Fig. 5: METTL3 O-GlcNAcylation targets MCM10 mRNA to maintain the tumorigenic behavior of hepatoma cells.

A Schematic representation of the METTL3-METTL14-WTAP complex. O-GlcNAcylation of METTL3 enhances its affinity for WTAP, without affecting its association with METTL14. B Flag-METTL3 (WT or 3A) was transfected with or without HA-WTAP into Huh-7 cells. Lysates were immunoprecipitated with anti-Flag antibody, followed by immunoblotting with specific antibodies. C Huh-7-shMETTL3 cells were transfected with Flag-METTL3-WT or -3A. m6A abundance in mRNAs from infected cells was measured using the dot-blot assay with an anti-m6A antibody, with mRNA loading confirmed by methylene blue staining (upper panels). Cell lysates were immunoblotted with the indicated antibodies (lower panels). D Screening for downstream targets of METTL3 by bioinformatic analysis. E Gene Ontology analysis of METTL3-knockdown differentially expressed genes. The mRNA levels of initially screened genes in Huh-7-shMETTL3 cells were measured using RT-qPCR (F), and the m6A modification levels of target mRNAs were assessed using MeRIP-qPCR (G) (n = 3 independent experiments). H Correlation analysis of METTL3 and MCM10 in the TCGA-LIHC cohort (Spearman correlation, P < 0.001). I MeRIP-qPCR assessment of the effect of METTL3 O-GlcNAcylation on m6A modification of MCM10 mRNA. J Immunoblotting to assess the effect of METTL3 O-GlcNAcylation on MCM10 protein levels. K–M Huh-7 cells were primarily infected with lentiviruses carrying shControl, shMETTL3, or shMCM10, followed by infection with either Ad-GFP or Ad-MCM10. All treatment groups were subjected to CCK-8 (K), Transwell (L, scale bar: 100 μm), and wound-healing (M, scale bar: 200 μm) assays. Results are derived from three independent experiments and are presented as mean ± SD. Data in (F, G) were analyzed using an unpaired, two-tailed Student’s t-test. Data in (I, K–M) were analyzed using one-way ANOVA followed by Tukey tests. *P < 0.05, **P < 0.01, ***P < 0.001.