SPI1 inhibits ferroptosis and immune escape of bladder cancer cells by promoting CRYAB expression

This study investigates the molecular mechanism by which SPI1 inhibits Ferroptosis and immune escape in bladder Cancer (BC) through upregulation of CRYAB expression. Bioinformatics was utilized to screen BC-related prognostic genes and their upstream regulators. RT-qPCR and Western blot (WB) were conducted to assess SPI1 and CRYAB expression in BC cells. CRYAB knockdown and SPI1/CRYAB overexpression were performed to explore their effects on cellular behaviors. Malignant phenotypes were evaluated through CCK-8, colony formation, scratch assays, and Annexin V-FITC/PI staining. Ferroptosis was assessed using Fe²⁺ and JC-1 staining, alongside biochemical analysis. THP-1 monocytes were differentiated into macrophages and co-cultured with BC cells; WB was performed to measure M2 macrophage polarization markers (CD86/CD206). T cell cytotoxicity assays were performed using activated CD3⁺ T cells from healthy donors co-cultured with BC cells to directly assess immune evasion. Dual-luciferase and ChIP-qPCR assays were used to explore the transcriptional regulation of CRYAB by SPI1. A xenograft mouse model was established to examine BC cell growth and immune escape in vivo. Bioinformatics identified CRYAB and SPI1 as key regulators in BC. Both were highly expressed in BC cells. CRYAB knockdown promoted Ferroptosis and inhibited malignant behaviors of BC cells, and suppressed epithelial-mesenchymal transition (EMT) and M2 macrophage polarization in co-culture systems. Furthermore, CRYAB or SPI1 knockdown sensitized BC cells to T cell-mediated killing and enhanced T cell effector molecule production (IFN-γ, TNF-α, Granzyme B), whereas CRYAB overexpression reversed the effect of SPI1 knockdown. Dual-luciferase and ChIP-qPCR assays confirmed that SPI1 directly transcriptionally regulates CRYAB. SPI1 knockdown enhanced Ferroptosis, inhibited EMT, and suppressed immune escape in BC cells, effects that were reversed by CRYAB overexpression. SPI1 inhibits Ferroptosis and immune escape in BC by promoting CRYAB expression, thereby facilitating BC progression. These findings provide new theoretical insights and potential therapeutic strategies to improve the prognosis of BC patients.