Ezh2 mRNA and protein levels in breast cancer cell lines and miR-214 expression and genomic locus deletion in breast tumors. (A) Schematic of miR-214 site on the Ezh2 3′UTR (top left panel) and its conservation across mammalian species (top right panel), Ezh2 RT–qPCR (lower left panel) and Ezh2, H3K27me3 and total histone H3 immunoblot (lower right panel) of the benign immortalized breast epithelial cell line MCF-10A and the MCF-7 and MDA-MB-231 breast cancer cell lines. (B) miRNA TaqMan RT–PCR of miR-214 in MCF-10A, MCF-7 and MDA-MB-231 breast cell lines. The three cell lines were cultured in the same growth medium. Shown are levels of miR-214 corrected to RNU6B used as an internal control (*P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0002). (C) High resolution comparative genomic hybridization analysis of breast cancers show a non-focal deletion in a region containing miR-214 in a subset of tumors (right panel) but not in the benign samples (left panel). (D) Tumor samples from (C) harboring a deletion in miR-214 show focal deletion for miR-101 in 3/6 tumor samples (left panel) but not in the benign samples (right panel). Horizontal green and blue lines correspond to the ratio profile for copy number changes in each sample and the red and green dots indicate the probes represented on the array platform (Agilent 244K array CGH). (D) Graph represents miR-214 deletion frequency in primary breast tumors and their distribution as a function of the presence or absence of a concomitant miR-101 deletion.

miR-214 overexpression reduces Ezh2 levels by targeting the 3′ UTR of Ezh2 in breast cancer cell lines. (A) Immunoblot of Ezh2 and phosphatase and tensin homolog (left panel) and Ezh2 mRNA by RT–qPCR (right panel in MCF-7 and MDA-MB-231 cells transiently transfected with increasing amounts (1,10 or 100 pmol, respectively) of wild-type or mutant (mut) miR-214 oligomers. (B) Immunoblot of Ezh2 in MCF-7 and MDA-MB-231 cells stably transfected with either a construct encoding the pre-miR-199 or the pre-miR-214. (C) miRNA TaqMan RT–PCR of mature miR-214 (right panel), and RT–qPCR of primary miR-214 and mutant miR-214 (mut) relative to control (left panel), in MCF-7 and MDA-MB-231 cells stably expressing either miR-214, mut or the control plasmid. (*P ≤ 0.05; **P ≤ 0.005; ***P ≤ 0.0002; NS, not significant. (D) Immunoblot (left panel) of Ezh2, phosphatase and tensin homolog, Bmi1, H3K27me3 and total histone H3 and Ezh2 mRNA RT–PCR (right panel) of MCF-7 and MDA-MB-231 cells stably transfected with either a construct encoding the pre-miR-214, the mutant form or an empty control vector. Shown is a representative experiment of three sets of stable transfectants, with β-actin as a control for equal loading of protein cell extract. (E) Luciferase activity of wild-type Ezh2-3′ UTR or the mutant, mut-Ezh2-3′ UTR reporter constructs in MCF-7 or MDA-MB-231 cells stably expressing either the wild-type pre-miR-214, the mutant form, mut or the control vector. The data represent the mean ± SD (**P ≤ 0.005) as determined from three independent experiments performed in triplicate. (F) Luciferase activity of wild-type Ezh2-3′ UTR or the pGl3 control vector in the presence of miR-214 or miR-101 or both oligomers in MCF-7 and MDA-MB-231. The data represent the mean ± SD as determined from two independent experiments done in triplicate.

miR-214 overexpression reduces proliferation of breast cancer cell lines. (A) Cell growth curves of MCF-7 MDA-MB-231 cells stably expressing miR-214, mut-miR-214 (mut) or control vector. The data represent average cell numbers ± SD determined at 24, 48 and 72 h from five experiments done in duplicates (*P ≤ 0.05, **P ≤ 0.005). (B) BrdU staining of MCF-7 (left panel) and MDA-MB-231 (data not shown). The ratio of BrdU/DAPI-stained cells calculated using counts from 10 random fields is presented as an average of three independent experiments ± SD (*P ≤ 0.05, **P ≤ 0.005) expressed as a percentage of the control vector cells (right panel). (C) Immunoblot of cyclin D,E, Ezh2 and β-actin in MCF-7 and MDA-MB-321 breast cancer cells stably expressing miR-214, mut-miR-214 (mut) or control vector. (D) RT–qPCR of Ezh2 downstream targets was performed in MCF-7 and MDA-MB-231 cells stably expressing miR-214, mut-miR-214 (mut) or control vector (*P ≤ 0.05, ***P ≤ 0.0002).

miR-214 expression blocks cell invasion. (A) Photographs of crystal violet-stained MDA-MB-231cells invading a laminin-coated membrane and migrating into the lower chamber containing fetal bovine serum (FBS). Shown is the reduced number of invading cells in MDA-MB-231 cells stably expressing miR-214 relative to mut or the control vector expressing-cells (left panel). Quantitative analysis of MDA-MB-231 invasion activity measured as fluorescence units in response to FBS (right panel). ***P ≤ 0.0002; NS, not significant. (B) RT–qPCR in MCF-7 and MDA-MB-231 cells for ECADH-1 transcript (left panel). Right panel shows ECAD immunoblot of MCF-7 cells-expressing miR-214 or mut. (C) RT–qPCR analyses of endogenous mature miR-214 in MCF-7 cells transfected with anti-miR-214 (left panel right panel) and increased invasion of MCF-7 overexpressing anti-miR-214 oligomer (right panel).