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. 2020 Aug 19;28(1):283–302. doi: 10.1038/s41418-020-0600-6

Fig. 3. MiR-326-5p regulates ADSC osteogenesis by targeting Itga5.

Fig. 3

a MiR-326-5p expression levels during ADSC osteogenic differentiation were assessed by qPCR and showed a gradual trend of downregulation (n = 3, *P < 0.05, statistical analysis was performed by one-way ANOVA). b Transfection efficiency of miR-326-5p as evidenced by fluorescence pictures. Scale bars: 200 µm. c Transfection efficiency of Lenti-miR-326-5p and Lenti-miR-326-5p inhibitor was evaluated by qPCR (n = 3, *P < 0.05 versus Lenti-miR-NC group, statistical analysis was performed by one-way ANOVA). d The mRNA expression of osteogenic marker genes Runx2, Col1a1, OPN and BSP was markedly inhibited in the Lenti-miR-326-5p-transfected cells whereas it was promoted by the Lenti-miR-326-5p inhibitor compared to Lenti-NC at osteogenesis day 7 (n = 3, *P < 0.05 versus Lenti-miR-NC group, statistical analysis was performed by one-way ANOVA). (e). Western blotting for the protein expression of Runx2, OSX, OPN and BSP at osteogenesis day 7. f Immunocytochemistry showed BSP was markedly inhibited in the Lenti-miR-326-5p-transfected cells whereas it was promoted by the Lenti-miR-326-5p inhibitor compared to Lenti-NC. (n = 3, *P < 0.05, statistical analysis was performed by one-way ANOVA, Scale bars: 100 µm). g The target gene prediction databases miRanda, miRwalk and Targetscan indicate that there are totally 213 target genes in intersection for miR-326-5p. h The protein levels of CRY2 and Mapk3 were not influenced by overexpression or knockdown of miR-326-5p in ADSCs. The protein level of Itga5 was reduced in Lenti-miR-326-5p-transfected ADSCs whereas it was promoted in Lenti-miR-326-5p inhibitor-transfected ADSCs at osteogenesis day 7. i qPCR results of Itga5 mRNA expression in Lenti-miR-326-5p-transfected ADSCs at osteogenesis day 7. j Western blot analysis at days 0, 2, 5, and 7 indicated that the protein expression of Itga5 was gradually increased. k–m. Immunocytochemistry analysis of Itga5 expression levels in Lenti-miR-326-5p-transduced ADSCs. Scale bars: 100 µm. n Schematic diagram depicting the constructed firefly luciferase reporter system and the predicted binding sites of miR-326-5p in the Itga5 3′UTR. o Luciferase reporter assay showed that co-transfection of miR-326-5p with the constructed Itga5 3'UTR-wt plasmid obviously decreased luciferase activity (*P < 0.05, statistical analysis was performed by one-way ANOVA). p Four Lenti-itga5-inhibitors were transduced into ADSCs, and Lenti-itga5-inhibitor-1 and Lenti-itga5-inhibitor-4 effectively knocked down the expression of Itga5 (n = 3, *P < 0.05 versus Lenti-NC group, statistical analysis was performed by one-way ANOVA). q Knockdown of Itga5 by Lenti-itga5-inhibitor-1 and Lenti-itga5-inhibitor-4 decreased the mRNA levels of Runx2, OSX, Col1a1 and OPN, while the expression of AdipoQ and SOX9 showed no obvious changes with Itga5 inhibition in ADSCs at osteogenesis day 7 (n = 3, *P < 0.05 versus Lenti-NC group, statistical analysis was performed by one-way ANOVA).