Circular RNA atlas in osteoclast differentiation with and without alendronate treatment

BACKGROUND: Alendronate (AL) is the most widely used bisphosphonate in the treatment of osteoporosis (OP). However, the role of circular RNAs (circRNAs) in the treatment of OP with AL remains unclear. METHODS: In this study, we showed that osteoclast (OC) precursors (OPCSs) could be induced into OCs with macrophage colony-stimulating factor (MCSF) and receptor activator of nuclear factor-κB ligand (RANKL) treatment. Subsequently, the OCs were treated with AL. OC differentiation-related biomarkers including RANK, tartrate-resistant acid phosphatase (TRAP), and cathepsin K (CTSK) were analyzed with TRAP staining, quantitative real-time (qPCR), and western blotting. Differentially expressed circRNAs (DECs) were identified among the OPCS, OC, and OC + AL groups. In addition, the expression levels of 10 DECs related to OC differentiation were verified by qPCR. RESULTS: TRAP staining showed that MCSF and RANKL treatment effectively induced OPCSs to differentiate into OCs. In addition, qPCR and western blot analysis revealed that the three biomarkers of OC (RANK, TRAP, and CTSK) were expressed significantly more in the OC group than those in the OPCS group. In contrast, the mRNA and protein expression levels of these three biomarkers decreased significantly in OCs treated with AL compared with those non-treated OCs. GO analysis of the DECs in the OPCS group vs. the OC group revealed that their functions were mainly related to cell, cell part, binding, and single-organism terms. KEGG analysis of the top 20 DECs in a comparison between the OPCS and OC groups showed that genes involved in mitogen-activated protein kinase signaling were the most common. Results of functional analyses of DECs in an OC vs. OC + AL comparison were similar to those in the OPCS vs. OC comparison. Finally, qPCR showed that, in the OC + AL vs. OC group comparison, the expression levels of seven and three DECs significantly decreased and increased, respectively. CONCLUSIONS: Having successfully induced OPCSs to differentiate into OCs, we showed that AL suppresses the differentiation of OPCS into OC and that 10 DECs were involved in the regulation of this process. This indicates that these DECs might be important to the treatment of OP.