Pathway Analysis of MicroRNA Expression Profile during Murine Osteoclastogenesis

Abstract

To design novel therapeutics against bone loss, understanding the molecular mechanisms regulating osteoclastogenesis is critical. Osteoclast formation and function are tightly regulated by transcriptional, post-transcriptional and post-translational mechanisms. This stringent regulation is crucial to prevent excessive or insufficient bone resorption and to maintain bone homeostasis. microRNAs (miRNAs) are key post-transcriptional regulators that repress expression of target mRNAs controlling osteoclast proliferation, differentiation, and apoptosis. Disruption of miRNA-mediated regulation alters osteoclast formation and bone resorption. Prior studies profiled miRNA expression in murine osteoclast precursors treated with RANKL for 24 hours. However, a more complete miRNA signature, encompassing early, mid and late stages of osteoclastogenesis, is wanting. An Agilent microarray platform was used to analyze expression of mature miRNAs in an enriched population of murine bone marrow osteoclast precursors (depleted of B220 ⁺ and CD3 ⁺ cells) undergoing 1, 3, or 5 days of RANKL-driven differentiation. Expression of 93 miRNAs, changed by >2 fold during early, mid, and late stages of osteoclastogenesis, were identified and sorted into 7 clusters. We validated the function and expression of miR-365, miR-451, and miR-99b, which were found in distinct clusters. Inhibition of miR-365 increased osteoclast number but decreased osteoclast size, while miR-99b inhibition decreased both osteoclast number and size. In contrast, overexpression of miR-451 had no effect. Computational analyses predicted mTOR, PI3 kinase/AKT, cell-matrix interactions, actin cytoskeleton organization, focal adhesion, and axon guidance pathways to be top targets of several miRNA clusters. This suggests that many miRNA clusters differentially expressed during osteoclastogenesis converge on some key functional pathways. Overall, our study is unique in that we identified miRNAs differentially expressed during early, mid, and late osteoclastogenesis in a population of primary mouse bone marrow cells enriched for osteoclast progenitors. This novel data set contributes to our understanding of the molecular mechanisms regulating the complex process of osteoclast differentiation.