are now seeking to promote osteoblastogenesis and resultant bone formation to directly counteract age-related bone loss. Targeting microRNA (miRNA) activity in adult osteoblasts may be a successful therapeutic strategy for age-related bone loss. We investigated the mechanism(s) by which miRNAs negatively regulate osteoblastogenesis and bone formation in vitro and in vivo . Methods: we performed a miRNA microarray screen followed by PCR validation in adult bone marrow-derived mesenchymal stem cells during the proliferation-to-mineralization transition to identify downregulated miRNAs, most notably miR-451. Primary human calvarial pre-osteoblasts were isolated and transfected with miR-451's agomir or antagomir for in vitro assays. Bioinformatics analysis and in vitro experiments verified YWHAZ as a miR-451 target gene. We next investigated the effects of YWHAZ knockdown on osteoblastic differentiation. To examine the effects of miR-451's antagomir in vivo , we injected ovariectomized (OVX) or sham-operated mice with miR-451's antagomir over a period of six weeks. We isolated stromal cells from murine bone marrow on week six for further ex vivo experimentation. Results: miR-451's antagomir stimulated pre-osteoblast differentiation into a more differentiated, mineralized phenotype. This phenotype was associated with upregulated RUNX2, ALP, and COL1A1 protein expression. miR-451's antagomir derepresses YWHAZ expression, thereby enhancing RUNX2 protein stability and promoting osteoblastic differentiation. When injected in vivo , miR-451's antagomir promotes osteoblastogenesis and mineralization, reversed OVX-induced bone loss, and increased bone strength in OVX and sham-operated mice. Conclusions: miR-451 suppresses osteoblastogenesis in vitro and in vivo . miR-451 inhibition may serve as an effective anabolic therapeutic strategy in senile osteoporosis patients.