Transcription Factor MYB Upregulates IQGAP3 to Mediate DNA Repair and Promote 5-FU Resistance in Gastric Cancer Cells

5-Fluorouracil (5-FU)-based chemotherapy is a first-line treatment for advanced gastric cancer (GC); however, the development of resistance remains a major limitation to its clinical efficacy. This study aims to investigate the role of the MYB/IQGAP3 axis in mediating 5-FU resistance in GC. Using bioinformatics, we analyzed expression profiles of IQGAP3 and MYB in GC tissues and pinpointed their binding sites. IHC was used to detect the expression of IQGAP3 in GC tissues. The signaling pathways potentially regulated by IQGAP3 were also investigated. Dual-luciferase and chromatin immunoprecipitation assays substantiated the regulatory link between MYB and IQGAP3. Expressions of IQGAP3, MYB, and drug-resistant genes were measured via qRT-PCR and western blot. The CCK-8 assay was implemented to gauge cell survival and the IC50 values. The colony formation assay assessed cell growth. Cell apoptosis was examined by flow cytometry. DNA damage was visualized by immunofluorescence staining. We detected a pronounced enhancement in the expression of IQGAP3 and MYB within GC tissues and cells and identified that IQGAP3 was involved in the regulation of mismatch repair and DNA repair (DNAR) pathways. Suppression of IQGAP3 led to increased sensitivity to 5-FU, as evidenced by a decreased IC50 value. Along with that, we observed increased apoptosis and restrained proliferation of GC cells, downregulated P-gp, MRP1, and GST-π protein levels, and hindered DNAR. The effects were inverted with the overexpression of IQGAP3. Furthermore, MYB could bind to IQGAP3 promoter to promote its transcription, and silencing IQGAP3 substantially negated the influence of MYB overexpression on GC cell DNAR and sensitivity to 5-FU. The upregulation of IQGAP3 by MYB mediates DNAR, thereby promoting 5-FU resistance in GC. This points to the therapeutic value of targeting MYB/IQGAP3 to reduce GC drug resistance and enhance the clinical efficacy of treatments.