Fig. 2

Characterization of circEZH2 and its clinical significance. A Schematic illustration of circEZH2 formation from EZH2 gene in chromosome 7, which was further validated by polymerase chain reaction (PCR) amplification and Sanger sequencing (right panel). B Relative expression of circSPON2 was determined in various human CRC cell lines (COLO205, HCT15, LoVo, SW620, HT-29 and HCT116) and normal colon cell line NCM460 by qRT-PCR assays. C Stability of circEZH2 and EZH2 mRNA was assessed by RNase R treatment followed by qRT-PCR in HCT116 and SW620 cells. D qRT-PCR analysis for the circEZH2 and EZH2 mRNA using the template cDNA reverse-transcribed by random primers and oligo dT primers using RNAs from HCT116 and SW620 cells. E qRT-PCR assay for the expression of circEZH2 and EZH2 mRNA in HCT116 and SW620 cells treated with Actinomycin D (2 μg/mL) at the indicated time points. F Fluorescence in situ hybridization (FISH) were performed with Cy3-labeled circEZH2 probes (red) to detect the location of circEZH2 in HCT116 cells. Scale bar = 10 μm. G Relative expression of circEZH2 in CRC tissues and matched adjacent normal tissues was analyzed by qRT-PCR assays (n = 124). The association between circEZH2 expression and tumor size (H), tumor stage (I), lymphatic metastasis (J) and distant metastasis (K). L Kaplan-Meier analysis (log-rank test) of overall survival according to circEZH2 expression level (P < 0.001). Data were showed as mean ± SD. *P < 0.05, **P < 0.01