Fig. 2

EV miR-218 directly targets Col1a1 and downregulates type I collagen in differentiated MC3T3. a Relative RNA level of intracellular and EV miR-218 normalized to U6 and miR-140-3p, respectively, in indicated cell lines. ***P < 0.001. b DiI-labeled EV was used to treat MC3T3 cells seeded in a chamber slide for 48 h. Cells were fixed and imaged with fluorescent microscope Zeiss Observer II. Scale bar indicates 100 μm. c Relative RNA level of miR-218 normalized to U6 in MC3T3 cells treated with indicated EV or PBS for 24 h in the presence of 10 μM DRB (5,6-dichloro-1-β -D-ribofuranosylbenzimidazole). d Relative RNA level of Col1a1, Col1a2 normalized to Actb in differentiated MC3T3 cells treated with indicated EVs. Osteoblast differentiation medium with EV was replenished every 3 to 4 days for 16 days. ***P < 0.001. e Western blot analyses of type I collagen in the CM from EV-treated differentiated MC3T3 at day 18. CM of miR-218 mimic-transfected differentiated MC3T3 was also examined. The top band (pro α1), second band (pC/pN α1) and third band (α1) indicates type I procollagen, type I procollagen with either N′ terminus or C′ terminus cleaved, and mature type I collagen with both N′ and C′ termini cleaved, respectively. Cellular β-actin was used as control. f P1NP in CM collected from EV-treated differentiated MC3T3 at day 3 was measured by ELISA. *P < 0.05. g and h Bone formation marker P1NP (g) and bone resorption marker CTX-1 (h) in the sera from EV-treated mice were measured by ELISA. *P < 0.05; n.s. not significant