Figure 2. Increased intra-osteoclast miR-214-3p associates with elevated serum exosomal miR-214-3p and reduced bone formation in OVX mice.

(a) A schematic diagram illustrating the experimental design. Serum and bone samples were collected from female C57BL/6 mice that were ovariectomized at 6-month-old and killed at 6 months (as baseline, OVX-6mon-BS), 9 months (OVX-9mon) and 12 months (OVX-12mon) after ovariectomy, respectively. (b) Real-time PCR analysis of the age-related changes in the levels of miR-214-3p in whole serum (left) and serum exosomes (middle left), and the levels of pri-miR-214-3p (middle), pre-miR-214-3p (middle right) and mature miR-214-3p in osteoclasts (right) in OVX mice in three age-subgroups, respectively. The relative miR-214-3p level in each group was normalized to the mean value of the OVX-BS group. Mouse snoRNA-202 was used as the internal control. (c) Representative images of new bone formation at distal femur metaphysis assessed by double labelling with calcein green and xylenol orange in three age-subgroups. Scale bar, 10 μm. (d) Bone histomorphometry analysis of the age-related changes in BFR/BS at distal femur in three age-subgroups, respectively. (e) Correlation analysis between serum exosomal miR-214-3p level and BFR/BS (left), between serum exosomal and intraosteoclast miR-214-3p levels (middle) and between intraosteoclast miR-214-3p level and BFR/BS (right), respectively. The n value for each group is indicated at the bottom of each histogram. All data are the mean±s.d. **P<0.01. One-way ANOVA with a post-hoc test was performed.