Advisory Committee Chair
Suresh K Verma
Advisory Committee Members
Prasanna Krishnamurthy
Hind Lal
Document Type
Thesis
Date of Award
2020
Degree Name by School
Master of Biomedical Engineering (MBE) School of Engineering
Abstract
Background: Transforming growth factor β (TGFβ) is a chemokine released during cardiac hypertrophy and remodeling. Evidence has shown that it plays a significant role in fibroblast differentiation/activation to myofibroblasts , which ultimately leads to adverse cardiac remodeling. It is previously suggested that m6A methylation plays important role in cardiac pathology in both ischemic and hypertrophic heart failure [1, 2], however, its role in fibroblast activation/differentiation is largely unknown. Therefore, in this study we investigated the relationship between m6A methylation and fibroblast activation using proinflammatory/profibrotic factor, TGFβ. Methods: Fibroblasts were isolated from neonatal rat heart and activated with TGFβ for 48 hours. RNA and protein were harvested from fibroblasts after treatment to verify activation of fibroblasts via qPCR and Western blot analyses. METTL3 expression in fibroblasts was inhibited using METTL3 siRNA silencer system before treatment with TGFβ. RNA and protein were isolated from treated fibroblasts and the expression of profibrotic genes (Col1a, FN, aSMA and Periostein), and METTL3 expression (both gene and protein) were measured using RT-qPCR and western blot techniques. The m6A RNA methylation was measured using Epigentek kit. Results and Conclusion: TGFβ treatment significantly activate the expression of profibrotic genes (aSMA, Col1a, FN). We also found that TGFβ treatment significantly increased canonical TGFβ signaling as shown by Smad2/3 phosphorylation. m6A methylation was significantly increased in TGFβ treated cells compared to the control. Inhibition of METTL3 in cells showed an inverse relationship to the fibroblast activation, i.e. fibroblasts with downregulated METTL3 showed less m6A RNA methylation, and did not express as many fibrotic factors compared to control fibroblasts. Therefore, our data suggests that TGFβ induced fibroblasts activation is partially dependent on RNA methylation.
Recommended Citation
Suleiman, Zainab Gbongbo, "Epitranscriptomic Regulation Of Fibroblast Activation In Diseased Heart" (2020). All ETDs from UAB. 931.
https://digitalcommons.library.uab.edu/etd-collection/931