miR-99a in bone homeostasis: Regulating osteogenic lineage commitment and osteoclast differentiation

The tight coupling between osteoblasts and osteoclasts is essential to maintain bone homeostasis. Deregulation of this process leads to loss and deterioration of the bone tissue causing diseases, such as osteoporosis. MicroRNAs are able to control bone-related mechanisms and have been explored as therapeutic tools. In this study, we investigated the potential of miR-99a-5p to modulate osteogenic differentiation, osteoclastogenesis, and the osteoblasts-osteoclasts crosstalk.

To achieve this goal, human primary Mesenchymal Stem/Stromal Cells (MSC) were differentiated into osteoblasts and adipocytes, and miR-99a-5p expression was evaluated by RT-qPCR. Knockdown and overexpression experiments were conducted to modulate miR-99a-5p expression in MC3T3 cells. Cell proliferation and cell death/apoptosis were evaluated by resazurin assay and flow cytometry, respectively. Proteomic analysis was used to identify the miR-99a-5p regulatory network, and ELISA to evaluate OPG levels in the cell culture supernatant. Conditioned media from MC3T3-transfected cells was used to culture RAW 264.7 cells and the effect on osteoclast differentiation was assessed. Human primary monocytes were isolated to induce osteoclastogenesis and evaluate miR-99a-5p expression. Finally, levels of miR-99a-5p were modulated in RAW 264.7 cells to understand the impact on osteoclastogenesis.

The results show that miR-99a-5p is significantly downregulated during the early stages of human primary MSCs osteogenic differentiation and during MC3T3 osteogenic differentiation. On the other hand, miR-99a-5p levels are increased during the initial stages of adipogenic differentiation. Inhibition of miR-99a-5p in MC3T3 pre-osteoblastic cells promoted osteogenic differentiation, whereas its overexpression suppressed the levels of osteogenic specific genes (Runx2 and Alpl), as well as mineralization, with no effect on proliferation or apoptosis. Proteomic analysis of miR-99a-5p-transfected cells showed that numerous proteins known to be involved in cell differentiation were altered, including osteogenic differentiation markers and extracellular matrix proteins. While inhibition of miR-99a-5p increased the Tnfrsf11b (OPG encoding gene)/Tnfsf11 (RANKL encoding gene) mRNA expression ratio, in addition to increasing OPG secretion, miR-99a-5p overexpression resulted in the opposite effect. The cell culture supernatant of miR-99a-5p-inhibited MC3T3 cells impaired the osteoclastogenic potential of RAW 264.7 cells by decreasing the number of multinucleated cells and reducing the expression of osteoclastogenic markers. Interestingly, miR-99a-5p expression is increased during osteoclasts differentiation, both in human primary monocytes and RAW 264.7. These results show that miR-99a-5p per se is a positive regulator of osteoclastogenic differentiation.

Globally, our findings show that miR-99a-5p inhibition promotes the commitment into osteogenic differentiation, impairs osteoclastogenic differentiation, and control bone cells communication. Ultimately, it supports miR-99a-5p as a target candidate for future miRNA-based therapies for bone diseases associated with bone remodeling deregulation.