Fig. 6

CircSLC6A6 is overexpressed in CRC. a The heatmap showed differentially expressed circRNAs form 12 paired fresh frozen CRC tissues in comparation with matched 12 adjacent normal mucosae tissues. b Schematic diagram showed the genomic loci of circSLC6A6. CircSLC6A6 was produced by 3-5 exons of SLC6A6; and the head-to-tail splicing junction of circSLC6A6 was confirmed by Sanger sequencing. c Divergent primer (circSLC6A6, ◀▶) and convergent primer (SLC6A6, ▶◀) were designed. The gel electrophoresis validated the existence of circSLC6A6. Divergent primers amplified circSLC6A6 in cDNA but not gDNA in RKO and HCT8 cells. GAPDH was used as a linear control. d The relative expression of circSLC6A6 and SLC6A6 mRNA in RKO and HCT8 cells were detected by qRT-PCR after the treatment of RNase R. e CircSLC6A6 was mainly located in the cytoplasm and determined by nuclear-cytoplasmic fractionation assay. f Relative expression of circSLC6A6 in CRC and FHC cell line were detected by qRT-PCR. g FISH assays helped to comfirmed that circSLC6A6 was mainly gathered in the cytoplasm of RKO and HCT8 cells. h Kaplan-Meier survival analysis (log-rank test) showed that CRC patients with high (32) circSLC6A6 expression were of lower DFS than that of low (31) expression of circSLC6A6. Using median circSLC6A6 expression as a cutoff value. All data are presented as the mean ± SEM (**P < 0.01, ***P < 0.001)