#2222 Suppression of miR-199a-3p ameliorates TGF-β-induced EMT in renal tubular cells

Abstract

Abstract Background and Aims Various kidney conditions, such as acute kidney injury and fibrosis, cause dysregulation of miRNAs. TGF-β is one of the most potent inducer of epithelial-to-mesenchymal transition (EMT) and fibrosis. We recently reported increased miR-199 expression in TGF-β-induced kidney fibrosis mouse model (doi: 10.3390/ijms242115520). However, the exact role of miR-199 in fibrosis and EMT development is still unknown. Method Primary tubular cells were isolated from a 4-week-old C57BL6 mouse and kept in DMEM/F12 medium containing 2% serum, supplemented with insulin/transferrin/selenium. Cells were treated for 24 hours with 30 nM miR-199-3p miRNA inhibitor (anti-miR), with or without TGF-β (10 ng/ml). The expression of profibrotic genes, as well as miR-199a-3p, was assessed by qPCR. Western blot was used to evaluate the protein expression. Results are presented as mean fold expression relative to calibrator. Statistical significance was assessed using the Kruskal-Wallis test, and the level of significance was set to p < 0.05. Results The miR-199a-3p expression in the anti-miR transfected control and TGF-β groups decreased by 93.5% and 85.5%, respectively, as compared to non-transfected groups (p < 0.05). Morphologically, cells transfected with miR-199a-3p and TGF-β1 showed no difference from cells exposed to only TGF-β1 but appeared more elongated compared to the control groups. However, the suppression of miR-199a-3p alleviated TGF-β1-induced fibrotic cell responses as shown by downregulated fibronectin mRNA (Fn1) and protein expression by 30.2% (p < 0.05) and by 4.3%, respectively. This was accompanied by reduced Acta2 (α-SMA) and Tgfb1 mRNA expressions by 30.2% and 17.5%, respectively (p < 0.05). Conclusion Our findings suggest that in murine renal tubular epithelial cells, miR-199a-3p suppresses TGF-β1-induced EMT by inhibiting FN1, Acta2, and Tgfb1 both at mRNA and protein levels. Further studies are needed to elucidate the underlying molecular interactions.