Fig. 1

circEZH2 is identified and characterized in BC. a Left, flowchart showing the screening criteria of upregulated circRNAs enriched in BCLM; right, heatmap revealing that 32 circRNAs was selected according to the step2 criteria and hsa_circ_0008324 was marked by red box. b RT-qPCR was used to testify the expression of four circRNA candidates among BC liver metastases and BC primary tumors. c Top, three exons of circEZH2 were highly conservative in different species according to NCBI; bottom, the consitutions of circEZH2 and Sanger-sequencing analysis identified the back-spliced site of circEZH2. d Divergent and convergent primers were used for amplification of circEZH2 in cDNA and gDNA while linear EZH2 and GAPDH were used as controls through PCR. e Following digestion with Rnase R, RT-qPCR was used to measure the change in circEZH2 and EZH2. f Nuclear-cytoplasmic fraction assays were performed to determine the subcellular expression of circEZH2. g FISH assays were performed to determine that circEZH2 was localized in the cytoplasm (Scale bar 20 μm). h The expression of circEZH2 and linear EZH2 were identified after treated with actinomycin D for 4 h, 8 h, 12 h, 24 h. i The expressions of circEZH2 among different BC cells were identified by RT-qPCR. j In primary BC tissues (n = 20) and liver metastatic tissues (n = 11), RT-qPCR was used to verify the expression of circEZH2. The results were analyzed by unpaired Student’s t test. k To determine the Kaplan-Meier survival of BC patients (n = 115), log-rank tests were used. l In BCLM sample, FISH analysis showed circEZH2 overexpression significantly (magnification, X4 scale bar, 200 μm and X20 scale bar, 50 μm). The data was revealed as the mean ± SD and all experiments were repeated at least three times, ns: no significant; * p < 0.05; ** p < 0.01; *** p < 0.001