Upregulation of miR-664a-3p Ameliorates Calcific Aortic Valve Disease by Inhibiting the BMP2 Signaling Pathway

Figure 1. miR-664a-3p is downregulated in human aortic valve calcification disease samples. (a) HE staining of the aortic valve from CAVD and normal clinical samples. Scale bar, 400 μm. (b) Western blotting and quantification of BMP2, RUNX2, MST2, and OSTERIX protein expression in CAVD and normal clinical samples. GAPDH was used for normalization. (c) Volcano plots showing the differential expression of microRNAs in the aortic valve from CAVD clinical samples compared to control samples. Pink dots indicate significantly upregulated microRNAs in the CAVD group. Green dots indicate significantly downregulated microRNAs. Blue dots indicate microRNAs with insignificant differences. (d) Heat map shows the clustering analysis results of the top 20 microRNAs for differential expression in three pairs of samples. The red (higher expression) or green (lower expression) color represents the normalized expression value of the indicated microRNAs. (e) The expression levels of four candidate microRNAs showing the most significant downregulation in CAVD clinical samples were verified by RT-qPCR in five additional pairs of samples. Data are presented as mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 2. miR-664a-3p inhibits osteogenic differentiation and calcification of valvular interstitial cells. (a) RNA-FISH revealed the localization of miR-664a-3p in VICs. miR-664a-3p is stained in red and the nucleus in blue. Scale bar, 20 μm. (b) RT-qPCR was conducted to detect the miR-664a-3p expression in VICs after overexpression with a mimic or knockdown by an inhibitor. (c) Western blotting analysis of RUNX2, MST2, and OSTERIX expression in VICs after alterations in the level of miR-664a-3p. Quantitative analysis of the protein bands are shown on the right. (d) The formation of calcium nodules after alterations in the level of miR-664a-3p was detected by Alizarin Red staining. Quantitative analysis is shown on the right. Scale bar, 50 μm. (e) ALP activity in VICs after alterations in the level of miR-664a-3p was detected and quantitated by ALP staining assay. Scale bar, 200 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 3. BMP2 is a target gene of miR-664a-3p. (a) Schematic representation of the predicted binding of miR-664a-3p to BMP2. Schematic diagram of the structure of the luciferase reporter system. (b) The relative luciferase activity of VICs coinfected with constructed luciferase reporters (BMP2-WT or BMP2-MUT) and miR-664a-3p mimic or inhibitor or negative control. (c) Western blotting analysis of BMP2 expression in VICs after alterations in the level of miR-664a-3p. Quantitative analysis of the protein bands are shown on the right. (d) Pearson's correlation analysis between BMP2 and miR-664a-3p in human CAVs (five dots in the red box) and human non-CAVs (five dots in the blue box). The horizontal and vertical coordinates indicate the mean Ct values of miR-664a-3p and BMP2 in RT-qPCR, respectively. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 4. Validation of the effect of BMP2 on osteogenic differentiation in valvular interstitial cells. (a) Detection of BMP2 expression by western blotting after overexpression or knockdown of BMP2 in VICs. Quantitative analyses of the protein bands are shown on the right. (b) RUNX2, MST2, and OSTERIX expression were detected by western blotting analysis after overexpression or knockdown of BMP2 in VICs. Quantitative analyses of the protein bands are shown on the right. (c) Alizarin Red staining was conducted to detect the formation of calcium nodules after alterations in the level of BMP2. The quantitative analysis is shown on the right. Scale bar, 50 μm. (d) ALP activity was detected and quantitated by ALP staining assay after alterations in the level of BMP2. Scale bar, 200 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 5. miR-664a-3p regulates aortic valve calcification via Bmp2 in calcific aortic valve disease mice. (a, b) RT-qPCR was performed to measure the miR-664a-3p and Bmp2 expression after upregulation of miR-664a-3p and Bmp2 separately or simultaneously in the aortic valves of mice. (c) Bmp2, Runx2, Mst2, and Osterix expressions were detected by western blotting after the upregulation of miR-664a-3p and Bmp2 levels separately or simultaneously in the aortic valves of mice. Quantitative analyses of the protein bands are shown on the right. (d) HE staining of the aortic valve of mice in different groups. Scale bar, 400 μm. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

Figure 6. The biological process and mechanism of action of this paper.