Fig. 7

miR-3653-3p reverses circ_0003865-regulated BMSC osteogenic differentiation by directly binding circ_0003865 and the GAS1 gene. a Relative expression of circ_0003865, miR-3653-3p, GAS1, and osteogenic marker genes in BMSCs as measured by qRT-PCR. b GAS1, ALP, RUNX2, and OPN protein levels in human BMSCs as quantitated by western blot analysis using GAPDH as the internal standard. c The proliferation in human BMSCs was tested by CCK-8 assay. d The expression of ALP in human BMSCs was analyzed by ALP staining. The top left are gross scanning images (scale bar: 1 mm), the lower left are enlarged images (magnification: 250×, scale bar: 160 μm), and the right is quantification of the left gross scanning images. e miR-3653-3p inhibitors repressed the osteogenic differentiation of human BMSCs transfected with circ_0003865 siRNAs compared with the si-CIRC group. The Alizarin Red staining was done to evaluate BMSC osteogenic differentiation. The top left are gross scanning images (scale bar: 1 mm), the lower left are enlarged images (magnification: × 250, scale bar: 160 μm), and the right is quantification of the left gross scanning images. f The association of circ_0003865 with miR-3653-3p as detected by an RNA pull-down assay. g, h The direct binding of miR-3653-3p with circ_0003865 and GAS1 gene as measured by the dual-luciferase reporter assay. Between miR-3653-3p and circ_0003865 (g). Between miR-3653-3p and GAS1 gene (h). si-CIRC: circ_0003865 siRNAs; miR-3653-3p In: miR-3653-3p inhibitors; GAS1: growth arrest-specific gene 1; CCK-8: cell counting kit-8; ALP: alkaline phosphatase; RUNX2: runt-related transcription factor 2; OPN: osteopontin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NC: negative control; *P < 0.05