Identification of 5‐Gene Prognostic Markers and Functional Verification of <scp>RRBP1</scp> in 5‐Fluorouracil Resistance of Colorectal Cancer by Multi‐Omics Analysis and Experimental Verification

Acquired resistance to 5-fluorouracil (5-FU) is a primary clinical challenge in colorectal cancer (CRC) treatment. Our study aimed to identify key factors predictive of 5-FU resistance and to elucidate their functional mechanisms by combining multi-omics analysis with experimental verification. The prognostic model was constructed based on the gene expression omnibus (GEO, GSE196900, GSE166555) and the cancer genome atlas (TCGA)-Colon Adenocarcinoma (COAD) datasets combined with regression analysis. Kaplan-Meier (K-M), receiver operating characteristic (ROC) curve, and nomogram were used to evaluate the predictive performance of the prognostic model. Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) were used for functional enrichment analysis. Single-cell RNA sequencing (scRNA-seq, GSE166555) and qRT-PCR analysis were used to analyze the tumor microenvironment and gene expression. In cell experiments, CCK-8 assay measured IC₅₀ value. Glycolysis metabolism was evaluated by detecting glucose consumption, lactic acid production, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR); cell stemness was evaluated by sphere formation assay. A 5-gene prognostic model was successfully constructed, which could effectively distinguish the high-/low-risk groups of CRC patients and was significantly correlated with overall survival. Ribosome binding protein 1 (RRBP1) is highly expressed in cancer tissues of non-responders to chemotherapy. It is also highly expressed in tumor epithelial cells, and its high expression is closely related to aneuploidy characteristics, up-regulation of oncogenes, and activation of pro-survival pathways. In vitro experiments confirmed that knockdown of RRBP1 significantly enhanced the sensitivity of CRC cells to 5-FU and inhibited cell proliferation. Mechanistically, RRBP1 knockdown effectively reversed the enhanced glycolysis activity and stem cell-like properties of 5-FU-resistant cells. This study established RRBP1 as a key CRC prognostic factor and 5-FU resistance driver, operating through the regulation of cell glycolysis and stemness. RRBP1 emerges as a new biomarker and therapeutic target for predicting the efficacy of 5-FU.