Novel insights into cuproptosis in alcoholic liver disease using bioinformatics analysis and experimental validation

Cuproptosis is crucial in the development of various liver diseases, yet its involvement in alcoholic liver disease (ALD) remains poorly understood. In this study, we screened cuproptosis-related genes (CRGs) regulating ALD and explored their potential molecular mechanisms. Bioinformatic methods were employed to screen CRGs in ALD, analyze their functional enrichment, signaling pathways, transcriptional regulation, relationship with the immune microenvironment and pathogenic genes, and corresponding single nucleotide polymorphism pathogenic regions, and construct transcription factor-miRNA-mRNA networks. Liver tissues from patients with ALD and NIAAA model mice were collected and assessed for copper deposition. CRG expression in the livers of patients was analyzed using RNA sequencing. CRG expression in liver tissues was also analyzed using RNA sequencing and verified using quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. Dihydrolipoamide S-acetyltransferase (DLAT), glutaminase (GLS), and cyclin-dependent protein kinase inhibitor 2A (CDKN2A) were identified as potential ALD-associated CRGs with significant diagnostic value. Consistent with the results of bioinformatics analysis, these genes were notably upregulated in liver tissues from both patients with ALD and NIAAA model mice. All three ALD-associated CRGs were regulated by E2F transcription factor 1 (E2F1) and showed a significant negative correlation with the expression of ADH1B and ALDH2, key enzymes in alcohol metabolism. They were positively correlated with PI3K/AKT/mTOR, IFN-γ response, and complement signaling pathways but negatively correlated with bile acid and xenobiotic metabolism. This study identified DLAT, GLS, and CDKN2A as the ALD-associated CRGs, providing valuable insights into their molecular mechanisms in ALD pathogenesis, which may inform diagnosis and treatment.