Identification of diagnostic markers and molecular clusters of cuproptosis-related genes in alcohol-related liver disease based on machine learning and experimental validation.

Background And Aims: Alcohol-related liver disease (ALD) is a worldwide burden. Cuproptosis has been shown to play a key role in the development of several diseases. However, the role and mechanisms of cuproptosis in ALD remain unclear.

Methods: The RNA-sequencing data of ALD liver samples were downloaded from the Gene Expression Omnibus (GEO) database. Bioinformatical analyses were performed using the R data package. We then identified key genes through multiple machine learning methods. Immunoinfiltration analyses were used to identify different immune cells in ALD patients and controls. The expression levels of key genes were further verified.

Results: We identified three key cuproptosis-related genes (CRGs) (DPYD, SLC31A1, and DBT) through an in-depth analysis of two GEO datasets, including 28 ALD samples and eight control samples. The area under the curve (AUC) value of these three genes combined in determining ALD was 1.0. In the external datasets, the three key genes had AUC values as high as 1.0 and 0.917, respectively. Nomogram, decision curve, and calibration curve analyses also confirmed these genes' ability to predict the diagnosis. These three key genes were found to be involved in multiple pathways associated with ALD progression. We confirmed the mRNA expression of these three key genes in mouse ALD liver samples. Regarding immune cell infiltration, the numbers of B cells, CD8 (+) T cells, NK cells, T-helper cells, and Th1 cells were significantly lower in ALD patient samples than in control liver samples. Single sample gene set enrichment analysis (ssGSEA) was then used to estimate the immune microenvironment of different CRG clusters and CRG-related gene clusters. In addition, we calculated CRG scores through principal component analysis (PCA) and selected Sankey plots to represent the correlation between CRG clusters, gene clusters, and CRG scores. Finally, the three key genes were confirmed in mouse ALD liver samples and liver cells treated with ethanol.

Conclusions: We first established a prognostic model for ALD based on 3 CRGs and robust prediction efficacy was confirmed. Our investigation contributes to a comprehensive understanding of the role of cuproptosis in ALD, presenting promising avenues for the exploration of therapeutic strategies.