Integrated expression and network-based characterization of H19-associated molecular alterations in Metabolic Dysfunction-Associated Fatty Liver Disease

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD) is characterized by hepatic lipid accumulation and metabolic dysregulation. However, reproducible noncoding RNA-associated molecular alterations in human liver tissue remain incompletely defined. We aimed to characterize hepatic H19 expression in MAFLD and to identify H19-associated molecular alterations related to lipid metabolism and proteostasis. Two public liver transcriptomic datasets and one in-house liver transcriptomic cohort were integrated using gene-symbol harmonization, cohort-specific analyses, within-cohort standardization, and random-effects meta-analysis. H19-associated candidate molecules were prioritised using differential expression analysis, correlation analysis, pathway annotation, effect-size assessment, and miRNA database integration. Findings were further evaluated in a lipid-loading L02 cell model and in clinical liver tissue samples using quantitative real-time PCR and tissue microarray immunohistochemistry. H19 expression was reproducibly reduced across MAFLD-related liver cohorts and was associated with lipid metabolism-related transcriptional alterations. FADS2 showed a consistent inverse association with H19 and was upregulated in MAFLD, supporting its prioritization as an H19-associated lipid metabolic candidate. Directional correlation analysis and pathway annotation prioritised HSPA5 as a proteostasis-related candidate associated with the H19–FADS2 expression pattern. Integration of LncACTdb3 and ENCORI identified miR-194-5p as a candidate H19-associated miRNA node supported by MAFLD-relevant module annotation and ENCORI-derived HSPA5/FADS2 target-site annotations. In L02 cells and clinical liver tissues, H19 and HSPA5 showed lower expression patterns, whereas miR-194-5p and FADS2 were increased. Tissue microarray immunohistochemistry showed lower HSPA5 protein expression and a trend toward higher FADS2 protein expression in MAFLD tissues. Reduced hepatic H19 expression is associated with lipid metabolism- and proteostasis-related molecular alterations in MAFLD. These findings support a candidate H19-associated expression framework involving miR-194-5p, FADS2, and HSPA5-related lipid-metabolic and proteostasis alterations. This framework should be considered hypothesis-generating and requires future perturbation-based validation.