Figure 5.

Expression of exogenous miR-218 reversed the inhibitory effects of myostatin-modified osteocyte-derived exosomes on osteoblastic differentiation. As shown in Fig. 1A, Ocy454 cells were differentiated for 12 days and were treated with 100 ng/ml myostatin or vehicle for 48 h followed by the isolation of exosomes released form Cyc454 cells. MC3T3 cells were then co-cultured with the myostatin-modified osteocytic exosomes in the presence of lentivirus-mediated exogenous expression of miR-218 or scramble control plasmid (miR-C) for 48 h. Total RNA or protein was isolated on day 3 and subjected to real-time PCR or Western blot analysis, respectively. A, efficacy of exogenous expression of miR-218 on inhibiting SOST mRNA expression in Ocy454 cells and IDG-SW3 cells. B, changes in mRNA levels of RunX2, osteocalcin, and OPG in MC3T3 cells were determined by real-time PCR. C, Western blotting was performed on total proteins from MC3T3 cells. Images shown were representative Western blottings. D, blots in C were quantified by scanning densitometry and normalized relative to β-tubulin. Data are expressed as the mean ± S.E. for three separate determinations. *, p < 0.05; **, p < 0.01, ***, p < 0.001 versus indicated group.