A & B, Stably transduced MDA-MB-231 or LM2-4142 cells ectopically expressing miR-145 (145) or vector alone (V) were subject to matrigel chamber assay as detailed in Materials and Methods. Invaded cells on the membrane were counted as follows. Since the distribution of cells on the membrane was not always even, we first took a picture at a low magnification and then enlarged the image in a computer screen with grids so that all of the cells on the entire membrane were counted. B & D, Lung metastasis as revealed by the experimental metastasis animal model. Stably transduced MDA-MB-231 (B) or LM2-4142 (D) cells ectopically expressing miR-145 (145) or vector alone (V) were introduced into female nude mice through tail vein as described in Materials and Methods. Values A and C are means ± SE of three independent experiments. **, p < 0.01; SC, scrambled oliog; anti-145, anti-miR-145.

A, Effect of miR-145 on MUC1-3'-UTR luciferase reporters. Top panels: alignment of MUC1 3'-UTR and miR-145, along with a mutant miR-145 or mutant MUC1-3'-UTR in which the sequences in red were deleted or mutagenized. Bottom panels: a luciferase reporter carrying the 3'-UTR of MUC1 (Luc-MUC1-UTR), deletion of miR-145 binding site at the 3'-UTR (Luc-MUC1-UTR-d) or mutant that the miR-145 binding site was mutated (Luc-MUC1-UTR-mt), was introduced into 293T cells along with miR-145 (145), miR-206 (206), miR-224 (224), mutant miR-145 (145-mt) or vector control (V) and the cells were harvested 24 h later for luciferase assays. miR-206 and miR-224 serve as a negative control. B, miR-145 suppresses, whereas anti-miR-145 enhances the endogenous protein levels of MUC1, as detected by western blot. Stably transduced MDA-MB-231 cells ectopically expressing miR-145 or transiently transfected with anti-miR-145 were used for western blot analysis. Top panels: Effect miR-145 or anti-miR-145 on large (L) and small (S) isoforms of MUC1. Bottom panels: stably transduced MDA-MB-231 cells with miR-224 reveal no suppression of MUC1. Topoisomerase I (topo I) and β-actin serve as loading controls. C, Immunofluorescence staining further confirms suppression of MUC1 by miR-145 in stably transduced MDA-MB-231 cells. The cells were grown on coverslips overnight and then subject to immunostaining with anti-MUC1 antibody, as described in Materials and Methods. All images with red signals (MUC1) were taken at the same fixed time. Merged pictures are overlays of both MUC1 red signals and nuclear staining by Hoechst dye (blue). Values in A are means ± SE of three independent experiments. **, p < 0.01; SC, scrambled oligo; anti-145, anti-miR-145.

A, Left panel: expression vector carrying Myc-tagged MUC1 with or without the 3'-UTR was introduced into 293T cells and western blot was performed 24 h after transfection. The membrane was probed with Myc tag antibody. Right panel: miR-145 suppresses MUC1 with the corresponding 3'-UTR. 293T cells were transfected with UTR (MUC1+UTR) or without UTR (MUC1 no UTR) along with vector alone or miR-145. The cells were harvested for extraction of protein 24 h after transfection. B, Top panels: Effect of miR-145 on morphology changes. Left three pictures: stably transduced MDA-MB-231 cells with vector pCDH, miR-145 (145) or miR-380 (380). We used miR-380 as a negative control here instead of miR-224 because our separate studies suggested that miR-224 is able to suppress invasion by targeting other metastatic genes (Zhu et al unpublished). Right two pictures: MDA-MB-231 cells infected with MUC1 along with vector control (V) or miR-145 (145). Images were taken before invasion assays. Bottom panels: Representative fields of invaded cells on the membranes after invasion assays. C, Quantitative analysis of invasion assays. D. Suppression of MUC1-mediated metastasis by miR-145 in experimental metastasis assays. MDA-MB-231 cells ectopically expressing MUC1 and vector (MUC1+V) or MUC1 and miR-145 (MUC1+145) were injected into female nude mice through tail vein as detailed in Materials and Methods. Values in C are means ± SE of three independent experiments. **, p < 0.01; *, p < 0.05.

A and B, miR-145 suppresses, while anti-miR-145 enhances β-catenin, cyclin D1 and cadherin 11. Stably transduced MDA-MB-231 cells ectopically expressing vector alone (V) or miR-145 (145); and MDA-MB-231 cells transfected with scrambled olig (SC) or anti-miR-145 were used for western blot analysis. C, Suppression of cadherin 11 by miR-145 in MDA-MB-231 cells as detected by immunohistochemistry. Stably transduced MDA-MB-231 cells ectopically expressing vector alone or miR-145 were used for immunohistochemistry analysis as detailed in Materials and Methods.

A, Effect of MUC1 siRNA on MUC1 protein as well as β-catenin, cadherin 11 and cyclin D1. B, Suppression of cell invasion by MUC1 siRNA. MDA-MB-231 cells were transfected with MUC1 siRNA, followed by invasion assay as detailed in Materials and Methods. Values are means ± SE of three separate experiments. **, p < 0.01.

A and B, Western blot reveals upregulation of MUC1 in breast tumors (T) compared to matched normal breast tissue (N). C, A negative correlation between MUC1 and miR-145 in breast tumor specimens after normalization with normal tissue. D, Expression of MUC1 in breast tumor specimens with different metastasis status by immunohistochemistry, a: infiltrating ductal carcinoma without metastasis, and b: infiltrating ductal carcinoma with metastasis.