MicroRNA-449c-5p inhibits osteogenic differentiation of human VICs through Smad4-mediated pathway

Figures

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  Figure 1. Characteristics and phenotypes of VICs. (a) The morphology of VICs (Left: low magnification; Right: high magnification). (b) Immunohistochemical staining of α-SMA (Left: low magnification; Right: high magnification). (c) Immunohistochemical staining of Vimentin (Left: low magnification; Right: high magnification). (d) Quantification of positive staining of α-SMA and Vimentin.

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  Figure 2. MiR-449c-5p is downregulated in human calcific aortic valves. (a) Heat map based on the differential expression of miRNAs between non-calcific and calcific aortic valves calculated by microarray. n = 3 in each group. (b) Verification of miR-449c-5p expression by qRT-PCR in non-calcific and calcific valves from 10 CAVD patients. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01.

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  Figure 3. Overexpression of miR-449c-5p inhibits VICs osteogenic differentiation while downexpression of miR-449c-5p promotes the process. (a) qRT-PCR analysis of miR-449c-5p expression in VICs transfected with miR-449c-5p mimic or inhibitor at day 2. (b) qRT-PCR analysis of Runx2 expression at day 7 after osteogenic differentiation. (c) ALP activity at day 14 after osteogenic differentiation. (d) Alizarin red staining at day 14 after osteogenic differentiation. (e) Quantitative analysis of alizarin red staining. n = 3 in each group. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01.

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  Figure 4. MiR-449c-5p directly targets Smad4. (a) Schematic of the putative miR-449c-5p target site in human Smad4 3′-UTR and the eleven mutated nucleotides are colored red. (b) Dual-luciferase assay after transfection of M-miR-449c-5p, NC-miR-449c-5p with wild-type Smad4 3′-UTR or mutant Smad4 3′UTR. n = 3 in each group. Data are presented as the mean ± SD. *P < 0.05.

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  Figure 5. Regulation of Smad4 by miR-449c-5p during VICs osteogenic differentiation. (a) qRT-PCR analysis of Smad4 relative mRNA expression at day 7 after osteogenic differentiation. (b,c) Western blot analysis of Smad4 at day 14 after osteogenic differentiation. (d,e) Western blot further confirmed Smad4 knockdown could block the effect of miR-449c-5p inhibitor during VICs osteogenic differentiation. n = 3 in each group. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01.

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  Figure 6. MiR-449c-5p inhibits aortic valve calcification in vivo. (a) The velocity profile in the aortic annulus measured by echocardiography six weeks after transfection in vitamin D3-treated mice. (b) Quantitative analysis of the velocity in the aortic annulus in each group. (c) Quantitative analysis of transvalvular gradients in each group. (d) qRT-PCR analysis of miR-449c-5p expression in the aortic valves of mice at day 5. (e) qRT-PCR analysis of Smad4 expression level in the aortic valves of mice at day 10. n = 7 in each group. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01.