Fig. 7
CircEZH2 interacts with m6A reader IGF2BP2 and blocks its ubiquitination-dependent degradation. A RNA pulldown was performed using biotin-labeled circEZH2 probe, followed by mass spectrometry analysis. B RNA pulldown assay was performed to verify the interaction between circEZH2 and IGF2BP2 in HCT116 and SW620 cells. C RBP immunoprecipitation (RIP) assay was performed using IGF2BP2 or IgG antibodies, followed by qRT-PCR assay for circEZH2 expression in HCT116 and SW620 cells. D Western blot was performed to detect the expression of IGF2BP2 in negative control (NC) and circEZH2-depleted (shRNA-1 and shRNA-2) HCT116 and SW620 cells. Hsp 70 served as a loading control. E qRT-PCR assay was performed to detect the mRNA levels of IGF2BP2 in negative control and circEZH2-depleted HCT116 and SW620 cells. F CircEZH2 knockdown accelerated the ubiquitination modification of IGF2BP2 in HCT116 cells transfected with ubiquitin (Ub) and treated with MG-132, which was detected by Western blot. G IHC staining of IGF2BP2 in tumors from control and circEZH2-OE AOM/DSS mice. Scale bar = 25 μm. H The mRNA level of IGF2BP2 in CRC (n = 275) and adjacent normal (n = 349) tissues was analyzed using TCGA database. I IHC staining of IGF2BP2 in CRC and adjacent normal tissues. Scale bar = 50 μm. J H score of IGF2BP2 in CRC and adjacent normal tissues (n = 124). K Spearman’s correlation analysis revealed a positive association between IGF2BP2 protein expression and circEZH2 expression in CRC tissues (r = 0.4886; P < 0.01). L Kaplan-Meier analysis (log-rank test) of overall survival according to IGF2BP2 expression level (P < 0.01). L Data were showed as mean ± SD. # P > 0.05, **P < 0.01