MicroRNA-499-5p inhibits transforming growth factor-β1-induced Smad2 signaling pathway and suppresses fibroblast proliferation and collagen synthesis in rat by targeting TGFβ-R1

• DOI: 10.1007/s11033-023-08755-0
• Publication Date: 2023-09-07T08:02:27Z
• Authors: Qing Zhao; Wentao Yang; Xiangdong Li; Hongtao Yuan; Jianping Guo; Yutang Wang; Zhaoliang Shan
• Keywords: Cell biology; Immunology; Western blot; Smad2 Protein; Cancer research; Signal transduction; Transfection; Gene; Biochemistry; Collagen Type I; Rats, Sprague-Dawley; Transforming Growth Factor beta1; Cell growth; In vitro; Transforming Growth Factor beta; Health Sciences; Atrial Fibrillation; Animals; RNA, Messenger; Luciferases; Biology; Internal medicine; Cell Proliferation; Immunology and Microbiology; Cardiac fibrosis; microRNA; FOS: Clinical medicine; Life Sciences; Blot; Fibroblasts; Innate Lymphoid Cells in Immunity; Fibrosis; Atrial fibrillation; Downregulation and upregulation; Rats; MicroRNAs; Chemistry; Molecular Mechanisms of Cardiac Remodeling and Repair; Medicine; Fibroblast; Original Article; Cardiology and Cardiovascular Medicine; Transforming growth factor; Receptors, Transforming Growth Factor beta; Signal Transduction

Abstract Background Atrial fibrosis has been recognized as a typical pathological change in atrial fibrillation. 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 rat were respectively transfected with miR-499-5p mimic, miR-499-5p negative control and miR-499-5p inhibitor, atrial fibroblasts without any treatment were also established. Cell counting kit-8 assay and transwell assay were used to detect the proliferation and migration of atrial fibroblasts in each group. Expressions of miR-499-5p, TGF-β1, smad2, α-SMA, collagen-I and TGFβ-R1 in mRNA and protein level were subsequently detected via quantitative real-time polymerase chain reaction 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 mimic group was upregulated, while it was downregulated in the miR-499-5p inhibitor group. Upregulated miR-499-5p expression led 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 TGF-β1, TGFβ-R1, smad2, α-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 Smad2 signaling and eventually suppressed the proliferation, migration and invasion of atrial fibroblasts and collagen synthesis by targeting TGFβ-R1.