Abstract | BACKGROUND AND AIMS: METHODS: Western blot, dot blot, and liquid chromatography with tandem mass spectrometry were used to determine the expression of m6A methyltransferase, METTL3, and METTL14, as well as global m6A modification. RNA-sequencing and m6A-seq were employed to screen differentially expressed genes and responsive m6A peaks. Nuclear factor κB (NF-κB)-mediated METTL3/METTL14 transactivation were validated with chromatin immunoprecipitation polymerase chain reaction and dual- luciferase reporter system, and the role of m6A in TGF-β1 upregulation was further verified in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice. RESULTS: Serum lipopolysaccharide (LPS) concentration is increased in high-fat diet-induced NASH rats. TGF-β1 upregulation is closely associated with METTL3/METTL14 upregulation and global m6A hypermethylation, in both NASH rat liver and LPS-activated KCs. LPS-responsive m6A peaks are identified on the 5' untranslated region (UTR) of TGF-β1 messenger RNA ( mRNA). NF-κB directly transactivates METTL3 and METTL14 genes. LPS-stimulated TGF-β1 expression is abolished in METTL3/METTL14-deficient KCs and myeloid lineage cell-specific METTL14 knockout mice. Mutation of m6A sites on the 5'UTR of TGF-β1 mRNA blocks LPS-induced increase of luciferase reporter activity. CONCLUSIONS: NF-κB acts as transcription factor to transactivate METTL3/METTL14 genes upon LPS challenge, leading to global RNA m6A hypermethylation. Increased m6A on the 5'UTR of TGF-β1 mRNA results in m6A-dependent translation of TGF-β1 mRNA in a cap-independent manner. We identify a novel role of m6A modification in TGF-β1 upregulation, which helps to shed light on the molecular mechanism of NASH progression.
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Authors | Yue Feng, Haibo Dong, Bo Sun, Yun Hu, Yang Yang, Yimin Jia, Longfei Jia, Xiang Zhong, Ruqian Zhao |
Journal | Cellular and molecular gastroenterology and hepatology
(Cell Mol Gastroenterol Hepatol)
Vol. 12
Issue 3
Pg. 839-856
( 2021)
ISSN: 2352-345X [Electronic] United States |
PMID | 33992834
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
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Copyright | Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved. |
Chemical References |
- 5' Untranslated Regions
- RNA, Messenger
- Transcription Factor RelA
- Transforming Growth Factor beta1
- N-methyladenosine
- Methyltransferases
- Adenosine
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Topics |
- 5' Untranslated Regions
- Adenosine
(analogs & derivatives, metabolism)
- Animals
- Base Sequence
- Diet, High-Fat
- Disease Models, Animal
- Gene Expression Regulation
- Gene Knockdown Techniques
- Kupffer Cells
(metabolism)
- Liver Cirrhosis
(etiology, metabolism, pathology)
- Methylation
- Methyltransferases
(deficiency, metabolism)
- Models, Biological
- Protein Biosynthesis
- RNA, Messenger
(genetics, metabolism)
- Rats
- Transcription Factor RelA
(metabolism)
- Transcriptional Activation
- Transforming Growth Factor beta1
(genetics, metabolism)
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