DNA methylation of RUNX3 promotes the progression of gallbladder cancer through repressing SLC7A11-mediated ferroptosis

Gallbladder cancer (GBC) is a type of rare but highly aggressive cancer with a dismal prognosis. Runt-related transcription factor 3 (RUNX3), a member of the runt-domain family, and its promoter methylation have been widely observed in a variety of human malignancies. However, the biological function and underlying mechanism of RUNX3 in GBC remain elusive. In this study, bisulfate sequencing PCR (BSP), Western blot, and qPCR were applied to identify the expression level and DNA methylation level of RUNX3 in GBC tissues and cells. The transcriptional relationship between RUNX3 and Inhibitor of growth 1 (ING1) was validated by dual-luciferase reporter assay and ChIP assay. A series of gain-of-function and loss-of-function assays were performed to detect the function and the regulatory relationship of RUNX3 in vitro and in vivo. RUNX3 was aberrantly downregulated in GBC cells and tissues caused by DNA Methyltransferase 1 (DNMT1)-mediated methylation, and downregulation of RUNX3 is associated with poor prognosis of GBC patients. Functional experiments reveal that RUNX3 can induce ferroptosis of GBC cells in vitro and in vivo. Mechanistically, RUNX3 induces ferroptosis by activating ING1 transcription, thereby repressing SLC7A11 in a p53-dependent manner. In conclusion, the downregulation of RUNX3 is mediated by DNA methylation, which promotes the pathogenesis of gallbladder cancer through attenuating SLC7A11-mediated ferroptosis. This study gives novel insights into the role of RUNX3 in the ferroptosis of GBC cells, which may contribute to developing potential treatment targets for GBC.