Fig. 9
CircEZH2/IGF2BP2 enhances the stability of CREB1 mRNA in CRC. A MeRIP-seq (accession number: GSE179042) analysis was performed to discover the potential m6A modification profile in CRC tissues. m6A sites were displayed in 5′-UTR, CDS and 3′-UTR. B Schematic diagram based on MeRIP-seq showed the remarkable m6A modification site in the mRNA of CREB1. C qRT-PCR assay was performed to detect the expression of RGMA, FCGR1A, B4GALT3, NEK9, CLDN2, RTN4RL1, ZNF74, CENPL, RAB44 and CREB1 mRNAs in negative control (NC) and circEZH2-depleted HCT116 and SW620 cells. D Symbol showed the m6A motif in CRC tissues. E Stability of CREB1 mRNA was assessed by RNase R treatment followed by qRT-PCR in HCT116 and SW620 cells. F RBP immunoprecipitation (RIP) assay demonstrated the CREB1 mRNA enrichment precipitated by anti-IgG or anti-IGF2BP2 antibodies in negative control and circEZH2-depleted HCT116 and SW620 cells. G RIP assays demonstrated the CREB1 mRNA levels precipitated by anti-IgG or anti-m6A antibodies in NC and circEZH2 shRNA, control and miR-133-OE and NC and IGF2BP2 siRNA HCT116 cells. H Relative expression of CREB1 mRNA in CRC tissues and matched adjacent normal tissues was analyzed by qRT-PCR assays (n = 124). I Spearman’s correlation analysis revealed a positive association between CREB1 mRNA expression and circEZH2 expression in CRC tissues (r = 0.5903; P < 0.01). J IHC staining of CREB1 in CRC and adjacent normal tissues. Scale bar = 50 μm. K H score of CREB1 in CRC and adjacent normal tissues (n = 124). L Spearman’s correlation analysis revealed a positive association between CREB1 protein expression and circEZH2 expression in CRC tissues (r = 0.2831; P < 0.01). M The expression levels of miR-133b and CREB1 in tumors from control and circEZH2-OE AOM/DSS models were determined by qRT-PCR assay. N IHC staining of CREB1 in tumors from control and circEZH2-OE AOM/DSS mice. Scale bar = 25 μm. Data were showed as mean ± SD. # P > 0.05, **P < 0.01