Figure 5.

MiR-20a initiates the BMP2 signaling pathway by targeting BAMBI and SMAD6. To detect the expression of putative miR-20a targets, MC3T3-E1 cells or mouse BMSCs transfected with 50 nM NC or miR-20a mimics were treated using 12 dyn/cm² FSS for 1 h. (A,C) Immunoblot analysis of BAMBI and SMAD6 protein in MC3T3-E1 on days 1 and 2 post-FSS treatment (A) and in mouse BMSCs on days 2 and 4 post- FSS treatment (C). (B,D) qRT-PCR analysis of BAMBI and SMAD6 mRNA expression in MC3T3-E1 at 12 and 24 h post-FSS treatment (B) and in mouse BMSCs at 24 and 48 h post-FSS treatment (D). (E,F) One putative target site of miR-20a predicted by the TargetScan program was contained in the BAMBI or SMAD6 mRNA 3'UTR. The mutated sites of the 3'-UTR of BAMBI or SMAD6 are shown as *. (G) Luciferase reporter assays in cos-7 cells, with co-transfection of WT or MUT BAMBI mRNA 3'-UTR and miR-20a. (H) Luciferase reporter assays in cos-7 cells, with co-transfections of WT or MUT SMAD6 mRNA 3'-UTR and miR-20a mimics. Data are presented as mean ± SD for n=3; **, P<0.01. BMP2, bone morphogenetic protein 2; qRT-PCR, real-time quantitative polymerase chain reaction; BAMBI, BMP and activin membrane-bound inhibitor; SMAD6, mothers against decapentaplegic family member 6; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; BMSCs, bone marrow stromal cells; C, transfection reagent only group; NC, miRNA negative control group; NC + F, negative control plus fluid shear stress group; miR-20a, miR-20a mimics group; WT, wild type; MUT, mutant type.