Figure 3. CCAT1 functions as a sponge for miR-218. (A) MiRcode and starBase were used to predict the miRNAs that could bind to CCAT1. Four miRNAs were identified: miR-130, miR-181, miR-216 and miR-218. (B) Relative expression of these four miRNAs in TNBC cells following transfection with si-CCAT1 or si-control. (C) Diagram showing the predicted miR-218 binding site in the CCAT1 sequence and the nucleotides that were mutated to impair binding. (D) Analysis of miR-218 expression in human TNBC and adjacent normal tissue by qRT-PCR. (E) Analysis of the relative expression of miR-218 in three TNBC cell lines (MDA-MB-231, MDA-MB-436, and MDA-MB-468) and in a human normal breast epithelial cell line (MCF-10A) by qRT-PCR. (F) Relative expression of CCAT1 after transfection of TNBC cells with either a miRNA mimic control, miR-218 mimic, inhibitor control, or miR-218 inhibitor. (G, H) Luciferase reporter assays demonstrating that overexpression of miR-218 repressed the luciferase activity in MDA-MB-231 and MDA-MB-468 cells transfected with CCAT1-Wt. *P < 0.05 compared to controls.