Identifying key genes against rutin on human colorectal cancer cells via ROS pathway by integrated bioinformatic analysis and experimental validation

Colorectal cancer (CRC) poses a significant global health challenge, characterized by substantial prevalence variations across regions. This study delves into the therapeutic potential of rutin, a polyphenol abundant in fruits, for treating CRC. The primary objectives encompass identifying molecular targets and pathways influenced by rutin through an integrated approach combining bioinformatic analysis and experimental validation. Employing Gene Set Enrichment Analysis (GSEA), the study focused on identifying potential differentially expressed genes (DEGs) associated with CRC, specifically those involved in regulating reactive oxygen species, metabolic reprogramming, cell cycle regulation, and apoptosis. Utilizing diverse databases such as GEO2R, CTD, and Gene Cards, the investigation revealed a set of 16 targets. A pharmacological network analysis was subsequently conducted using STITCH and Cytoscape, pinpointing six highly upregulated genes within the rutin network, including TP53, PCNA, CDK4, CCNEB1, CDKN1A, and LDHA. Gene Ontology (GO) analysis predicted functional categories, shedding light on rutin's potential impact on antioxidant properties. KEGG pathway analysis enriched crucial pathways like metabolic and ROS signaling pathways, HIF1a, and mTOR signaling. Diagnostic assessments were performed using UALCAN and GEPIA databases, evaluating mRNA expression levels and overall survival for the identified targets. Molecular docking studies confirmed robust binding associations between rutin and biomolecules such as TP53, PCNA, CDK4, CCNEB1, CDKN1A, and LDHA. Experimental validation included inhibiting colorectal cell HT-29 growth and promoting cell growth with NAC through MTT assay. Flow cytometric analysis also observed rutin-induced G1 phase arrest and cell death in HT-29 cells. RT-PCR demonstrated reduced expression levels of target biomolecules in HT-29 cells treated with rutin. This comprehensive study underscores rutin's potential as a promising therapeutic avenue for CRC, combining computational insights with robust experimental evidence to provide a holistic understanding of its efficacy.