Integrative Mendelian Randomization and Single-Cell Transcriptomic Analyses Reveal Ferroptosis-Related Protective Genes and Tumor Microenvironmental Remodeling in Colorectal Cancer

Background Ferroptosis is closely linked to tumor progression, therapeutic vulnerability, and tumor microenvironmental remodeling in colorectal cancer (CRC). However, most existing ferroptosis-related studies in CRC rely on transcriptomic signatures or prognostic models, while the genetic relevance and cell type-specific microenvironmental features of ferroptosis-related regulators remain insufficiently defined. This study aimed to identify ferroptosis-related genes genetically associated with CRC risk and to characterize their functional and cellular contexts through integrative bulk and single-cell transcriptomic analyses. Methods Bulk transcriptomic datasets from GEO were used to identify differentially expressed genes between CRC and normal tissues, which were intersected with FerrDb-derived ferroptosis-related genes. Protein–protein interaction analysis was performed to characterize candidate ferroptosis-related genes. Two-sample Mendelian randomization using eQTLGen cis-eQTL data and CRC GWAS summary statistics was conducted to prioritize genes genetically associated with CRC risk, followed by sensitivity analyses for heterogeneity, horizontal pleiotropy, and single-SNP influence. A diagnostic nomogram was constructed and evaluated by ROC, calibration, and decision curve analyses. Candidate gene expression was validated in independent GEO cohorts. Gene set enrichment analysis, immune infiltration analysis, and ceRNA network construction were performed to explore potential biological functions and regulatory mechanisms. Single-cell RNA-seq data were further analyzed to define cell type-specific expression patterns, endothelial pseudotime trajectories, and cell–cell communication changes in CRC. Results We identified 1,439 differentially expressed genes between cancer and normal tissues, of which 70 overlapped with ferroptosis-related genes and were retained for downstream analyses. Protein–protein interaction analysis highlighted several hub genes, including IL6, IL1B, PTGS2, HMOX1, MAPK3, and CDKN1A. MR prioritized four protective genes—AKR1C2, MAPK3, MAP3K11, and CDKN1A—whose genetically predicted higher expression was associated with reduced CRC risk. These associations remained robust across sensitivity analyses, with no evidence of substantial heterogeneity, horizontal pleiotropy, or single-SNP bias. A four-gene diagnostic nomogram achieved favorable discrimination, calibration, and clinical utility, with an AUC of 0.863. Independent GEO cohorts consistently confirmed downregulation of all four genes in CRC tissues and revealed broadly positive intergene correlations. Functional enrichment linked these genes to cell-cycle regulation, MYC-driven proliferation, inflammatory signaling, TNFα/NF-κB activity, and epithelial–mesenchymal transition. Immune infiltration analysis demonstrated extensive microenvironmental remodeling, with altered dendritic cell, NK-cell, cytotoxic, and interferon-related signatures closely associated with gene expression. A ceRNA network suggested potential post-transcriptional regulation. Single-cell analysis of 53,253 cells further revealed cell type-specific dysregulation across epithelial, immune, stromal, and endothelial compartments. Endothelial pseudotime and cell–cell communication analyses indicated tumor-associated endothelial remodeling and enhanced epithelial–stromal–immune crosstalk. Conclusions This integrative study identifies AKR1C2, MAPK3, MAP3K11, and CDKN1A as ferroptosis-related protective genes in CRC. These genes were consistently downregulated in tumors, genetically associated with reduced colorectal cancer risk, and showed promising diagnostic value. Functional, immune, and single-cell analyses further linked them to proliferative signaling, immune remodeling, endothelial transition, and altered intercellular communication. These findings provide potential genes and mechanistic clues for CRC risk stratification and tumor microenvironment characterization.