Fig. 5: Methionine inducing NR4A2 expression via the METTL3-m⁶A-IGF2BP2 cascade.

A, B Influence of methionine on NR4A2 expression at mRNA and protein levels in ESCC cells and the non-transformed esophageal epithelial cell line Het-1A. Cells were cultured for 24 h. B Effect of methionine administration via drinking water on the protein abundance of NR4A2 in the neoplastic cells of esophagi in the 4-NQO-induced ESCC mouse model. Treatment with the methylation inhibitor DAA at 10 μM for 24 h affecting methionine-elicited RNA m⁶A methylation (C) and NR4A2 expression at mRNA and protein levels (D) in ESCC cells. E Perturbation of methionine-elicited NR4A2 expression at both mRNA and protein levels in ESCC cells by METTL3 ablation. F NR4A2 mRNA degradation curves over time in ESCC cells treated with or without methionine in the presence of the transcription inhibitor actinomycin D. G mRNA degradation curves of NR4A2 over time in ESCC cells with or without METTL3 deletion under the treatment of actinomycin D. Cells were cultured in complete medium. H RIP-qPCR revealing the association of NR4A2 mRNA with IGF2BPs in KYSE150 cells. I mRNA degradation curves of NR4A2 over time in ESCC cells with or without IGF2BP2 deletion under the treatment of actinomycin D. Cells were cultured in complete medium. J Disturbance of NR4A2 protein levels in ESCC cells by IGF2BP2 deletion. Cells were cultured in complete medium. K Schematic diagram showing three predicted m⁶A motifs within NR4A2 mRNA with high confidence, and construction of three NR4A2 mutants containing these motifs. Mut1 and Mut2 represent full-length coding sequence mutants of NR4A2 with A-T substitutions (shown in red) within m⁶A consensus. Mut3 represents a 715-bp fragment of the 3′-UTR of NR4A2 with an A-T substitution (shown in red) within m⁶A consensus. L mRNA degradation curves of wild-type NR4A2 (NR4A2-WT), NR4A2 Mutant 1 (NR4A2-Mut1), and NR4A2 Mutant 2 (NR4A2-Mut2) in the presence of methionine, along with NR4A2-WT in the absence of methionine, under the treatment of actinomycin D. M Relative F-luc mRNA levels of NR4A2-WT or NR4A2 Mutant 3 (NR4A2-Mut3) reporters in HEK 293T cells with or without methionine treatment. NC non-target control, WT wild-type, Mut mutant, F-luc firefly luciferase, R-luc renilla luciferase. Error bars represent mean ± SEM. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001 (Student’s t test).