MiRNA-1202 promotes the TGF-β1-induced proliferation, differentiation and collagen production of cardiac fibroblasts by targeting nNOS

Abstract

Background: Atrial fibrillation (AF) is a clinically common arrhythmia that affects human health. Myocardial fibrosis serves as an important contributor to AF. Recently, miRNA-1202 have been reported to be up-regulated in AF. However, the role of miRNA-1202 and its mechanism in myocardial fibrosis remain unclear.

Methods: Human cardiac fibroblasts (HCFs) were used to construct a fibrosis model by TGF-β1 induction. The expression of miR-1202 was measured by qRT-PCR. Cell proliferation was assessed by CCK-8 assays. Protein expression levels were measured by western blot. Collagen accumulation was measured by ELISA. The relationship between miR-1202 and nNOS was investigated by luciferase reporter assays.

Results: MiR-1202 expression was obviously increased in HCFs and was both time- and dose-independent. MiR-1202 could increase the proliferation and collagen I, collagen III, and α-SMA levels with or without TGF-β1. MiR-1202 could also increase TGF-β1 and p-Smad2/3 protein levels in comparison to the control group. However, they were obviously decreased after inhibitor transfection. MiR-1202 targets nNOS for negative regulation of HCFs fibrosis by decreasing cell differentiation, collagen deposition and the activity of the TGF-β1/Smad2/3 pathway. Co-transfection of miR-1202 inhibitor and siRNA of nNOS inhibited nNOS protein expression, thereby enhancing the HCFs proliferation. Furthermore, co-transfection of the miR-1202 inhibitor and siRNA of nNOS significantly promoted collagen I, collagen III, TGF-β1, Smad2/3 and α-SMA protein expression and Smad2/3 protein phosphorylation. These findings suggested that miR-1202 promotes HCFs transformation to a pro-fibrotic phenotype by targeting nNOS through activating the TGF-β1/Smad2/3 pathway.

Fig 1. The miR-1202 expression levels in HCFs.

(A) The miR-1202 expression level was dose-dependent. (B) The miR-1202 expression level was time-dependent. All of the data are shown as the mean ± SD. Compared with the control group, n = 3, *P < 0.05; **P < 0.01, ***P < 0.001.

Fig 2. Effects of miR-1202 on the proliferation of HCFs treated with or without TGF-β1.

(A) The miR-1202 levels in TGF-β1-induced HCFs cells; (B) The cell viability in TGF-β1-induced HCFs; (C) The cell viability in HCFs without TGF-β1 treatment. All of the data are expressed as the mean ± SD. Compared with the control group, n = 3, *P < 0.05; **P < 0.01, ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 3. Effects of miR-1202 on the differentiation and collagen synthesis of HCFs treated with or without TGF-β1.

The protein expression levels of α-SMA, collagen I and collagen III in HCFs treated with TGF-β1 (A) or in HCFs without TGF-β1 (B) were assessed by western blotting. All of the data are expressed as the mean ± SD. Compared with the control group, n = 3, *P < 0.05; **P < 0.01, ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 4. Effects of miR-1202 on collagen synthesis of HCFs treated with or without TGF-β1.

The expression levels of collagen I and collagen III in HCFs treated with TGF-β1 (A) or in HCFs without TGF-β1 (B) were assessed by ELISA assay. All of the data are expressed as the mean ± SD. Compared with the control group, n = 3, *P < 0.05; **P < 0.01, ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 5. Effects of miR-1202 on the TGF-β1/Smad2/3 pathway.

All of the data are shown as the mean ± SD. Compared with the control group. n = 3, *P < 0.05; **P < 0.01; ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 6. nNOS is a target of miR-1202.

(A) Left: Binding sites for miR-1202 in the 3’-UTR of nNOS mRNA; Right: Luciferase reporter assay showing reduced luciferase reporter activity in HCFs containing the nNOS-WT 3’-UTR fragment. (B) nNOS protein expression in HCFs infected with miR-1202 mimic or miR-1202 inhibitor was measured after 48 h. All of the data are expressed as the mean ± SD. Compared with the control group. n = 3, *P < 0.05; **P < 0.01; ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 7. Effect of nNOS on TGF-β1 induced proliferation, differentiation and collagen synthesis of HCFs.

(A) The nNOS levels in TGF-β1-induced HCFs; (B) The cell viability of TGF-β1-induced HCFs; (C) The protein expression levels of α-SMA, collagen I and collagen III in TGF-β1-treated HCFs were assessed by western blotting; (D) The expression levels of collagen I and collagen III in TGF-β1-treated HCFs were assessed by ELISA assay. All of the data are shown as the mean ± SD. Compared with the control group. n = 3, *P < 0.05; **P < 0.01; ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 8. Effects of nNOS on the TGF-β1/Smad2/3 pathway.

All of the data are shown as the mean ± SD. Compared with the control group. n = 3, *P < 0.05; **P < 0.01; ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 9. Effect of miR-1202 and nNOS co-transfection on TGF-β1 induced proliferation, differentiation and collagen synthesis of HCFs.

(A) nNOS protein expression in TGF-β1-induced HCFs; (B) The cell viability in TGF-β1-induced HCFs; (C) The protein expression levels of α-SMA, collagen I and collagen III in TGF-β1-treated HCFs were assessed by western blotting; (D) The expression levels of collagen I and collagen III in TGF-β1-treated HCFs were assessed by ELISA assay. All of the data are expressed as the mean ± SD. Compared with the control group. n = 3, *P < 0.05; **P < 0.01; ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.

Fig 10. Effect of miR-1202 and nNOS co-transfection on the TGF-β1/Smad2/3 pathway.

All of the data are expressed as the mean ± SD. Compared with the control group. n = 3, *P < 0.05; **P < 0.01; ***P < 0.001. The analysis is not able to detect small significant differences due to small sample size.