MicroRNA‑331 inhibits isoproterenol‑induced expression of profibrotic genes in cardiac myofibroblasts via the TGFβ/smad3 signaling pathway

Figure 1. Direct interaction between miR-331 with TGFβ-R1 3′UTR sequence. (A) Schematic representation showing a miR‑331 recognition site (MRE) within both mouse and human TGFβ-R1 3′UTRs. (B) Shows dual luciferase assay result that supports direct interaction between miR-331 and TGFβ-R1 3′UTR sequence. Co-transfection of HEK293-T cells with miR-331 overexpressing cassette along with the luciferase: TGFβ-R1 3′UTR sequence construct significantly reduced luciferase activity. As a control, when non-target 3′UTR (AXIN1) was used in the assay, made no significant change in the luciferase activity compared with mock transfected cells. (C) Shows RT-qPCR results for detection of some genes expression involved in the TGFβ signaling pathway in both MCF and HCF cells under miR-331 overexpression. Downregulation of TGFβ-R1, TGFβ1 and SMAD3 expression was evident in this assay. Assays were performed in triplicate. Means ± SEM was shown. The asterisk mark (*) represents a p value < 0.05.

Figure 2. Induction of cardiac fibrosis in mouse model and in vitro condition using Isoproterenol. (A) Representative Masson’s Trichrome stain of mouse left heart sections demonstrated fibrotic tissues after ISO injection, compared with the saline group (×400) (n = 3). (B) The volume fraction of collagen was markedly increased in ISO treated group of mice, compared with the saline group. Collagen volume was measured using ImageJ software. (C,D) Shows the upregulation of fibrosis-related genes including, COL1A1, COL3A1, FN1, ELN, TGF-β1, SMAD2, SMAD3, MMP9, IL-10, IL-6, and TNFα at the mRNA levels in the mouse heart at days 3, 7, 14 and 28 following the ISO treatment (each group n = 5). (E,F) Profibrotic and proinflammatory genes expression were upregulated in both ISO-treated neonatal mouse and human cardiac myofibroblasts (CMyoFbs) after 24 and 48 h. Assays were performed in triplicate. Means ± SEM are shown. The asterisk mark (*) represents a p value < 0.05.

Figure 3. The miR‑331 expression alteration in the cardiac cells of mouse model and cultured cardiac myofibroblasts following ISO‑treatment. (A) RT‑qPCR results indicated that miR‑331 level has been reduced in the cardiac fibrosis tissues compared with the saline-treated tissues at days 3, 7, 14 and 28 (each group n = 5). (B) The miR‑331-5p relative expression has been downregulated in both ISO‑treated neonatal mouse and human cardiac myofibroblasts compared with the control group after 24 and 48 h. Assays were performed in triplicate. Means ± SEM was shown. The asterisk mark (*) represents a p value < 0.05.

Figure 4. Effect of miR-331 overexpression on some fibrosis-related genes expression in cardiac myofibroblasts following ISO treatment. (A,B) Shows a successful overexpression of miR-331 both in mouse cardiac myofibroblasts and human cardiac myofibroblasts. (C,D) Shows the downregulation of fibrosis-related genes including, COL1A1, COL3A1, FN1, ELN, TGF-β1, SMAD2, SMAD3, MMP9, IL-10, IL-6, and TNFα at the mRNA levels in ISO-treated cardiac myofibroblasts under miR-331 overexpression. Assays were performed in triplicate. (E,F) Overexpression of miR-331 significantly downregulated the level of both Smad3 and Col1A1 proteins, detected by western blot analysis. Protein band quantification was performed using Image-J software in order to ensure the result’s significance. Means ± SEM was shown. The asterisk mark (*) represents a p value < 0.05.

Figure 5. Effects of miR-331overexpression on cell cycle status and cell migration of mouse cardiac myofibroblasts. (A) Shows Flow cytometry cell cycle analysis of mouse cardiac myofibroblasts, following the overexpression of miR-331 compared with mock transfected cells. Significantly, sub-G1 cell population has been increased and G1 population fraction has been decreased, following the overexpression of this miRNA. (B) RT-qPCR results confirmed that miR-331 overexpression has resulted in significant downregulation of CCND1 and CCND2 genes at the mRNA level compared with mock transfected cells. (C) A wound-healing assay in mouse cardiac myofibroblasts transfected with either miR-331 overexpressing cassette or mock vector at 0, 24, and 48 h post-scratching (×40 magnification). (D) Quantitative analysis of scratch wound closure revealed miR-331 transfected cells showed no significant cell migration 48 h after of transfection. However, mock transfected cells showed significant cell migration since 24 h and started to progressively increase till 48 h after transfection. Assays were performed in triplicate. Means ± SEM was shown. The asterisk mark (*) represents a p value < 0.05.

Figure 6. Schematic representation of TGFβ pathway regulation by miR-331. In this model, miR-331 by targeting some genes (TGFβ-R1, TGFβ-1, SMAD2, and SMAD3) results in reduced TGFβ signaling pathway activity which in turn, effects the fibrotic genes expression of cardiac myofibroblasts and other related biological processes, including cell proliferation and migration.