METTL16 suppresses ferroptosis in cholangiocarcinoma by promoting ATF4 via m⁶A modification

<b>Background:</b> N6-methyladenosine (m⁶A) modification is the most common post-transcriptional modifications, which is critical for the metabolism of ferroptosis-related RNAs. Yet, the impact of m⁶A modification on ferroptosis in cholangiocarcinoma (CC) is far from clear. <b>Methods:</b> Public databases and tissue arrays were applied to explore the clinical relevance of METTL16 in CC. Then, the effects of METTL16 on growth and ferroptosis were studied <i>in vitro</i> and <i>in vivo</i>. Mechanistically, RNA-sequencing, methylated RNA immunoprecipitation, dual-luciferase reporter assays and RNA stability assays were used to identify the METTL16/ATF4 axis in ferroptosis in CC. <b>Results:</b> Clinically, we find that METTL16 is overexpressed and associated with a poor prognosis in patients with CC. Functionally, METTL16 protects against ferroptosis by maintaining mitochondrial homeostasis, including mitochondrial structure, membrane potential and energy products. It also decreases cellular metabolism of Fe²⁺ and lipid peroxide, thereby promoting cell growth <i>in vitro</i> and <i>in vivo</i>. Mechanistically, ATF4 is a novel target of METTL16 and METTL16 enhances the m6A level and expression of ATF4 mRNA by inhibiting its decay, which further prevented ferroptosis in CC via m6A modification. <b>Conclusions:</b> Our findings highlighted the role of METTL16/ATF4 in ferroptosis, which sheds light on potential therapeutic strategies for CC.