miR-218-5p Promotes Osteoblast Differentiation and Inhibits Osteoclast Formation through the ROBO1/DKK-1 Pathway during Bone Remodeling

Abstract

Aim: To study the effect and mechanism of miR-218-5p on osteoblast differentiation and osteoclast formation. Methods: TargetScan was employed to predict the target miRNA, which was tracked by luciferase reporter assays. MC3T3-E1 cells and receptor activator of nuclear factor-κB ligand (RANKL)-treated RAW 264.7 cells were co-transfected with miR-218-5p mimic, miR-218-5p inhibitor, and si-roundabout guidance receptor 1 (ROBO1). The expression of miR-218-5p was evaluated using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). MC3T3-E1 cells were stained with Alizarin red and alkaline phosphatase (ALP) kits, and ALP activity was assessed. Pit formation and F-actin ring immunofluorescence assays were conducted in RANKL-treated RAW 264.7 cells. Protein levels of ROBO1, Dickkopf-1 (DDK-1), ALP, runt-related transcription factor 2 (RUNX2), bone morphogenetic protein-2 (BMP-2), tartrate-resistant acid phosphatase (TRAP), and nuclear factor of activated T-cells 1 (NFATC1) were determined using western blot analyses. Results: miR-218-5p targeted ROBO1 in the 3′-UTR. miR-218-5p and si-ROBO1 significantly decreased the levels of ROBO1 and DDK-1. miR-218-5p and si-ROBO1 increased calcified nodule formation, ALP activity, and levels of ALP, RUNX2, and BMP-2 but suppressed pit formation and F-actin ring formation and decreased levels of TRAP, F-actin, and NFATC1. In addition, miR-218-5p inhibitor weakened the effects of si-ROBO1 on osteoblast differentiation and osteoclast formation. Conclusions: Collectively, miR-218-5p promotes osteoblast differentiation and inhibits osteoclast formation through the ROBO1/DKK-1 pathway, which provides a potential therapeutic strategy for bone remodeling.