MiR‐21‐3p Inhibits Adipose Browning by Targeting FGFR1 and Aggravates Atrial Fibrosis in Diabetes

Figures

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  Figure 1. Atrial fibrosis and browning of pericardial adipose tissue were analyzed 3 months after the modeling. (a) H&E staining of atrial myocardial tissue samples, scale bar = 50 μm. (b) Masson's staining of atrial myocardial tissue samples, scale bar = 50 μm. (c) TGF-β1, CTGF, collagen I, and collagen III expressions in atrial myocardial tissue were analyzed by western blot and quantified by Image J. (d) Representative images and quantification of UCP1 expression were analyzed by immunohistochemistry in sections containing both pericardial adipose tissue and atrial myocardium from the hearts of mice. Hollow arrows indicate pericardial adipose tissue; solid arrows indicate atrial myocardium. Scale bar = 200 μm. (e) Representative images and quantification of UCP1 expression were analyzed by immunohistochemistry in collected pericardial adipose tissue, scale bar = 50 μm. (f) UCP1 expression in collected pericardial adipose tissue was analyzed by western blot and quantified by Image J. (g) FGFR1, FGF21, and PPARγ expression in collected pericardial adipose tissue were analyzed by western blot and quantified by Image J. The data are presented as the mean ± SD of six mice per group. ∗P <…

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  Figure 2. MiR-21-3p plays a major role in adipose browning. (a) Adipocytic differentiation of preadipocytes isolated from WT or miR-21 KO mice and oil red O staining of mature adipocytes after differentiation induction, scale bar = 50 μm. Preadipocytes from miR-21 KO mice were induced to differentiate into mature adipocytes and then transfected with mimics NC, miR-21a-3p mimics, or miR-21a-5p mimics. (b) The relative miR-21-3p and miR-21-5p expressions were detected by qRT-PCR in mature adipocytes. (c) UCP1 expression was analyzed by western blot and quantified by Scion Image software. (d) Representative images and quantification of UCP1 expression were detected by immunofluorescence in mature adipocytes, scale bar = 50 μm. UCP1 (red) is shown; nuclei are stained by DAPI (blue). The data are presented as the mean ± SD of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Figure 3. MiR-21-3p inhibits adipose browning to promote HG-induced myocardial fibrosis. Preadipocytes from miR-21 KO mice were induced to differentiate into mature adipocytes and then transfected with mimics NC, miR-21-3p mimics, or miR-21-5p mimics. Mature adipocyte coculture with atrial fibroblasts from WT mice treated with LG (5.5 mM) and HG (30 mM) for 72 h. (a) TGF-β1, CTGF, collagen I, and collagen III expressions in atrial fibroblasts were analyzed by western blot and quantified by Scion Image software. (b) Representative images and quantification of collagen I and collagen III expressions in atrial fibroblasts were analyzed by immunohistochemistry, scale bar = 100 μm. (c) ELISA was used to analyze IL-6, TNF-α, and MCP-1 concentrations in coculture supernatants. (d) Western blot was used to analyze UCP1 expression in mature adipocytes and quantified by Scion Image software. (e) Representative images and quantification of UCP1 expression in mature adipocytes were detected by immunofluorescence, scale bar = 50 μm. UCP1 (red) was shown; nuclei were stained by DAPI (blue). The data are presented as the mean ± SD of three independent experiments. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001.

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  Figure 4. MiR-21-3p regulated the FGFR1/FGF21/PPARγ pathway to participate in adipose browning by targeting FGFR1. (a) Putative miR-21-3p binding sites in the FGFR1 3′-UTR. (b) A luciferase reporter plasmid containing wild-type or mutant FGFR1 was cotransfected into 293 cells with miR-21-3p mimics or control mimics. Luciferase activity was determined at 48 h after transfection. Preadipocytes extracted from WT mice were induced to differentiate into mature adipocytes and then transfected with mimics NC, miR-21-3p mimics, inhibitor NC, or miR-21-3p inhibitor. (c, d) qRT-PCR and western blot were determined to analyze FGFR1 expression. Preadipocytes extracted from WT mice were induced to differentiate into mature adipocytes and then transfected with shRNA NC, FGFR1 shRNA, pcDNA-NC, or pcDNA-FGFR1. (e) The relative UCP1 expression was detected by qRT-PCR. (f) Western blot was used to analyze UCP1 expression and quantified by Scion Image software. (g) Representative images and quantification of UCP1 expression were detected by immunofluorescence, scale bar = 50 μm. UCP1 (red) was shown; nuclei were stained by DAPI (blue). Preadipocytes from miR-21 KO mice were induced to differentiate into…

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  Figure 5. MiR-21-3p regulates the adipose browning-associated pathway FGFR1/FGF21/PPARγ to promote HG-induced myocardial fibrosis by targeting FGFR1. Preadipocytes from miR-21 KO mice were induced to differentiate into mature adipocytes, and were then transfected with mimics NC+pcDNA-NC, miR-21-3p mimics, pcDNA-FGFR1, or miR-21-3p mimics+pcDNA-FGFR1. Mature adipocyte coculture with atrial fibroblasts from WT mice treated with LG (5.5 mM) and HG (30 mM) for 72 h. (a) TGF-β1, CTGF, collagen I, and collagen III expressions in atrial fibroblasts were analyzed by western blot and quantified by Scion Image software. (b) Representative images and quantification of collagen I and collagen III expressions in atrial fibroblasts were analyzed by immunohistochemistry, scale bar = 100 μm. (c) Western blot was used to analyze FGFR1, FGF21, and PPARγ expression in mature adipocytes, and the results were quantified by Scion Image software. (d) ELISA was used to analyze TNF-α, IL-6, and MCP-1 concentration in coculture supernatants. (e) Western blot was used to analyze UCP1 expression in mature adipocytes, and the results were quantified by Scion Image software. (f) Representative images and quantification…

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  Figure 6. Schematic representation of the mechanisms by which miR-21-3p regulates the adipose browning-associated pathway FGFR1/FGF21/PPARγ to promote HG-induced myocardial fibrosis by targeting FGFR1.