Figure 5. CircCCNB1 functions as a miR-449a sponge and suppresses miR-449a activity. (A) FISH was performed to observe for localization of CircCCNB1 (red) and miR-449a (green) in 2BS or IMR-90 cells. Scale bar, 20μm, red arrowhead indicates co-localization. (B) Schematic illustration of the CircCCNB1-wild-type (wt) and CircCCNB1-mutant (mut) dual luciferase reporter vectors. (C) Relative luciferase activities were measured in 293T cells after transfection with CircCCNB1-wt or CircCCNB1-mut and miR-449a mimics or miR-NC, respectively, *P<0.05. (D and E) RNA pull-down in 2BS cells, followed by qRT-PCR to detect the enrichment of CircCCNB1 and miR-449a, **P<0.01. (F) Overexpression of CircCCNB1 by qRT-PCR, **P<0.01. (G) Relative miR-449a expression in CircCCNB1-overexpressed 2BS or IMR-90 cells by qRT-PCR, *P<0.05. (H) Relative miR-449a expression in CircCCNB1 knockdown 2BS or IMR-90 cells by qRT-PCR. *P<0.05.