Fig. 3

hsa_circ_0000231 functions as a sponge for miR-375 in CRC. A The miR-375 binding site on hsa_circ_0000231 predicted by targetScan and miRanda. B FISH assay was performed to observe the cellular location of hsa_circ_0000231 (red) in cells (magnification, × 200, scale bar, 50 μm). C BaseScope assay was performed to observe the cellular location of hsa_circ_0000231 (red) in CRC tissues (magnification, × 100, scale bar, 100 μm). It was found that most of the expression of hsa_circ_0000231 was located in the cytoplasm. D Relative expression of miR-375 in CRC tissues (Tumor) and adjacent non-tumor tissues (Normal) was tested by qRT-PCR (n = 160). The result indicated that the expression of miR-375 was markedly downregulated in CRC tissues compared with adjacent nontumor tissues. E Pearson correlation analysis of hsa_circ_0000231 and miR-375 expression in 160 CRC tissues. The result indicated that the expression of miR-375 negatively correlated with hsa_circ_0000231. F Schematic illustration of hsa_circ_0000231-WT and hsa_circ_0000231-Mut luciferase reporter vectors. G The relative luciferase activities were detected in SW480 cells after transfection with hsa_circ_0000231-WT or hsa_circ_0000231-Mut and miR-375 mimics or miR-NC, respectively. H and I Anti-AGO2 RIP was executed in SW480 cells after transfection with miR-375 mimic or miR-NC, followed by western blot and qRT-PCR to detect AGO2 protein, hsa_circ_0000231 and miR-375, respectively. J and K RNA pull-down was executed in SW480 cells, followed by qRT-PCR to detect the enrichment of hsa_circ_0000231 and miR-375. Data were showed as mean ± SD, *p < 0.05, **p < 0.001