Fig. 4.

CCND2 is directly targeted by miR-375 and indirectly regulated by hsa_circ_0000231. A Schematic illustration of CCND2 3’UTR-WT and CCND2 3’UTR-Mut luciferase reporter vectors. B Relative expression of CCND2 was detected by qRT-PCR in cells transfected with indicated vectors, miRNAs or inhibitors. C and D Western blot assay was performed to determine the relative expression of CCND2 transfected with indicated vectors, miRNAs or inhibitors. E Schematic illustration of CCND2 3’UTR-WT and CCND2 3’UTR-Mut luciferase reporter vectors. F The relative luciferase activities were detected in SW480 cells after transfected with CCND2 3’UTR-WT or CCND2 3’UTR-Mut and miR-375 mimics or miR-NC, respectively. G The luciferase activity was recovered after transfection with hsa_circ_0000231 in the miR-375 + CCND2 3’UTR-WT group. 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