Figure 5

miR-568 targets nuclear factors of activated T cells 5 (NFAT5) to regulate metastasis of breast cancers. (A) Predicted binding sites of miR-568 on the 3′ UTR of NFAT5 mRNA. (B) qRT-PCR assay of breast cancer cell lines. Relative levels compared with GAPDH (for NFAT5) and U6 (for miR-568) were plotted. (C) Cells were transfected with miR-568 mimics, antagomiR-568, or scrambled control, followed by RT-PCR and western blot analysis. (D) Luciferase activity measured 24 h after co-transfection of HEK293 cells with miR-568 mimics and a pGL3 construct containing wild-type or mutant NFAT5 3′UTR regions encompassing the three predicted binding sites of miR-568. Data were normalized to cells co-transfected with scrambled miRNA and pGL3 vector. (E) qRT-PCR assays of MDA-MB-231 cells after transfection with indicated siRNAs. Relative levels compared with untransfected cells were plotted. (F) Transwell assays of cells transfected with the mimics or inhibitor of miR-568 or respective control. (G) Western blotting using the lysates of MDA-MB-231 cells transfected with control or miR-568 mimics. (H) H&E staining of primary tumors and the lung (left), and recorded incidence of lung metastases (right) by xenograft tumors derived from MDA-MB-231 cells modified with control (vector) or pre-miR-568-expressing recombinant lentiviruses. Arrows show the tumor boundary with black arrows indicating an invasive interface between the tumor and normal tissues. (I) qRT-PCR assay of clinically normal mammary tissues and breast cancer samples in Figure 1F. miR-568 levels normalized to U6 are plotted. All data are representative photographs or represent the mean ± SD of three replicates. *P <0.05.