Role of microRNA-1 on fibrosis induced by high glucose cultured cardiac fibroblasts

Abstract

Objective To investigate the effect of microRNA-1 (miR-1) on the fibrosis of cardiac fibroblasts induced by high glucose. Methods The primary cultured fibroblasts from 1-3 days old Sprague-Dawley (SD) rats, were randomly divided into 4 groups (n = 3): normal glucose + lentivector-vehicle (CON+Lv-Vehicle group), normal glucose + lentivector-miR-1 (CON+Lv-miR1 group), high glucose + lentivector-vehicle (HG+Lv-Vehicle group), high glucose + lentivector-miR-1 (HG+Lv-miR1 group). Fibroblasts were cultured in glucose concentration 5.5 mmol/L and 25 mmol/L DMEM culture, and were injected lentiviral vector carrying miR-1 silencer sequence or the same volume of lentiviral vector. After 12 hours, the medium was replaced with fresh complete medium. After 3 days when transfection efficiency was up to 90%, the cellular miR-1 content was detected by real-time reverse transcription-polymerase chain reaction (qRT-PCR). The secretion of collagen Ⅰ and Ⅲ were measured by enzyme linked immunosorbent assay (ELISA). Expression of collagen Ⅰ and Ⅲ, matrix metalloproteinase 2 and 9 (MMP-2, MMP-9), autophagy related protein LC3B-Ⅱ, P62/SQSTM1 and Cathepsin D were assessed by Western Blot. Results Compared with the CON+Lv-Vehicle group, the content of miRNA in the CON+Lv-miR1 group had no statistical significance. Compared with the CON+Lv-Vehicle group, high glucose increased the amount of miR-1 (2-ΔΔCt: 1.82±0.17 vs. 1.00±0.04), collagen Ⅰ and Ⅲ secretion (mg/L: 14.55±0.33 vs. 7.28±0.22, 157.50±13.22 vs. 61.25±8.54) and expression (gray value: 432.35±56.00 vs. 100.00±15.00, 320.35±47.00 vs. 100.00±15.00), the level of MMP-2, MMP-9 and the expression of autophagy related protein LC3B-Ⅱ and P62/SQSTM1 (gray value: 249.0±21.0 vs. 100.0±15.0, 142.3±20.0 vs. 100.0±16.0, 178±19 vs. 100±14, 378.3±20.0 vs. 100.0±15.0), decreased the expression of lysosomal associated protein Cathepsin D (gray value: 60±14 vs. 100±10), and the differences were statistically significant (all P < 0.01). Compared with the HG+Lv-Vehicle group, the amount of miR-1 in the HG+Lv-miR1 group was significantly decreased (2-ΔΔCt: 1.21±0.10 vs. 1.82±0.17), collagen Ⅰ and Ⅲ secretion (mg/L: 10.68±0.54 vs. 14.55±0.33, 87.25±13.55 vs. 157.50±13.22) and expression (gray value: 179.41±45.00 vs. 432.35±56.00, 173.41±50.00 vs. 320.35±47.00), the level of MMP-2, MMP-9 and the expression of autophagy related protein LC3B-Ⅱ and P62/SQSTM1 (gray value: 172.0±23.0 vs. 249.0±21.0, 90.0±17.0 vs. 142.3±20.0, 138±15 vs. 178±19, 265.0±17.0 vs. 378.3±20.0) in the HG+Lv-miR1 group were decreased and the expression of lysosomal associated protein Cathepsin D was higher (gray value: 110±17 vs. 60±14), and the differences were statistically significant (all P < 0.05). Conclusions The expression of miRNA-1 was up-regulated in cardiac fibroblasts cultured in high glucose, and miRNA-1 silencing inhibited cardiac fibroblast induced fibrosis in high glucose. The mechanism may be related to the recovery of autophagy flux, up-regulation of Cathepsin D expression and inhibition of collagen production. Key words: High glucose damage; Cardiac fibroblast; Fibrosis; MicroRNA; Autophagy