Figure 6. MiR-218 directly targets ZFX in TNBC cells. (A) Diagram showing the predicted miR-218 binding site in the ZFX sequence and the nucleotides that were mutated to impair binding. (B) Analysis of relative ZFX mRNA expression in human TNBC and adjacent control tissue by qRT-PCR. (C) Quantitative RT-PCR analysis of the relative expression of ZFX in three TNBC cell lines (MDA-MB-231, MDA-MB-436, and MDA-MB-468) and in a normal human breast epithelial cell line (MCF-10A). (D, E) Western blot analysis of ZFX protein expression in TNBC cells following transfection with either a miR-218 mimic or miRNA mimic control. (F) Relative ZFX mRNA expression following transfection of TNBC cells with either a miR-218 mimic or miRNA mimic control. (G, H) Luciferase reporter assays demonstrating that overexpression of miR-218 suppresses luciferase activity in MDA-MB-231 and MDA-MB-468 cells transfected with ZFX-Wt. *P < 0.05 compared to controls.