NSUN2 promotes glycolysis and stemness of retinoblastoma cells by m5C modification of PFKFB3.

Retinoblastoma (RB) is a cancerous tumor that often develops in children. NSUN2, a m5C writer, has been reported to promote RB progression. The objective of this study was to analyze the function of NSUN2 in RB, especially revealing m5C-modified mRNA mediated by NSUN2. RB cellular behaviors, including glycolysis and stemness, were evaluated using Seahorse analysis, western blotting and tumorsphere formation assay. The assessment of m5C methylation of PFKFB3 involved methylated RNA immunoprecipitation, luciferase reporter experiment, and RNA stability assay. A tumor-bearing mouse model was created to investigate the in vivo function of NSUN2. The results showed that both m5C levels and NSUN2 expression showed higher levels in RB cell lines, while knocking down NSUN2 suppressed cell viability, glycolysis, and stemness. Furthermore, the stability of PFKFB3 mRNA was enhanced by NSUN2 through direct binding and m5C modification. Importantly, PFKFB3 overexpression reversed the inhibitory impact resulting from NSUN2 silencing on glycolysis, stemness, and tumor growth. In conclusion, NSUN2 acts as an oncogene in RB. It enhances glycolysis and supports stemness through the stabilization of PFKFB3 via m5C modification. The findings highlight a novel NSUN2-PFKFB3 axis that might serve as a therapeutic target in RB.