Fig. 4

Activation of BMSC osteogenic differentiation by circ_0003865 knockdown. a A schematic illustration of circ_0003865 formation from the back-splicing of ANKRD12 gene pre-mRNA. Exons 4–8 of the ANKRD12 gene contributed to the formation of circ_0003865. b Detection of circ_0003865 formation in human BMSCs by RT-PCR. The existence of circ_0003865 in human BMSCs was validated by RT-PCR using a divergent primer pair in combination with cDNA samples as the template. Opposite-directed primers and gDNA were used as negative controls. c Characterization of the splice junction of circ_0003865 in human BMSCs by Sanger DNA sequencing. The samples amplified by RT-PCR using divergent primers were subjected to Sanger DNA sequencing. d Relative expression of circ_0003865 and osteogenic differentiation marker genes in BMSCs transfected with circ_0003865 siRNAs. qRT-PCR was performed to detect circRNA and mRNA expression in BMSCs. e ALP, RUNX2, and OPN protein levels in BMSCs transfected with circ_0003865 siRNAs. Protein levels were determined by western blot analysis using GAPDH as the internal standard. f No significant change of proliferation in human BMSCs by circ_0003865 knockdown. The CCK-8 assay was used to detect the proliferation in BMSCs. g Increased ALP expression in human BMSCs by circ_0003865 knockdown. The ALP staining method was used to measure in BMSCs. 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. h The enhanced osteogenic differentiation of human BMSCs by circ_0003865 knockdown. The osteogenic differentiation of human BMSCs was evaluated using the Alizarin Red staining method. 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. si-NC: negative control siRNA; si-hsa_circ_0003865: circ_0003865 siRNA; M: marker; gDNA: genomic DNA; CCK-8: cell counting kit-8; ALP: alkaline phosphatase; RUNX2: runt-related transcription factor 2; OPN: osteopontin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05