DNA Methylation and Target Gene Expression in Fatty Liver Progression From Simple Steatosis to Advanced Fibrosis

Metabolic dysfunction-associated steatotic liver diseases (MASLD), also known as non-alcoholic fatty liver diseases (NAFLD), have become a leading risk factor for hepatocellular carcinoma (HCC) in Western countries. NAFLD progresses from simple steatosis to HCC, with advanced liver fibrosis (ALF) and metabolic dysfunction-associated steatohepatitis (MASH) or non-alcoholic steatohepatitis (NASH) representing the two preceding high-risk stages. We analysed changes in the DNA methylation landscape from simple steatosis to ALF or NASH and determined their relevance in gene regulation and HCC survival. Methylomic datasets generated from applying the Illumina 450K BeadChip on human MASLD/NAFLD liver samples were analysed using integrative data analyses to identify differentially methylated regions (DMRs) associated with ALF (F3/4 vs. F0/1) or non-fibrotic NASH (NASH-F0/1 vs. NAFLD-F0/1). Gene Set Enrichment Analysis (GSEA) of genes associated with fibrosis-DMRs showed enrichment in xenobiotic metabolism, UV response and hypoxia pathways. Expression of 25 DMR-associated genes showed significant associations with HCC survival outcomes, including 16 genes with fibrosis-DMRs and 2 with NASH-DMRs mapped to their promoter regions. Binding motifs of seven transcription factors (TFs) were enriched in fibrosis-DMRs. Four DMR-associated genes (ESR1, TYW3, CLGN and TUBB) displayed an inverse relationship between promoter methylation and gene expression during human MASLD progression, which was further validated in a mouse MASLD model. We propose a model in which changes in promoter DNA methylation during NAFLD progression regulate gene expression, impacting HCC survival outcomes.