RPS2 and EEF1B2 are associated with glycolysis-related malignant phenotypes in COAD.

Lactate metabolism-related reprogramming is closely associated with colon adenocarcinoma (COAD) progression, but related key molecules remain unclear. This study integrated the colorectal cancer single-cell RNA sequencing dataset GSE231559, which served as a single-cell reference and integrated it with bulk RNA-seq data from TCGA-COAD to screen lactate metabolism-related candidate genes. Epithelial cells showed higher lactate metabolism-related transcriptional scores. An overlap-based screening strategy was applied, encompassing three gene sets: epithelial markers associated with lactate metabolism, dysregulated transcripts in the TCGA colon adenocarcinoma cohort, and genes within the co-expression module correlated with the lactate score. This analysis prioritized 14 genes for subsequent investigation. RPS2 and EEF1B2 were selected for further validation. Public database analysis and immunohistochemistry of paired tissues from 50 patients with COAD showed that RPS2 and EEF1B2 were highly expressed in tumor tissues. In HT-29 cells, siRNA-mediated knockdown of RPS2 or EEF1B2 reduced proliferation and migration, increased apoptosis, and decreased HK2, PFK1, PKM2, and LDHA expression, with stronger changes after double knockdown. RPS2 knockdown also increased residual glucose levels and reduced lactate release. Co-immunoprecipitation showed that RPS2 and EEF1B2 were detected in the same protein complex, and EEF1B2 overexpression partially rescued the effects of RPS2 knockdown. These findings suggest that RPS2 and EEF1B2 are associated with glycolysis-related malignant phenotypes in HT-29 cells.