ERα regulates miR-191/425 cluster.

<p>(A) Schematic representation of E2 modulated miRNA genes (p-value<0.05 and fold change >1.2) after 6 days of hormone starvation and 6 h of E2 treatment. Blue (down) and yellow (up) columns represent all miRNA genes regulated after hormone starvation. (B) Mature miR-191, miR-425 and DALRD3 mRNA levels were determined by qRT-PCR (* p-value<0.05) after E2 treatment (10 nM). (C) Schematic representation of miR-191/425-DALRD3 transcription unit. Green, red and gray boxes depict CpG islands, miR-191/miR-425, and DALRD3 exons, respectively. Prom1 and prom2 regions are represented as red and orange squares, respectively. The red lines across miR-191/425 locus define the location of the amplicons that were used for the chromatin immunoprecipitation experiments. For ChIP experiment, MCF7 cells were hormone starved for 6 days and then treated with E2 (10 nM) for 3 h, 6 h and 24 h. The amount of ChIP-enriched DNA was quantified by quantitative PCR (qPCR), and the results are shown as relative enrichment to the input. (D) Luciferase assays were performed on prom1 and prom2 in HEK293 cells 24 h after transfection (* p-value<0.001). (E) Luciferase assays for prom1 and prom2 after estrogen stimulation. MCF7 cells were hormone starved for 4 days and then transfected with prom1 and prom2 plasmids. 24 hr post-transfection, cells were treated with E2 (10 nM) and luciferase activity assessed at the reported time points (* p-value<0.05). Error bars indicate s.d. and p-values were obtained with two-sided Student's t-test.