Figure 6: miR-21 deficiency blocks ovariectomy (OVX)-induced osteopenia by inhibiting osteoclastogenesis through targeting programmed cell death 4 (PDCD4).
From: miR-21 deficiency inhibits osteoclast function and prevents bone loss in mice

(a) Representative micro-CT images demonstrating bone phenotypes of ovariectomized WT and miR-21−/− mice. Mice were sacrificed at 1 month post OVX. Orange frames indicate the region of interest analyzed for trabecular bone mass in the distal femoral metaphysis (up). Cortical bone mass was analyzed in the midshaft of femora (bottom). Bars: 500 μm. (b,c) Corresponding parameters showed that miR-21 deficiency prevented both trabecular (b) and cortical (c) bone loss induced by OVX. BV/TV, bone volume per tissue volume. Ct.Th, cortical thickness. (d) Tartrate resistant acid phosphotase (TRAP) staining of the trabecular bone in histological sections of ovariectomized WT and miR-21−/− mice. Bars: 25 μm. (e,f) The corresponding parameter of TRAP and the serum bone resorption marker detected by enzyme-linked immunosorbent assay (ELISA) showed that miR-21 deficiency blocked OVX-induced osteoclastogenesis and bone resorption. Oc.S/BS, osteoclast surface per bone surface (e). CTX-1, cross linked C-telopeptide of type 1 collagen (f). (g) ELISA detection of serum ratio of receptor activator of nuclear factor κB ligand (RANKL) over osteoprotegerin (OPG). No significant difference was detected between ovariectomized WT and miR-21−/− mice. (h,i) Representative images (h) and the corresponding parameter (i) demonstrated that miR-21 deficiency blocked OVX-induced resorption activity of osteoclasts (OCs). Bone marrow macrophages (BMMs) were harvested, seeded on dentine slices and cultured. Resorption pits were stained with toluidine blue. M, macrophage colony-stimulating factor (M-CSF). RL, RANKL. Tt.Ar, total area. Bars: 100 μm. (j) Western blot analysis of mature osteoclasts derived from ovariectomized WT and miR-21−/− mice. miR-21 deficiency inhibited OVX-induced osteoclastogenesis through promoting the PDCD4 protein level, a functional target of miR-21, which suppressed the phosphorylation level of c-fos. Cropped blots are displayed with only brightness adjusted equally across the entire images. Data represents mean ± standard errors of the mean. n = 6 per group. Statistical significance was evaluated by two-tailed Student’s t test for two-group comparison, and one way analysis of variation (ANOVA) with Newman-Keuls post-hoc tests for multiple comparisons. *P < 0.05. NS, not significant (P > 0.05).