Fig. 5: MiRNA-1224-5p inactivates the ADCY2-dependent Rap1 signaling pathway.
a Gene expression levels of BMMs using agomiRNA-1224 and antagomiRNA-1224 24 h after transfection. The expression of 460 genes was upregulated (red), and the expression of 670 genes was downregulated (blue). Each group was treated with 30 ng/ml M-CSF. b Heatmap showing genes related to osteoclasts. c RT-PCR quantitatively confirmed that the expression of key genes in osteoclasts was upregulated. d KEGG pathway analysis predicts that the function of the Rap1 signaling pathway will change. e Heatmap shows genes related to the Rap1 pathway. f Western blot analysis showed that 6 h after treatment with agomiRNA-1224 in BMMs, the level of Rap1 decreased significantly. g Western blotting confirmed that treatment of BMMs with agomiRNA-1224 decreased the RANKL-induced protein expression of CDC42, p-Rac1, p-Rho-A, FAK, and Rac1 for 24 h. h Representative immunohistochemical staining showing that agomiRNA-1224 injection decreased ADCY2 and Rap1 levels in vivo. Femurs were collected within 24 h of miRNA after injection. Scale bar, 50 µm. i Western blotting confirmed that treatment of BMMs with agomiRNA-1224 decreased the expression of NFATC1 protein induced by RANKL for 48 h. j ChIP experiment results show that agomiRNA-1224 induces ADCY2 to occupy the Nfatc1-binding region together. k Western blot analysis of Nfatc1. The removal of ADCY2 weakened the expression of Nfatc1 induced by agomiRNA-1224. l ChIP experiment results show that removing ADCY2 abolishes the binding region of Rap1 and Nfatc1 triggered by agomiRNA-1224. The results are presented as the mean ± standard deviation, N = 3; #P ≥ 0.05; *P < 0.05, **P < 0.01; ***P < 0.001 by t test.
