Fig. 5: m6A modification promotes the miR-133a repression on its targets via IGF2BP2.

Neonatal mouse cardiomyocytes (CMs) were transfected with miR-133a and Si-RNAs. A Representative western blotting (up) and quantification (down) showing the miR-133a repression on CDC42 protein level could be reversed by Igf2bp2, but neither HuR nor Ythdf2 knockdown in CMs. B Western blotting assay indicating overexpression of IGF2BP2 could further decrease CDC42 with miR-133a transfection. C Luciferase reporter activities were quantitated in CMs and indicated miR-133a activity could increase by knockdown of Ago2 or Igf2bp2, but neither HuR nor Ythdf2. D Inhibiting Cdc42 mRNA decay by silencing Ago2 or Igf2bp2 in Actinomycin D treated CM cells. E RNA immunoprecipitated-qRT-PCR assay using IGF2BP2 antibody suggested that the enrichment of IGF2BP2 on Cdc42 mRNA was increased by Fto knockdown, but had no effect by Ago2 silencing. F AGO2-RNA immunoprecipitated-qRT-PCR assay showing the enrichment of AGO2 on Cdc42 was increased by Fto knockdown but decreased by Igf2bp2 knockdown. G Co-immunoprecipitation and western blotting showing the interaction of AGO2 and IGF2BP2 in CMs cells, representative of three independent experiments. H Carton model of IGF2BP2 binding to m6A-modified site promotes the miR-133a-AGO2-RISC complex accumulation on its targets, which would enhance the decreasing of their stability and translation. Data are mean ± SD (n = 4 per group). *p < 0.05; ** P < 0.01 was determined by one-way ANOVA followed by Tukey’s test in (A, C, E, and F). *p < 0.05 was detected by Student’s t test in (B).