Fig. 3

TLR4 activation enhances miRNA-218 in OCPs, which suppresses osteoclast differentiation by directly inhibiting RANK expression. A The mouse OCPs were incubated with LPS for 4 h and miR-218–2 expression was measured by qPCR. B WT or Tlr4^−/− OCPs were incubated with 10 ng/mL LPS for 4 h and miR-218–2 expression was determined using qPCR. C WT or Slit3 knockdown OCPs were incubated with 0, 1, or 10 ng/mL LPS for 4 h and miR-218–2 expression was then assayed. D Schematic representation of miR-218–2-3p targeting sites in the Tnfrsf11a 3’-UTR. E, F OCPs were transfected with or without the miR-218–2-3p mimic and miR-218–2-3p inhibitor (100 nM), and then incubated with or without 10 ng/mL LPS for 4 h and subjected to qPCR analysis of the relative Tnfrsf11a mRNA expression levels (E). Moreover, these transfected cells were incubated with or without LPS for 24 h and then the RANK protein expression level was analyzed by immunoblotting and quantified with the ImageJ software (F). G Schematic representation of firefly luciferase constructs containing the CMV promoter, luciferase coding region, and a fragment of the Tnfrsf11a 3’-UTR (G, left). NIH3T3 cells were co-transfected with miR-218–2-3p, firefly luciferase constructs (Tnfrsf11a 3’-UTR WT, Mut1, Mut2, or Mut1 + 2), and Renilla luciferase control for the dual-luciferase assay. The relative luciferase activity represents the dual luciferase activity ratio (i.e., firefly/Renilla luciferase). WT, wild type; Mut 1, mutation at site 1; Mut 2, mutation at site 2; Mut1 + 2, mutation at both sites 1 and 2 (G, right). H The transfected OCPs with or without miR-218–2-3p mimic or miR-218–2-3p inhibitor were cultured with M-CSF and RANKL in the presence or absence of LPS (10 ng/mL), and in the presence or absence of anti-miR (Qiagen). These cells were then fixed, stained for TRAP (H, left), and the number of TRAP⁺ MNCs containing three or more nuclei (MNCs; ≥ 3 nuclei) were counted under a light microscope (H, right). Scale bar, 100 μm. Data are shown as mean with s.d., *P < 0.05, **P < 0.005, ***P < 0.0001. All representative data from three independent experiments are shown