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Fig. 6 | Stem Cell Research & Therapy

Fig. 6

From: Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p

Fig. 6

Inhibition of miR-3653-3p expression by circ_0003865 and the effects of miR-3653-3p on GAS1 expression and BMSC osteogenic differentiation. a The interaction networks between circ_0003865 with microRNAs and target genes. The microRNAs and target genes were predicted to interact with circ_0003865 using the miRanda database. b Relative expression of representative microRNAs and target genes in BMSCs treated with MEL. The expression of microRNAs and mRNAs were detected by qRT-PCR. c Elevated miR-3653-3p and decreased GAS1 expression in human BMSCs by circ_0003865 knockdown. Relative expression of microRNAs and GAS1 was measured by qRT-PCR. d circ_0003865 abrogates the suppression of GAS1 expression and elevates miR-3653-3p expression in BMSCs caused by MEL treatment. Relative expression of microRNAs and GAS1 was measured by qRT-PCR. e Influence of miR-3653-3p overexpression on circ_0003865, GAS1, and osteogenic biomarker gene expression in human BMSCs. Relative expression was evaluated by qRT-PCR. f ALP, RUNX2, OPN, and GAS1 protein levels in human BMSCs transfected with miR-3653-3p mimics. Western blot analysis was conducted to measure the protein levels in BMSCs. g Proliferation in human BMSCs transfected with miR-3653-3p mimics was detected by CCK-8 assay. h In situ expression of ALP in human BMSCs transfected with miR-3653-3p mimics was detected 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. i The enhancement of osteogenic differentiation of human BMSCs by miR-3653-3p mimics. Alizarin Red staining was conducted to evaluate the osteogenic differentiation of human 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 image. MEL: melatonin; GAS1: growth arrest-specific gene 1; SFRP2: secreted frizzled-related protein 2; CCK-8: cell counting kit-8; ALP: alkaline phosphatase; RUNX2: runt-related transcription factor 2; OPN: osteopontin; NC: negative control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *P < 0.05

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