Fig. 7.

METTL3 mediates TAM polarization and PD-L1 expression via the miR-146b/p110β/PI3K/AKT axis in vitro and in vivo. (A) BMDMs from WT mice were transfected with MO-Mettl3 for 24 h and then stimulated with IL-4 plus IL-10 for 4 h. Arg-1 and Ym-1 mRNA levels were determined by qPCR. (B) BMDMs from WT mice were transfected with MO-Mettl3 for 24 h and then stimulated with IL-4 plus IL-10 for 24 h. TGFβ1 and IL-10 protein levels were determined by ELISA. (C-D) BMDMs from WT mice were transfected with MO and then treated with IL-4 plus IL-10. WT mice were implanted with MC38 cells mixed with in vitro cultured M2 macrophages (n = 6). Tumor growth and weight were monitored. (E–F) BMDMs from WT mice were transfected with MO-Mettl3 for 12 h, transfected with miR-146b mimic for another 12 h, and then stimulated with IL-4 plus IL-10 for 4 h or 24 h. The mRNA levels of Arg-1 and Ym-1 were determined by qPCR. TGFβ1 and IL-10 protein levels were determined by ELISA. (G) BMDMs from WT mice were transfected with MO-Mettl3 for 12 h and then stimulated with IL-4 plus IL-10 for the indicated times. The expression of p110β, pAkt/Akt, pC/EBPβ, and C/EBPβ was determined by western blotting. (H) BMDMs from WT mice were transfected with MO-Mettl3 for 12 h, and then stimulated with IL-4 plus IL-10. C/EBPβ DNA-binding activity and Arg-1 activity were determined. (I-J) BMDMs from WT mice were transfected with MO-Mettl3 for 12 h, preincubated with an AKT inhibitor (GSK2141795) or a PI3K inhibitor (LY294002) for an additional 30 min and then treated with IL-4 plus IL-10. Arg-1 and Ym-1 mRNA levels were determined by qPCR. TGFβ1 and IL-10 protein levels were determined by ELISA. (K) BMDMs from WT mice were transfected with MO-Mettl3 for 12 h and then stimulated with IL-4 plus IL-10. PD-L1 expression was determined by western blotting. (L) BMDMs from WT mice were transfected with MO-Mettl3 for 12 h, transfected with miR-146b mimic for an additional 12 h, and then stimulated with IL-4 plus IL-10. PD-L1 expression was determined by western blotting. (M) BMDMs from WT mice were transfected with MO-Mettl3 for 12 h; preincubated with GSK2141795, LY294002 or TGX221 for an additional 30 min and then treated with IL-4 plus IL-10. PD-L1 expression was determined by western blotting. (N) Schematic overview of the administration of anti-PD-1 antibodies in the MC38 model (n = 6). Mice were treated with anti-PD-1 antibodies (250 μg/mouse) or control IgG twice/week for 2 weeks. (O) Representative photograph showing tumor formation. (P) Tumor growth and weight were monitored. All experiments were repeated three times independently. The data represent the mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant