MicroRNA‐26a Protects the Heart Against Hypertension‐Induced Myocardial Fibrosis

Figures

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  Figure 1. Expression patterns of miR‐26a in spontaneously hypertensive rats (SHRs) and miR‐26a+/− mice. A–C, 8‐ and 16‐week‐old male Wistar‐Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs). A, Systolic blood pressure in WKYs and SHRs. B and C, microRNA‐26a (miR‐26a) relative expression in plasma (B) and myocardium (C) in WKYs and SHRs. Data are mean±SD (n=6 per group); **P<0.01. D through F, 18‐ to 20‐week‐old male miR‐26a+/− mice and wild‐type (WT) miR‐26a+/+ mouse littermates. D, Representative images of hematoxylin and eosin (HE) and Masson’s staining in myocardial tissues from miR‐26a+/− and WT mice. Scale bars, 50 μm. E, Collagen volume fraction (CVF) of myocardium with Masson’s staining. F, Relative expression of miR‐26a in myocardial tissue of miR‐26a+/− and WT mice. Data are mean±SD (n=4 per group); *P<0.05; **P<0.01. Statistical significance was determined using Student t test for 2‐group comparison, 1‐way ANOVA followed by Tukey multiple comparisons test for multiple group comparisons and Mann–Whitney U test for Masson’s staining analyses.

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  Figure 2. microRNA‐26a (miR‐26a) regulates blood pressure in spontaneously hypertensive rats (SHRs). SHRs (n=8 per group) were injected by tail vein with recombinant adeno‐associated virus (rAAV‐) miR‐26a, rAAV‐GFP, or saline, and WKYs (n=6) were injected by tail vein with saline as normal controls. A, Viral fluorescence in myocardium after administration with various virus‐expressing vectors. Scale bars, 50 μm. B, Relative mRNA levels of miR‐26a in myocardium in rats. C, Systolic blood pressure of each group before and after the intervention. Data are mean±SD. *P<0.05 vs WKY, # P<0.05 vs rAAV‐GFP and SHR‐Ctrl. Statistical significance was determined using one‐way ANOVA followed by Tukey multiple comparisons test.

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  Figure 3. microRNA‐26a (miR‐26a) has a protective effect on hypertensive myocardial fibrosis in spontaneously hypertensive rats (SHRs). Myocardial morphological analysis and relative expression of fibrotic indicators in SHRs and Wistar‐Kyoto rats (WKYs) with different treatment groups. A, Representative images of morphological staining in myocardial tissues from WKYs and SHRs, Scale bars, 50 μm. B, Quantification of myocardial fibrosis. C, Relative protein levels of MMP2, CTGF, TGFβRI, Smad3, Smad4, and p‐Smad3 in myocardium from WKYs and SHRs with different treatments. Data represent the end of the experiment, Data are mean±SD (n≥3). *P<0.05 vs WKY, # P<0.05 vs rAAV‐GFP and SHR‐Ctrl. Statistical significance was determined using 1‐way ANOVA with Tukey test and Masson’s staining analysis was determined by Kruskal–Wallis test. CTGF indicates connective tissue growth factor; CVF, collagen volume fraction; MMP2, matrix metalloproteinase 2; p‐Smad3, phospho‐Smad3; and TGFβRI, transforming growth factor‐β receptor I.

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  Figure 4. microRNA‐26a (miR‐26a) inhibited AngII‐induced extracellular matrix release from cardiac fibroblasts (CFs). AngII‐induced CFs were transfected with miR‐26a mimic, inhibitor, or corresponding NC. The control group was a blank control without any treatment, and the AngII group received AngII only. A, Relative miR‐26a expression in different treatment groups in CFs. B and C, Relative mRNA expression of Col I, Col III, MMP2, and TGFβRI in CFs. D, Relative protein levels of TGFβRI, MMP2, Col I, and Col III in CFs from different treatment groups. Data are mean±SD (n=3). *P<0.05 vs control, # P<0.05 vs AngII, mimic NC+AngII and inhibitor NC+AngII, Δ P<0.05 vs inhibitor+AngII. Statistical significance was determined using 1‐way ANOVA with Tukey test. AngII indicates angiotensin II; Col I, Col III, collagen I, collagen III; MMP2, matrix metalloproteinase 2; NC, negative control; and TGFβRI, transforming growth factor‐β receptor I.

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  Figure 5. microRNA‐26a (miR‐26a) protects myocardium from cell proliferation in spontaneously hypertensive rat (SHRs). Myocardial stromal fibroblasts proliferation index and related protein expression in SHRs and Wistar‐Kyoto rats (WKYs) with different treatments. A, The expression of Ki‐67 in myocardial stromal fibroblasts detected by immunohistochemistry. Scale bars, 50 μm. B, Myocardial stromal fibroblast proliferation index defined as the proportion of Ki‐67+ nuclei per total nuclei sample (5 rats ×10 scope field count in each group). C, Relative protein levels of EZH2 and p21 in myocardium from WKYs and SHRs with different treatments. Data are mean±SD (n≥3). *P<0.05 vs WKY, # P<0.05 vs rAAV‐GFP and SHR‐Ctrl. Statistical significance was determined using 1‐way ANOVA with Tukey test. EZH2 indicates enhancer of zeste homolog 2.

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  Figure 6. microRNA‐26a (MiR‐26a) inhibited AngII‐induced proliferation of cardiac fibroblasts (CFs). A and B, Comparisons of cell cycles among groups in CFs detected by propidium iodide (PI) single staining. A, Cell cycle in each group. B, Proportion of cells in the G1, S, and G2 phases in each group. Data are mean±SD (n=3), *P<0.05. (C) CCK‐8 assays of the effect of miR‐26a on growth of CFs. Data are from 5 independent experiments, with at least 3 replicates in each independent experiment. *P<0.05 vs control, # P<0.05 vs AngII, mimic NC+AngII and inhibitor NC+AngII, Δ P<0.05 vs inhibitor+AngII. Statistical significance was determined using 1‐way ANOVA with Tukey test. AngII indicates angiotensin II, and NC, negative control.

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  Figure 7. EZH2, CTGF, and Smad4 are physiological targets of microRNA‐26a (miR‐26a). MiR‐26a inhibits EZH2, CTGF and Smad4 expression and targets the 3′ UTR of EZH2, CTGF and Smad4. A, E, and I, Relative protein levels of EZH2, CTGF and Smad4 after the introduction of miR‐26a into CFs. C, G, and K, Relative mRNA levels of EZH2, CTGF and Smad4 in CFs. Data are mean±SD (n=3). *P<0.05 vs control, # P<0.05 vs AngII, mimic NC+AngII and inhibitor NC+AngII, Δ P<0.05 vs inhibitor+AngII. Statistical significance was determined using 1‐way ANOVA with Tukey test. B, F, and J, Prediction of miR‐26a targets was conducted in silico and EZH2, CTGF, and Smad4 were selected as candidates. D, H, and L, Luciferase reporter gene detection results. Luciferase activities were analyzed by dual‐luciferase reporter assay. Data are mean±SD (n=3), *P<0.05 vs WT NC group and MUT miR‐26a mimic group. Statistical significance was determined using 2‐way ANOVA followed by Bonferroni posttest. AngII indicates angiotensin II; CTGF, connective tissue growth factor; EZH2, enhancer of zeste homolog 2; MUT, mutation type; NC, negative control; and WT, wild type.

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  Figure 8. Proposed model for the role of microRNA‐26a (miR‐26a) in hypertensive MF. Reduced miR‐26a level increases CTGF and MMP2 levels, leading to production of collagens and ECM. Downregulation of miR‐26a upregulates the expression of EZH2 and downregulates that of p21, leading to CF proliferation. Excessive deposition of ECM and CF proliferation are the main pathological basis of myocardial fibrosis. Meanwhile, downregulation of miR‐26a upregulates Smad4, which promotes fibrosis regulated by the TGFβ/Smad signal pathway. CTGF indicates connective tissue growth factor; ECM, extracellular matrix; EZH2, enhancer of zeste homolog 2; MF, myocardial fibrosis; MMP2, matrix metalloproteinase 2; p, phosphorylation; R, receptor; and TGF, transforming growth factor.