Inhibition of miR-101-3p prevents human aortic valve interstitial cell calcification through regulation of CDH11/SOX9 expression

Figures

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  Fig. 1. miR-101-3p expression was upregulated in the calcified human aortic valves. A, B Photomicrographs of freshly isolated normal and calcified human aortic valves. C, D Alizarin red staining of normal and calcified human aortic valves, red staining indicating calcification (indicated by arrows). E, F Masson’s Trichrome staining of collagen deposition (blue staining indicated by arrows) of normal and calcified human aortic valves. G Small RNA deep sequencing results of miR-101-3p of normal and calcified human aortic valves. Data = means ± SEM. ***P < 0.001 vs Normal, n = 3. H qPCR analysis of miR-101-3p of normal and calcified human aortic valves. Scale bar = 200 μm. *P < 0.05 vs Normal, n = 3

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  Fig. 2. miR-101-3p mimic promoted HAVICs calcification and inhibition of miR-101-3p protected HAVICs from calcification in CM. Alizarin red staining (A–F) of HAVICs cultured in osteogenic-conditioned media (10 mM βGP, 5 mM CaCl2) for 14 days after transfected with miR-101-3p mimic, inhibitor and negative control. G Quantification of alizarin red staining, absorbance normalized by protein concentration. *P < 0.05, ***P < 0.001 and ****P < 0.0001 vs Control; ++++P < 0.0001 vs Mimic; ^P < 0.05, ^^^^P < 0.0001 vs Control, CM; ####P < 0.0001 vs Inhibitor. H Western blot analysis of Fetuin A protein expression in HAVICs cultured in CM for 48 h after miR-101 mimic/inhibitor transfection. β-actin serves as internal loading control. I, J Quantification of Fetuin A protein expression normalized by β-actin. Data = means ± SEM. ****P < 0.0001 vs Control; ^P < 0.05 and ^^^^P < 0.0001 vs Mimic or Inhibitor groups; ++++P < 0.0001 vs Control, CM. n = 3 repeats

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  Fig. 3. miR-101-3p regulated CDH11 and Sox9 protein expression and osteogenesis markers BMP2/Runx2 in HAVICs. A Western Blots against antibodies of Cadherin-11 (CDH11) (ab147215), Sox9 (AB5535, Millipore), BMP2 (ThermoFisher, 710022), and Runx2 (Santa Cruz sc-12488) in HAVICs at 72 h after transfection with negative control and various doses of miR-101-3p mimic. B–E Quantification of CDH11, Sox9, BMP2, and Runx2 protein expression normalized with β-actin. F Western Blots against antibodies of Cadherin-11 (CDH11) and Sox9 in HAVICs at 72 h after transfection with negative control and various doses of mir-101-3p inhibitor. G, H Quantification of CDH 11 and Sox9 protein expression normalized with β-actin. Data = means ± SEM. *p < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 vs Control. n = 3 repeats

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  Fig. 4. CDH11 and SOX9 protein expression were decreased in calcified human aortic valves (HAVs). A Immunohistochemical staining against the antibody of Cadherin-11 (CDH11) (ab147215) on calcified human aortic valves and age-matched normal human aortic valves (Positive staining indicated by red arrows). Negative control staining without the secondary antibody. B Semi-quantification of area fraction of CDH11 staining on human aortic valves. ***P < 0.001 vs Normal. C, D Western blot analysis of CDH11 protein expression in human aortic valves normalized with β-actin. E, F Western blot analysis of SOX9 protein expression in human aortic valves normalized with β-actin. In A, scale bar = 500 μm for the upper panel and 200 μm for the lower panel. Data = means ± SEM. **P < 0.01, and ****P < 0.0001 vs Normal. n = 3

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  Fig. 5. Mimic of miR-101 downregulated CDH-11 and SOX9 expression and promoted osteogenesis in HAVICs treated by CM after transfection. A Western blot against antibodies of CDH11, SOX9, Runx2, and OPN in HAVICs transfected with miR-101-3p mimic for 48 h and then treated with CM for additional 48 h. β-actin serves as internal loading control. B–E Quantification of CDH11, SOX9, Runx2, and OPN normalized with β-actin. Data = means ± SEM. *p < 0.05, **P < 0.01 and ****P < 0.0001 vs Control; +++P < 0.001 vs Control, CM; ^P < 0.05 and ^^^P < 0.001 vs Mimic. n = 3 repeats

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  Fig. 6. Inhibition of miR-101 rescued downregulation of CDH-11 and SOX9 expression and prevented osteogenesis in CM-treated HAVICs. A Western blot against antibodies of CDH11, SOX9, Runx2, and OPN in HAVICs transfected with miR-101-3p inhibitor for 48 h and then treated with CM for additional 48 h. β-actin serves as internal loading control. B–E Quantification of CDH11, Sox9, Runx2 and OPN protein expression normalized with β-actin. Data = means ± SEM. **P < 0.01 and ***P < 0.001 vs Control; +P < 0.05, ++P < 0.001 and ++++P < 0.0001 vs Control, CM. n = 3 repeats

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  Fig. 7. miR-101 mediated ASPN expression in CM treated HAVICs through regulation of CDH11 and Sox9. A Western Blot against antibodies of ASPN in HAVICs transfected with miR-101-3p mimic or inhibitor for 48 h and then treated with CM for 48 h. β-Actin serves as internal loading control. B, C Quantifications of ASPN protein expression normalized with β-actin. *P < 0.05 and ****P < 0.0001 vs Control; +P < 0.05 and ++P < 0.01 vs Control, CM; ^^P < 0.01 vs Mimic. D Western Blot against CDH11, Sox9 and ASPN in HAVICs co-transfected with SiRNAs of CDH11 (10 nM) and Sox9 (1 nM) for 72 h. E–G Quantifications of WBs normalized by β-actin. Data = means ± SEM. ***P < 0.001 and ****P < 0.0001 vs Control. n = 3 repeats