Elevated IgE promotes cardiac fibrosis by suppressing miR-486a-5p

Figures

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  Figure 1. Effects of IgE-FcεR1 signaling on CFs. (A) FcεR1a expression levels in CFs of WT and FcεR1-KO mice measured by immunoblot. (B-D) FcεR1a expression levels after IgE treatment (5 μg/mL) for 0, 3, and 24 h measured by qPCR (B) and immunoblot (C-D). (E-F) mRNA expression of key fibrotic genes (a-SMA, Col1a1 and Col3a1) in IgE-stimulated FcεR1-WT (E) and FcεR1-KO (F) CFs at 0, 3, and 24 h. (G) Representative western blot of α-SMA, COL1A1, and COL3A1 in FcεR1-WT CFs after IgE treatment for 0, 3, and 24 h. (H) Statistical analysis of α-SMA, COL1A1, and COL3A1 protein expression in FcεR1-WT CFs, normalized to GAPDH (fold change versus 0 h). (I) Representative western blot of α-SMA, COL1A1, and COL3A1 in FcεR1-KO CFs after IgE treatment. (J) Statistical analysis of the relative protein expression of α-SMA, COL1A1, and COL3A1 in FcεR1-KO CFs. Data are mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates no significant difference in One-way ANOVA with Bonferroni's post hoc test.

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  Figure 2. Effects of CF FcεR1 deletion on Ang II-induced cardiac fibrosis. (A) Representative images of Masson staining of the heart tissues from Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. Images were taken at 200X magnification. Scale bars, 50 μm. (B) Quantification of myocardial interstitial fibrosis by Masson staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (C) Representative images of Sirius Red staining of the heart tissues from Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. Images were taken at 400X magnification. Scale bars, 50 μm. (D) Quantification of myocardial interstitial fibrosis by Sirius Red staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (E) qPCR analysis of periostin (Postn) and a-SMA expression in the heart tissues of the four indicated groups. (F) Representative immunoblot analysis showing the expression of COL1A1 and COL3A1 in the hearts of Ang II- or saline-infused FcεR1-Flox and FcεR1-cKO mice. (G) Statistical analysis of COL1A1 and COL3A1 expression normalized to GAPDH. Total n…

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  Figure 3. IgE alters miRNAs profile in CFs and down-regulates miR-486a-5p. (A) Heatmap illustrates differentially expressed miRNAs. The expression is normalized as row z-scores, up-regulation is indicated in red and down-regulation is indicated in blue. Each row represents a miRNA. (B) Three-way Venn diagram indicating the total number of miRNAs predicted to directly target Col1a1 and Col3a1. The numbers of shared miRNAs are indicated in the intersections of the Venn diagram. Two miRNAs in the intersections are shown as black stars in (A). (C) Two-way Venn diagram indicating the miRNAs that may be involved in fibrosis. Seven miRNAs in the intersection are shown as red stars in (A). (D) qPCR analysis of eight candidate miRNAs from the intersections shown in (B-C). The expression levels of miRNAs in FcεR1-WT (left panel) and FcεR1-KO (right panel) CFs after IgE stimulation were normalized to U6. Three independent experiments were performed. (E) Expression of miR-486a-5p in IgE-stimulated FcεR1-WT and FcεR1-KO CFs at 0, 3, and 24 h measured by qPCR (fold change versus 0 h) in three independent experiments. Results are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, n.s indicates no…

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  Figure 4. MiR-486a-5p directly regulates Smad1 in CFs. (A) Potential miR-486a-5p targets from Targetscan7.1 and miRanda databases. (B) Diagram showing the predicted miR-486a-5p binding site on Smad1 3' UTRs, the sequence marked in red shows a mutation of the miR-486a-5p matched sequence. (C) Activity of the Smad1 3' UTR luciferase reporter was measured 24 h after transfection of miR-485a-5p mimic or scrambled controls in 293T cells. Renilla luciferase was used as the internal control. Three independent experiments were performed. (D) The expression of Smad1 mRNA was reduced by transfection the miR-485a-5p mimic into CFs. (E-F) Representative immunoblot (E) and quantification (F) of SMAD1 and phospho-SMAD1 expression after miR-486a-5p overexpression in CFs. (G) The expression of Smad1 mRNA was increased by transfection of the of miR-485a-5p inhibitor into CFs. (H-I) Representative immunoblot (H) and quantification (I) of SMAD1 and phospho-SMAD1 expression after miR-486a-5p knockdown in CFs. GAPDH was used for normalization. Quantification results are shown as mean ± SD. *p < 0.05, **p < 0.01, ****p < 0.0001, n.s indicates no significant difference using Student's t-test.

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  Figure 5. IgE promotes collagen expression via miR-486a-5p/Smad1 in CFs. (A-B) Representative immunoblot (upper panel) and quantification (lower panel) of SMAD1 protein expression in IgE-stimulated WT (A) and FcεR1-KO (B) CFs at indicated lengths of time (0, 3, 24 h). (C-D) Representative immunoblot (C) and quantification (D) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with small interference RNA (siRNA) against Smad1 or scrambled control. (E-F) Representative immunoblot (E) and quantification (F) showing protein levels of α-SMA, COL1A1, and COL3A1 in CFs transfected with Smad1 ORF clone or scrambled control. (G-H) Representative immunoblot (G) and quantification (H) of SMAD1, COL1A1, and COL3A1 protein expression in CFs transfected with miR-486a-5p mimic and/or IgE. (I-J) Immunoblot (I) and quantification (J) showing protein levels of SMAD1, COL1A1, and COL3A1 in CFs transfected with siRNA against Smad1 and/or miR-486a-5p inhibitor. Data are mean ± SD from 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s indicates no significant difference in One-way ANOVA with Bonferroni's post hoc test (A-B), Student's t-test (D, F) or…

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  Figure 6. Effects of miR-486a-5p overexpression on Ang II-induced cardiac fibrosis. (A) Representative images of Masson staining of the heart tissues from lenti-miR486 or scramble-treated Ang II- or saline-infused mice. Images were taken at 200X magnification. Scale bars, 50 μm. (B) Quantification of myocardial interstitial fibrosis by Masson staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (C) Representative images of Sirius Red staining of the heart tissues from lenti-miR486 or scramble-treated Ang II- or saline-infused mice. Images were taken at 400X magnification. Scale bars, 50 μm. (D) Quantification of myocardial interstitial fibrosis by Sirius Red staining. A total of nine fields from three sections (three fields from each section) per mouse were randomly selected for analysis. (E) qPCR analysis of Postn and a-SMA expression in the hearts of lenti-miR486 or scramble-treated Ang II- or saline-infused mice. (F) qPCR analysis of miR-486a-5p expression in the heart tissues of the 4 indicated groups. (G-H) Representative immunoblot (G) and quantification (H) showing the expression of SMAD1 and SMAD2 in…

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  Figure 7. Expression of miR-486-5p in the serum samples of HF patients. (A) qPCR analysis of miR-486-5p expression in the serum samples of HF patients (n = 57) and age- and sex-matched healthy subjects (n = 87) (fold change versus healthy subjects). The results are shown as mean ± SEM. Statistical analysis were performed using the Mann-Whitney U test, ****p < 0.0001. (B) Pearson correlation analysis of the relationship between serum IgE and serum NT-proBNP in HF patients (n = 57).