Fig. 4

Estrogen promotes the binding of ER-α in the miR-196a promoter region in ER+ BC cells. a GEO dataset GSE25021 was reanalyzed to identify the binding sites and enrichment intensity of ERα in the promoter region of miR-196a, and the call peaks results were displayed by IGV. b The JASPAR program was employed to seek the seed binding sites of ERα in the promoter region of miR-196a. c, d The MCF7 and MDA-MB-231 cells were cultured with estrogen-free medium for 72 h t, then treated with 10 nM E2 or equal amount of solvent ethyl alcohol (Eth) for 24 h. The ChIP assay was performed to determine the binding sites of ERα in the promoter region of miR-196a. The enrichment of miR-196a in cell lysates pulled down by ERα antibody was analyzed by qRT-PCR and normalized to the value of ERα + Eth group. ** indicates significant difference at P < 0.01. e, f The wild type (AATGTCAGGACGGTCTT) or mutant (ACTCTAAGGACCGCCCT) seed binding site of miR-196a promoter region was subcloned into pGL3 luciferase reporter vector and verified by sequencing. The cells were pretreated as above, transfected with the wild type or mutant reporter construct, pRL-TK plasmid, and treated with Eth or E2. After 24 h, the dual-luciferase assay was performed to analyze the luciferase activities of each group. ** indicates significant difference at P < 0.01