Integrated bulk and single-cell RNA sequencing analysis reveals cuproptosis-related proteins in Crohn's disease

Crohn's disease (CD) is a chronic inflammatory bowel disorder with a rising annual incidence. Recent studies have linked CD to disrupted copper homeostasis. We aimed to explore the role of cuproptosis-related proteins in CD using bioinformatics.

Integrated microarray dataset (including 494 cases total, including CD inflammatory tissue and control tissue) and single-cell RNA sequencing data (GSE184291, comprising 25,121 cells from inflammatory and non-inflammatory tissue). Combined with the cuproptosis gene set, batch correction, differential expression analysis, enrichment analysis, WGCNA, and PPI network analysis were performed. A CD diagnostic model based on cuproptosis-related genes was established and validated on an external dataset. Single-cell analysis further characterized cellular heterogeneity, the distribution of diagnostic markers across cell populations, and cell communication features.

Four cuproptosis-related protein markers-CD274, PDK1, CP, and SLC31A2-were identified to be upregulated in CD and validated via RT-qPCR. These markers were not only enriched in metabolism-related pathways but were also strongly connected to immune cells, with epidermal growth factor receptor (EGFR) being a potential central node. The diagnostic model demonstrated robust performance, with an area under the curve of 0.789 in an internal validation and 0.806 in an external validation. The single-cell analysis provided initial, hypothesis-generating insights into the immune microenvironment in CD.

This study identified four potential protein markers for CD: CD274, PDK1, CP, and SLC31A2. Our findings suggest a correlation among cuproptosis, metabolism, and immunity. Further studies are required to experimentally validate these associations and explore the potential of markers as therapeutic targets.