MicroRNA-499-5p inhibits Transforming Growth Factor β1-Induced Smad Signaling pathway and suppresses atrial fibroblasts in rat by targeting TGFβ-R1

Abstract

Abstract Background Artial fibrosis has been recognized as a typical pathological change in atrial fibrillation (AF). Although present evidence suggests that microRNA-499-5p (miR-499-5p) plays an important role in the development of atrial fibrosis, the specific mechanism is not fully understood. Therefore, this study attempted to assess the influence of miR-499-5p on atrial fibroblasts and explore the potential molecular mechanism. Methods Atrial fibroblasts from Sprague Dawley (SD) rat were respectively transfected with miR-499-5p mimics, miR-499-5p negative control and miR-499-5p inhibitors, atrial fibroblasts without any treatment were also established. Cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the proliferation and migration of atrial fibroblasts in each group. Expressions of miR-499-5p, TGFβ1, smad2, a-SMA, collagen-I and TGFβ-R1 in mRNA and protein level were subsequently detected via Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. Furthermore, the prediction of the binding sites of miR-499-5p and TGFβ-R1 was performed via the bioinformatics online software TargetScan and verified by Dual Luciferase Reporter. Results By utilizing miR-499-5p-transfected atrial fibroblasts model, expression of miR-499-5p in the miR-499-5p mimics group was upregulated, while it was downregulated in the miR-499-5p inhibitors group. Upregulated miR-499-5p expression led to to a significant decrease in the proliferative and migratory ability of cultured atrial fibroblasts, while downregulated miR-499-5p expression led to a significant increase in the proliferative and migratory ability of cultured atrial fibroblasts. Additionally, upregulated miR-499-5p expression made a significant rise in TGF-β1-induced mRNA and protein expression of TGFβ1, TGFβ-R1, smad2, a-SMA and collagen-I in atrial fibroblasts. Furthermore, results from the Dual Luciferase Reporter conformed that miR-499-5p may repress TGFβ-R1 by binding the 3'UTR of TGFβ-R1 directly. Conclusions MiR-499-5p is able to inhibit the activation of Transforming Growth Factor β-Induced Smad Signaling and eventually suppressed the proliferation, migration and invasion of atrial fibroblasts by targeting TGFβ-R1.