Network pharmacology and single-cell sequencing analysis of gastrodin in anticancer effects against colorectal cancer by regulating key target genes and the tumor immune microenvironment.

BACKGROUND: Gastrodin is a natural compound with anti-inflammatory, antioxidant, and antitumor activities; however, its therapeutic role and molecular mechanisms in colorectal cancer (CRC) remain unclear. In this study, we explored the anti-CRC mechanisms of gastrodin through integrated bioinformatics and experimental validation. METHODS: The potential targets of gastrodin were obtained from public databases. Transcriptomic data of CRC were retrieved from TCGA and GEO datasets, and the differentially expressed genes were screened. The CRC-related genes were subjected to gene-set variation analysis and weighted gene co-expression network analysis, and the core genes were identified by the LASSO regression, support vector machine, and random forest algorithms, followed by SHAP analysis. Single-cell RNA sequencing (scRNA-seq), immune infiltration analysis, consensus clustering, molecular docking, and in vitro experiments were further performed. RESULTS: A total of 628 potential gastrodin targets and 15 overlapping CRC-related genes were identified. Five core hub genes, including NFE2L3, UBE2S, CA9, CCNB1, and CFD, were screened, among which NFE2L3 showed the highest SHAP importance score. The scRNA-seq data indicated cell-type-specific expression patterns of these genes in CRC tissues. Immune infiltration analysis further suggested that gastrodin may regulate the CRC immune microenvironment by regulating the activated B cells, activated CD4⁺ T cells, and other immune cells. Molecular docking revealed favorable binding affinity between gastrodin and the five core proteins. Moreover, NFE2L3 was highly expressed in CRC tissues and cell lines, and its knockdown significantly inhibited the proliferation, migration, and invasion of the tumor cells. CONCLUSION: Gastrodin may suppress CRC progression by targeting key genes, particularly NFE2L3, and modulating the tumor immune microenvironment. Furthermore, our study has identified the potential biomarkers and therapeutic targets for CRC.