Hypoxia Triggers ALYREF-Mediated m5C Methylation of KIF20A to Activate KIF20A/BUB1 for Generating Ferroptosis Resistance in Cervical Cancer Cells.

Ferroptosis resistance is a key player in cervical cancer (CC) development. Hypoxia is a negative factor affecting CC treatment by inducing ferroptosis resistance. Our study aimed to investigate the detailed mechanisms of hypoxia-induced ferroptosis resistance in CC cells. The mRNA and protein levels were assessed using RT-qPCR and western blot. Immunofluorescence staining was used to analyze the co-localization of Budding uninhibited by benzimidazole 1 (BUB1) and Kinesin family member 20A (KIF20A) in CC cells. Fe²⁺, MDA, and GSH levels were measured by commercial kits. Cell viability was determined by CCK-8. The molecular interactions were analyzed by RIP or Co-IP. MeRIP was adopted to measure the m⁵C level of KIF20A. We found that hypoxia facilitated ferroptosis resistance in CC cells. Hypoxia led to the upregulation of KIF20A, and KIF20A knockdown weakened hypoxia-mediated ferroptosis resistance in CC cells. Mechanistically, Aly/REF export factor (ALYREF) stabilized KIF20A mRNA stability via m⁵C modification. In addition, KIF20A upregulated BUB1 in CC cells by directly interacting with BUB1. Rescue experiments indicated that BUB1 overexpression partially reversed the inhibitory effect of KIF20A knockdown on hypoxia-mediated ferroptosis resistance in CC cells. In conclusion, hypoxia triggers ALYREF-mediated m⁵C methylation of KIF20A to activate the KIF20A/BUB1 axis, thereby inducing ferroptosis resistance in CC cells.