MiRNAs Expression Profiling in Raw264.7 Macrophages after Nfatc1-Knockdown Elucidates Potential Pathways Involved in Osteoclasts Differentiation

SIMPLE SUMMARY: Bone diseases are a worldwide health public problem, and their management has required extensive studies on bone homeostasis. Among all the various mechanisms involved, understanding those underlying osteoclastogenesis is of paramount importance. Here, we tried to elucidate the possible role of miRNAs differentially expressed in RANKL-stimulated RAW264.7 cells expressing the transcription factor NFATc1, the master regulator of osteoclasts generation, and in NFATc1-silenced RAW264.7 cells, which are depleted of NFATc1 by 80%. We performed miRNAs PCR array followed by bioinformatic analysis to discover new possible miRNAs and their targets involved in this process. The results were interesting and suggested that relatively unknown miRNAs (miR-880 and miR-295) control the phosphorylation/dephosphorylation of proteins/transcription factors such as ERK-p38 and NFATc1, by enzymes (DYRKs and DUSPs). In addition, our results confirmed the role of some miRNAs, already known for their involvement in the process of mature osteoclasts formation. This study contributes to a more complete overview of the early stages of osteoclast formation, including cell migration and fusion. ABSTRACT: Differentiation of macrophages toward osteoclasts is crucial for bone homeostasis but can be detrimental in disease states, including osteoporosis and cancer. Therefore, understanding the osteoclast differentiation process and the underlying regulatory mechanisms may facilitate the identification of new therapeutic targets. Hereby, we tried to reveal new miRNAs potentially involved in the regulation of early steps of osteoclastogenesis, with a particular focus on those possibly correlated with NFATc1 expression, by studying miRNAs profiling. During the first 24 h of osteoclastogenesis, 38 miRNAs were differentially expressed between undifferentiated and RANKL-stimulated RAW264.7 cells, while 10 miRNAs were differentially expressed between RANKL-stimulated cells transfected with negative control or NFATc1-siRNAs. Among others, the expression levels of miR-411, miR-144 and members of miR-29, miR-30, and miR-23 families changed after RANKL stimulation. Moreover, the potential role of miR-124 during osteoclastogenesis was explored by transient cell transfection with anti-miR-124 or miR-124-mimic. Two relatively unknown miRNAs, miR-880-3p and miR-295-3p, were differentially expressed between RANKL-stimulated/wild-type and RANKL-stimulated/NFATc1-silenced cells, suggesting their possible correlation with NFATc1. KEGG enrichment analyses showed that kinase and phosphatase enzymes were among the predicted targets for many of the studied miRNAs. In conclusion, our study provides new data on the potential role and possible targets of new miRNAs during osteoclastogenesis.