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While the benefits of exercise are thought to involve epigenetic mechanisms, the precise role of RNA m6A methylation remains unclear. This study investigates how treadmill exercise modulates RNA m6A methylation to prevent MASLD in a high-fat diet (HFD)-induced mouse model. Male C57BL\/6 mice were fed either a standard diet (SD) or HFD for 12 weeks, with a subset of HFD-fed mice undergoing treadmill exercise (HFD + Ex). Liver pathology and biochemical markers were assessed. RNA sequencing (RNA-Seq) and methylated RNA immunoprecipitation sequencing (MeRIP-seq) were performed to identify differentially expressed genes (DEGs) and m6A methylation changes. Key candidate gene Paqr7 was validated through siRNA-mediated knockdown in AML-12 cells to assess its role in lipid metabolism. Treadmill exercise alleviated MASLD-related pathology and biochemical abnormalities. RNA-Seq identified 984 DEGs in the HFD vs. SD comparison and 544 in the HFD + Ex vs. HFD comparison. Intersection analysis identified 155 genes upregulated in MASLD and downregulated following exercise. MeRIP-seq revealed 225 hypermethylated and 208 hypomethylated m6A peaks in HFD + Ex vs. HFD groups. Integrative analysis highlighted Adra2b, Lipa, and Paqr7 as key exercise-responsive genes. Silencing Paqr7 through siRNA-mediated knockdown reduced lipid accumulation and suppressed lipogenic gene expression, suggesting its role in exercise-mediated MASLD improvement. Treadmill exercise prevents MASLD by modulating RNA m6A methylation, with Paqr7 emerging as a potential regulator of lipid metabolism. These findings highlight epigenetic modulation as a key mechanism in exercise-induced liver protection.<\/jats:p>","DOI":"10.3390\/ijms26125810","type":"journal-article","created":{"date-parts":[[2025,6,17]],"date-time":"2025-06-17T10:45:26Z","timestamp":1750157126000},"page":"5810","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Treadmill Exercise-Induced RNA m6A Methylation Modification in the Prevention of High-Fat Diet-Induced MASLD in Mice"],"prefix":"10.3390","volume":"26","author":[{"given":"Xueli","family":"Liu","sequence":"first","affiliation":[{"name":"Fujian Key Laboratory of Developmental and Neural Biology, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanming","family":"Zhong","sequence":"additional","affiliation":[{"name":"School of Physical Education and Sport Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuqian","family":"Guo","sequence":"additional","affiliation":[{"name":"Fujian Key Laboratory of Developmental and Neural Biology, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianhua","family":"Xu","sequence":"additional","affiliation":[{"name":"School of Physical Education and Sport Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaobing","family":"Wang","sequence":"additional","affiliation":[{"name":"Provincial University Key Laboratory of Sport and Health Science, School of Physical Education and Sport Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yiping","family":"Liu","sequence":"additional","affiliation":[{"name":"Provincial University Key Laboratory of Sport and Health Science, School of Physical Education and Sport Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Lv","sequence":"additional","affiliation":[{"name":"Fujian Key Laboratory of Developmental and Neural Biology, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0019-6459","authenticated-orcid":false,"given":"Xi","family":"Zheng","sequence":"additional","affiliation":[{"name":"Fujian Key Laboratory of Developmental and Neural Biology, College of Life Sciences, Fujian Normal University, Fuzhou 350117, China"},{"name":"School of Physical Education and Sport Sciences, Fujian Normal University, Fuzhou 350117, China"},{"name":"Provincial University Key Laboratory of Sport and Health Science, School of Physical Education and Sport Sciences, Fujian Normal University, Fuzhou 350117, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,17]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2212","DOI":"10.1016\/S0140-6736(20)32511-3","article-title":"Non-Alcoholic Fatty Liver Disease","volume":"397","author":"Powell","year":"2021","journal-title":"Lancet"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1556","DOI":"10.1002\/hep.31158","article-title":"Inadequate Physical Activity and Sedentary Behavior Are Independent Predictors of Nonalcoholic Fatty Liver Disease","volume":"72","author":"Kim","year":"2020","journal-title":"Hepatology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1851","DOI":"10.1053\/j.gastro.2020.01.052","article-title":"Nonalcoholic Fatty Liver Disease 2020: The State of the Disease","volume":"158","author":"Cotter","year":"2020","journal-title":"Gastroenterology"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"2263","DOI":"10.1001\/jama.2015.5370","article-title":"Nonalcoholic Fatty Liver Disease: A Systematic Review","volume":"313","author":"Rinella","year":"2015","journal-title":"JAMA"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"829","DOI":"10.1016\/j.jhep.2017.05.016","article-title":"Treatment of NAFLD with Diet, Physical Activity and Exercise","volume":"67","author":"Trenell","year":"2017","journal-title":"J. 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