Identifying the importance of PCK1 in maintaining ileal epithelial barrier integrity in Crohn's disease.

ABSTRACT

BACKGROUND: Crohn's disease (CD) is marked by disruption of intestinal epithelial barrier, with unclear underlying molecular mechanisms. This study aimed to investigate key genes regulating the intestinal barrier in CD patients. METHODS: Differential gene expression analysis and gene set enrichment analysis were conducted to identify potential key genes involved in CD within the GEO database. Single-cell RNA sequencing from ileum samples in GSE134809 of 59,831 inflamed and uninflamed cells from 11 CD patients and microarray data from ileal tissues in GSE69762 (3 controls and 4 CD patients) and GSE75214 (11 controls and 51 CD patients) with GSE179285 (49 uninflamed and 33 inflamed from CD patients) as the validation set. Protein-protein interaction and logistic regression analyses identified key downregulated genes in CD. A key gene was then investigated through immunohistochemistry of ileal tissues from 5 CD patients and in the Caco-2 cell line with RNA interference and treatment with IFN-γ and TNF-α to stimulate inflammation. RESULTS: Single-cell RNA-seq identified 33 genes and microarray identified 167 genes with significant downregulation in inflamed CD samples. PCK1 was identified and validated as one of the most promising candidate genes. Reduced PCK1 expression was evident in inflamed ileal tissues. In vitro, knockdown of PCK1 resulted in decreased cell viability, increased apoptosis, and reduced nectin-2 production, while combination of IFN-γ and TNF-α significantly reduced PCK1. CONCLUSIONS: PCK1 is downregulated in inflamed ileal tissues of CD patients and may be a key factor in maintaining epithelial integrity during inflammation in Crohn's disease.