Fig. 1

circROBO1 is validated and characterized in BC cells. a The cluster heat map showed that circROBO1 was in the upregulated expressed circRNAs in 3 pairs of human BC primary tissues and liver metastatic tissues. The red and green strips indicate upregulated and downregulated circRNAs. b The volcano plot showed that circROBO1 was one of the upregulated expressed circRNAs. c Schematic illustration of circROBO1 formation by the circulation of exon5 to exon8 in ROBO1 genes. The back-spliced junction sequences of circROBO1 were validated by Sanger sequencing. d PCR was conducted to detect the existence of circROBO1 and ROBO1 from gDNA and cDNA in BC cells respectively. Divergent primers amplified circROBO1 in cDNA rather than in gDNA while GAPDH was used as internal reference. e RT-qPCR was performed to determine the abundances of circROBO1 and linear ROBO1 mRNA in BC cells treated with Rnase R. f Oligo dT primers and random hexamers were used for the reverse transcription. The relative RNA levels were normalized by the value of random hexamer primers. g Nuclear-cytoplasmic fraction assay indicated that circROBO1 was localized in the cytoplasm of BC cells mostly. GAPDH was used as a cytoplasmic control and U6 was considered as a nuclear control. h The localization of circROBO1 was observed in BC cells by FISH assays (Scale bar=20μm). I The expression of circROBO1 and ROBO1 of BC cells were quantified by RT-qPCR after treatment for 4h, 8h, 12h, 24h. j RT-qPCR was performed to detect the expression of circROBO1 in different BC cells, normalized by GAPDH. k The expression of circROBO1 in paired BC primary tissues and liver metastatic tissues were analyzed by RT-qPCR and paired Student’s test, normalized by GAPDH (n=20). l Kaplan-Meier survival analysis of BC patients according to the expression level of circROBO1 using log-rank test (n=133). m RNA FISH assays in human BC liver metastatic specimen showing significant circROBO1 overexpression. The data are showed as the mean ± SD, NS (no significance) *P < 0.05 **P < 0.01, ***P < 0.001 and all above experiments have been repeated for three times