Fig. 3

miR-196a expression is induced by E2 and miR-196a is a gene signature associated to ER-α. a, b ER+ BC cells MCF7 and ER- BC cells MDA-MB-231 were cultured with estrogen-free medium for 72 h, then treated with 10 nM E2 or equal amount of solvent Eth for 0, 3, 6, 12 or 24 h. The expression levels of miR-196a were analyzed by qRT-PCR and normalized to the values of the Eth control. U6 levels were used as an internal control. Data were presented as means ± SD from three independent experiments with triple replicates per experiment. * and ** indicate significant difference compared to the 0 h group at P < 0.05 and P < 0.01, respectively. c, d Cells were treated as above, then transfected with 50 nM ERα siRNAs, ERβ siRNAs or negative control siRNAs (siNC). After 24 h, the cells were treated with 10 nM E2 or Eth, the expression levels of miR-196a were analyzed by qRT-PCR and U6 levels were used an internal control, and normalized to the value of siNC+Eth group. ** indicates significant difference between the siNC+Eth group and the siERα+Eth group at P < 0.01. ## indicates significant difference between the siNC+E2 and the siERα+E2 group and at P < 0.01. e, f GSEA program were employed to analyze the ERα signal enrichment scores between miR-196a high and low expression group using the TCGA BRCA dataset. g Primary human breast cancers of TCGA BRCA dataset were classified to high or low ERα signature score group, and the expression levels of miR-196a were analyzed in the two groups. The details were described in methods and materials. ** indicates significant difference between ERα signature high score and low score group. h Pearson’s correlation analysis was used to determine the correlation between the expression levels of ERα (ESR1 signature score) and miR-196a expression levels