miRNA Expression Profiling Uncovers a Role of miR-139-5p in Regulating the Calcification of Human Aortic Valve Interstitial Cells

Figures

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  FIGURE 1. miRNA expression profile was altered during aortic valve calcification. (A) Schematic description of the workflow illustrating the two-stage-approach involving independent samples for discovery and validation. (B) Overall count distribution of miRNAs in each sample in the RNA-sequencing experiment. (C) Pearson correlation coefficient of overall miRNA expression levels between every two samples. (D) Volcano plot revealing miRNA-sequencing results comparing CON (n = 3 biologically independent samples) versus CAV (n = 5 biologically independent samples). Individual miRNAs are displayed by the p-value and the corresponding fold change. (E) Heatmap of the 338 differentially expressed miRNAs. Red color indicated higher expression level; blue color indicated lower expression level. CON, control; CAV, calcified aortic valves.

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  FIGURE 2. Pathway analysis of the predict target genes of differential expressed miRNAs. (A) The GO biological process (BP) function of the predict target genes of differential expressed miRNAs was analyzed and the top 10 terms were listed. (B) KEGG pathway of the predict target genes of differential expressed miRNAs was analyzed and the top 10 pathways were listed.

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  FIGURE 3. The verification of downregulated candidate miRNAs in calcified aortic valves. (A) The expression levels of osteogenic genes ALPP, RUNX2 and BMP3 in valves from CON patients (n = 5 biologically independent samples) and CAV patients (n = 4 biologically independent samples) were accessed by qRT-PCR and normalized with GAPDH. (B,C) The expression levels of hsa-miR-181a-2-3p (miR-181a), hsa-miR-490-5p (miR-490), hsa-miR-204-5p (miR-204), hsa-miR-1180-3p (miR-1180), hsa-miR-149-5p (miR-149), hsa-miR-139-5p (miR-139) (B) and hsa-miR-374c-3p (C) in valves from CON patients (n = 5 biologically independent samples) and CAV patients (n = 4 biologically independent samples) were accessed by qRT-PCR and normalized with U6. (D) Spearman’s correlation test was used to analyze the correlation of the expression of hsa-miR-204-5p, hsa-miR-149-5p, hsa-miR-139-5p, hsa-miR-181a-2-3p and hsa-miR-490-5p with the expression levels of RUNX2 in aortic valves. Unpaired Student’s t-test, *p < 0.05, **p < 0.01. CON, control; CAV, calcified aortic valves.

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  FIGURE 4. Both miR-149-5p and miR-139-5p were downregulated in osteogenic differentiated VICs significantly. (A) Primary cultured VICs and VECs were immunofluorescence stained with Periostin (green) and CD31 (red), respectively and the nucleus was stained with DAPI (blue) (the scale bar = 50 μm). (B) The expression levels of hsa-miR-149-5p (miR-149) and hsa-miR-139-5p (miR-139) in VECs and VICs were accessed by qRT-PCR and normalized with U6 (n = 4 samples from independent wells in each group). (C) Alizarin red staining was used to access the matrix calcium deposition in VICs at 15 days after OM induction. (D) The expression levels of hsa-miR-149-5p and hsa-miR-139-5p in control and osteogenic differentiated VICs were accessed by qRT-PCR and normalized with U6 (n = 4 samples from independent wells in each group). Unpaired Student’s t-test, *p < 0.05, **p < 0.01.

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  FIGURE 5. miR-139-5p overexpression could inhibit the osteogenic differentiation of VICs. (A) The expression levels of miR-139-5p in negative control (NC) mimics and miR-139-5p mimics (miR-139 mimic) transfected VICs were accessed by qRT-PCR and normalized with U6 (n = 4 samples from independent wells in each group). Unpaired Student’s t-test, **p < 0.01. (B) Alizarin red staining was used to access the matrix calcium deposition in NC mimics and miR-139-5p mimics transfected VICs at 15 days after OM induction and the right graph was the percentage of Alizarin red positive area per field (n = 3 wells in each group and three to four fields per well were counted, the scale bar = 200 μm). Unpaired Student’s t-test, **p < 0.01. (C–E) The expression levels of BGLAP (C), ALPP (D) and RUNX2 (E) in NC mimics and miR-139-5p mimics transfected VICs were accessed by qRT-PCR and normalized with GAPDH (n = 4 samples from independent wells in each group). (F,G) The expression levels of FZD4 (F) and CTNNB1 (G) in NC mimics and miR-139-5p mimics transfected VICs were accessed by qRT-PCR and normalized with GAPDH (n = 4 samples from independent wells in each group). One-way ANOVA, *p < 0.05, **p < 0.01.