Figure 4.

miRNA-92a-3p directly targets IBSP

(A) Prediction of the miRNA-92a-3p target gene. Using four online miRNA target gene prediction sites and visualizing the intersection of the Venn diagrams identifies ISBP as a target gene at all four sites. (B) The predicted luciferase (Luc) reporter gene construct contains WT IBSP 3′ UTR (WT 3′ UTR) or the same region after site-directed mutagenesis (3′ UTR-Mut). (C) Luciferase activity after treatment with antagomiR-92a-3p to investigate the effect of endogenous miRNA-92a-3p in MC3T3-E1 cells on IBSP 3′ UTR Muts (WT IBSP 3′ UTR, lucu-UTR-mut). (D and E) After transfection of the control group, agomiR-92a-3p, agomiR-NC, antagomiR-92a-3p, and antagomiR-NC, (D) qPCR, and (E) western blot analyses were used to detect the expression of IBSP. (F) Expression of IBSP in the control group, Fracture group, Fracture+TBI group. (G) Expression of IBSP in bone callus tissue of Fracture and Fracture+TBI mice. (H) Immunohistochemistry showed decreased expression of IBSP protein in the Fracture+TBI callus (the red arrow shows the positive IBSP immunohistochemical staining). (I and J) Transfection of MC3T3-E1 subclone 14 cells with siRNA (control), siRNA-NC, and siRNA-IBSP, and subsequent evaluation of Col1a1, ALP, OCN, and Runx2 using (I) western blot and (J) RT-PCR analysis. n = 3; data are presented as mean ± SD (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; # no significance).