Figure 3.

MiR-20a up-regulates the BMP2 signaling pathway during FSS-induced osteogenic MC3T3-E1 cell differentiation. Transfection of MC3T3-E1 cells was performed using 50 nM NC, miR-20a mimics, or 100 nM anti-miR-20a, after which they were subjected to 12 dyn/cm² FSS for 1 h. (A-C) The transcriptional levels of BMP2, SP7, and RUNX2 in miR-20a-overexpressed cells were evaluated using qRT-PCR analysis at 12 h pf. (D-F) Transcriptional levels of BMP2, RUNX2, and SP7 in miR-20a-inhibited cells were evaluated using qRT-PCR analysis at 12 h pf. (G,H) Protein levels of BMP2 as well as RUNX2 were measured by immunoblot analysis at 24 h pf. in miR-20a-overexpressioned cells (G) and miR-20a-inhibited cells (H). Data are expressed as mean ± SD for n=3; *, P<0.05; **, P<0.01; ***, P<0.001. FSS, fluid shear stress; pf., post-FSS treatment; BMP2, bone morphogenetic protein; RUNX2, Runt-related transcription factor 2; SP7, SP7 transcription factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qRT-PCR, real-time quantitative polymerase chain reaction; C, transfection reagent only group; NC, miRNA negative control group; NC + F, negative control plus fluid shear stress group; miR-20a + F, miR-20a mimics plus fluid shear stress group; inhibitor-NC, inhibitor negative control group; anti-miR-20a + F, anti-miR-20a mimics plus fluid shear stress group.