The lncRNA MIR4435 ‐ 2HG Modulates Bladder Cancer Progression and Ferroptosis Through the IQGAP3 /Ras/ ERK / CREB Pathway

ABSTRACT Bladder cancer (BCa) is characterized by high incidence and recurrence rates, and its development is closely related to ferroptosis. However, the regulatory mechanism of MIR4435‐2HG in BCa and ferroptosis remains unclear. This study investigated the role of the long non‐coding RNA (lncRNA) MIR4435‐2HG in BCa progression and ferroptosis regulation through the IQ motif containing GTPase activating protein 3 (IQGAP3)/rat sarcoma (Ras)/extracellular signal‐regulated kinase (ERK)/cAMP response element‐binding protein (CREB) axis. Bioinformatics analysis of TCGA‐BLCA data (402 tumors, 19 normals) was integrated to establish clinical relevance. Bladder cancer T24 cells were transfected with an overexpression vector (OE‐MIR4435‐2HG) or a knockdown vector (sh‐MIR4435‐2HG). Various assays, including cell counting kit‐8 (CCK‐8), scratch, transwell, flow cytometry, enzyme‐linked immunosorbent assay (ELISA), and Western blotting, were subsequently utilized to assess cellular behavior and protein expression. Finally, a subcutaneous xenograft model was employed for in vivo validation. TCGA analysis revealed MIR4435‐2HG overexpression in tumors (log2FC = 3.21, p < 0.0001), association with reduced survival (HR = 1.68, p = 0.006), and enrichment in Basal subtypes. Overexpression of MIR4435‐2HG promoted the migration, invasion, and proliferation of bladder cancer cells, with increased Ki‐67 and cytoskeleton‐associated protein 4 (CKAP4) protein expression, whereas MIR4435‐2HG knockdown had the opposite effects. MIR4435‐2HG overexpression activated the IQGAP3/Ras/ERK/CREB signaling axis, reducing the protein levels of IQGAP3, Ras, ERK, and CREB while increasing the expression of ferroptosis markers, specifically malondialdehyde (MDA), lipid peroxidation (LPO), and reactive oxygen species (ROS). MIR4435‐2HG knockdown reversed these changes. In vivo, overexpression of MIR4435‐2HG significantly enhanced tumor growth, whereas its suppression markedly reduced tumor volume and weight. The ferroptosis activator RSL3 further attenuated tumor progression in both the knockdown and overexpression models, and combined treatment with the ERK inhibitor SCH772984 synergistically suppressed tumor growth in MIR4435‐2HG‐overexpressing xenografts. Consistent with the in vitro findings, MIR4435‐2HG modulated the IQGAP3/Ras/ERK/CREB axis and ferroptosis markers (ROS, MDA, LPO, glutathione peroxidase 4 (GPX4)) in vivo, reinforcing its role in promoting malignancy and ferroptosis resistance. Treatment of OE‐MIR4435‐2HG‐transfected cells with SCH772984 further reduced protein levels and cell viability. Notably, RSL3 diminished IQGAP3, Ras, ERK, CREB, and GPX4 levels across the experimental conditions. These findings indicate that MIR4435‐2HG is a pivotal regulator of bladder cancer progression and ferroptosis via the IQGAP3/Ras/ERK/CREB axis, suggesting that MIR4435‐2HG is a potential therapeutic target for intervention.