Targeting SYK with miR-512-3p provides therapeutic potential in myocardial fibrosis

Figures

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  Fig. 1. SYK expression is significantly upregulated in activated myocardial fibroblasts. (A) Immunofluorescence staining of heart tissue sections showing SYK (p-SYK, red) and α-SMA (green) expression in cardiac fibroblasts from mice treated with isoproterenol (ISO) or normal saline (NS). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (B–D) Western blot analysis of total SYK and phosphorylated SYK (p-SYK) levels in HL-1 mouse myocardial fibroblasts following Ang II stimulation at the indicated time points. GAPDH served as a loading control. Quantification of p-SYK (C) and total SYK (D) levels relative to those observed at 0–12 h (n = 3). Data are presented as the means ± SEMs. (E) Representative phalloidin immunofluorescence images of HL-1 cells treated with PBS or Ang II (12 h) showing cytoskeletal remodelling associated with myofibroblast activation. F-actin (red), nuclei (blue). Scale bar: 50 μm.

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  Fig. 2. Silencing SYK significantly inhibited Ang II-induced activation of myocardial fibroblasts. (A) Quantitative RT‒PCR analysis of SYK mRNA expression following transfection with three different SYK-targeting siRNAs (SYK-siRNAs 1, 2, and 3) or a control siRNA (Ctrl-siRNA) (n = 3). (B) Representative phalloidin immunofluorescence staining of HL-1 cells transfected with Ctrl-siRNA or SYK-siRNA and treated with PBS or Ang II for 12 h, showing cytoskeletal remodelling indicative of myofibroblast activation. F-actin (red), nuclei (blue). Scale bar: 50 μm. (C) Western blot analysis of COL1A1 protein expression in HL-1 cells under the indicated conditions. GAPDH served as a loading control. (D) Quantification of COL1A1 expression levels normalized to those of GAPDH. The data are presented as the means ± SEMs (n = 3).

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  Fig. 3. MiR-512-3p effectively inhibits the expression of SYK in myocardial fibroblasts. (A) SYK target prediction combined with expression abundance analysis to identify candidate miRNAs. (B) Changes in the expression of candidate miRNAs in the ISO model (n = 3). (C) Western blot analysis to evaluate SYK expression levels after transfection with candidate miRNAs. (D) Statistical analysis of SYK expression levels (n = 3). (E) Sequence analysis to identify the binding sites of miR-512-3p in the 3’-UTR of SYK. The data are presented as the means ± SEMs.

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  Fig. 4. MiR-512-3p inhibits HL-1 activation by targeting SYK. (A) Alamar blue assay evaluating the inhibitory effect of miR-512-3p on the proliferation of HL-1 cells (n = 3). (B) Immunofluorescence staining was used to assess the activation of myocardial fibroblasts. (C,D) Western blot analysis demonstrated the ability of 3’-UTR MUT SYK to reverse the miR-512-3p-mediated inhibition of SYK expression (n = 3). (E) Immunofluorescence analysis revealed that the antifibrotic effect of miR-512-3p was reversed by 3’-UTR MUT SYK. The data are presented as the means ± SEMs (n = 3).

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  Fig. 5. MiR-512-3p inhibits ISO-induced heart dysfunction and myocardial fibrosis. (A) Representative images of left ventricular (LV) M-mode echocardiograms. (B) The LV ejection fraction (EF), (C) left ventricular fractional shortening (FS), (D) posterior wall diameter (LVPWd) and (E) anterior wall diameter (LVAWd) were calculated via echocardiographic analysis. (F) Representative images of haematoxylin‒eosin (H&E)-stained heart tissue. Representative images of (G) Sirius Red-stained heart tissue and (H) quantitative analysis of the relative fibrosis area. The data are presented as the means ± SEMs (N = 3–7 mice per group as individual data indicated in each bar).