NAT10 promotes the glycolysis of retinoblastoma through ac4C modification of PFKFB3 mRNA.

Retinoblastoma (RB) is the most prevalent primary intraocular malignancy in children. N-acetyltransferase-like protein 10 (NAT10)-mediated N4-acetylcytidine (ac4C) modification is involved in regulating cancer progression. This study aimed to elucidate the role and underlying mechanisms of NAT10 in RB progression. The cell viability was detected by CCK-8 assay. The cell glycolysis was evaluated by glucose uptake, lactate production, extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) assays. The underlying molecular mechanism was investigated by dot blot, western blot, quantitative real-time PCR, RNA immunoprecipitation (RIP), ac4C RIP, and dual-luciferase reporter assay. Results showed elevated NAT10 and ac4C levels in RB cells. Genetic modulation of NAT10 bidirectionally regulated glycolysis, with 2‑deoxy‑D‑glucose (2‑DG) attenuating NAT10-induced glycolytic activation. Mechanistically, NAT10 bound PFKFB3 and stabilized its mRNA via ac4C modification. PFKFB3 overexpression rescued glycolytic suppression caused by NAT10 knockdown. In vivo, NAT10 promoted tumor growth. Collectively, NAT10 contributes to RB progression by enhancing glycolysis through ac4C-mediated PFKFB3 mRNA stabilization, identifying the NAT10-ac4C-PFKFB3 axis as a potential therapeutic target.