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. 2016 Mar 7;7:10872. doi: 10.1038/ncomms10872

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(a) A schematic diagram illustrating the experimental design. Serum and bone samples were collected from elderly woman patients with or without fractures, and divided into 60–69, 70–79 and 80–89 groups, respectively, according to age. (b) Real-time PCR analysis of the age-related changes in miR-214-3p levels in whole serum (left), serum exosomes (middle) and bone specimens (right) from elderly patients with or without fractures, respectively. The relative miR-214-3p level in each group was normalized to the mean value of the 60–69 group. Human RUN6B was used as the internal control. All data are the mean±s.d. *P<0.05, **P<0.01. Two-way analysis of variance (ANOVA) with a Turkey's multiple comparisons test was performed. Both the time effect (age), the group effect (with or without fractures) and the time-by-group interaction effect were all statistically significant for all the examined parameters. (c) Real-time PCR analysis showing the age-related changes in BGLAP mRNA levels in bone specimens from elderly patients with fractures, respectively. The relative BGLAP mRNA level in each group was normalized to the mean value of the 60–69 group. Human GAPDH mRNA was used as the internal control. Data are the mean±s.d. **P<0.01. One-way ANOVA with a post-hoc test was performed. (d) Western blot analysis of the osteoclast marker proteins (CTSK, TRAcP5 and Sema4D) in the lysates of serum exosomes isolated from elderly patients with or without fractures. OCs, the lysates of osteoclasts differentiated from human peripheral blood mononuclear cells. Exo 1, the lysates of serum exosomes from elderly patients without fractures. Exo 2, the lysates of serum exosomes from elderly patients with fracture. (e) Correlation analysis between exosomal and intra-osseous miR-214-3p levels (left), between exosomal miR-214-3p level and intra-osseous BGLAP mRNA level (middle) and between intra-osseous miR-214-3p level and intra-osseous BGLAP mRNA level (right), respectively, in elderly patients with fractures. The n value for each group is indicated at the bottom of each histogram.