miR-191/425 cluster effects in ERα-positive breast cancer cells.

<p>(A) MTT assay in MCF7 cells transfected with anti-miR-191/425 and scrambled (CTR) control oligonucleotide in normal culture condition (+E2) and MCF7 cells transfected with miR-191/425 and scrambled (CTR) control in hormone starvation for 7days (−E2) (* and ** indicate p-value<0.05 and 0.01, respectively). (B) Cell cycle analyses of MCF7 cells transfected with anti miR-191/425 and scrambled control (CTR) oligonucleotide in normal culture condition. Cells were harvested 72 h following transfection, fixed, stained with propidium iodide, and analyzed by flow cytometry; the data are representative of three independent experiments. (C) Cell cycle analyses of MCF7 cells transfected with miR-191/425 and scrambled control (CTR) oligonucleotide. Briefly, MCF7 cells were hormone deprived for 3 days and then transfected with miRs and control oligonucleotide. Cells were harvested 48 h following transfection, fixed, stained with propidium iodide, and analyzed by flow cytometry; the data are representative of three independent experiments. (D) <i>In vivo</i> growth kinetic of MCF7 cells transfected with anti-miR-191/425 and scrambled control oligonucleotide. Briefly, MCF7 were transfected in 10 cm plates by using 2-O-methyl anti miR-191 and miR-425 oligonucleotides (100 nM); 48 h after transfection, cells were detached and injected in nude mice previously implanted (two weeks before injection) with estradiol pellets. p-value was calculated on one experiment performed with 6 mice for each group. (E) Levels of EGR1 and p21 were measured by western blot analyses 72 h after transfection of anti-miR-191, anti-miR-425 and scrambled oligonucleotide control in MCF7 cells. Densitometric values are reported in red below each band and represent the average of three independent experiments. GAPDH was used as a loading control. (F) Schematic representation of miR191 binding site located in the 3′UTR (in green) of the target gene EGR1. The blue line defines the 3′UTR fragment cloned into pGL3 control plasmid and the arrow head indicates the position of the miRNA binding site, whose sequence is reported below with the mutagenesis strategy used to generate the disruption between miRNA and mRNA of the target gene. Luciferase assays for wildtype and disrupted miRNA binding sites: (top panel) reduced luciferase activity after miR-191 overexpression; (lower panel) increased luciferase activity after miR191 enforced expression on mutated plasmid. In the lower panel, luciferase activity values for wildtype and mutated plasmids are represented as a ratio between the relative luciferase activity of miR-transfected cells with the relative luciferase activity of control-transfected cells. * represent p-value<0.01. (G) Western blot for EGR1 protein after estrogen treatment of miR-191 knocked-down MCF7 cells. Briefly, MCF7 cells were hormone deprived for 2 days and then transfected by using 2-O-methyl anti miR-191 (100 nM); 48 h after transfection cell were stimulated with E2 (10 nM) and collected at the reported time points. Densitometric values are reported in red below each band and represent the average of three independent experiments. GAPDH was used as a loading control. (H) qRT-PCR for EGR1 and p21 mRNAs after miR-191 inhibition in E2 stimulated MCF7 cells. Gene expression levels are reported as relative expression to GAPDH levels. Error bars indicate s.d. *, ** represent p-value of 0.05 and 0.01, respectively.