Fig. 3.

Relative levels of hsa-miR-196a2* and hsa-miR-196a2 in MCF-7 cells, time course of the E2 stimulated increase in hsa-miR-196a2* and the requirement for ERα and ERK2, prediction of miR-196a2* target genes, and impact of ERα and ERK2 knockdown on target gene expression. a Relative levels of hsa-miR-196a2* and hsa-miR-196a2 in control MCF-7 cells. b Time course monitoring changes in the level of hsa-miR-196a2* and hsa-miR-196a2 after treatment with 10 nM E2 for the times indicated. c Impact of ERα or ERK2 knockdown on the response of hsa-miR-196a2* to E2 treatment. MCF-7 cells were transfected with siCtrl, siERα, or siERK2 for 48 h and then treated with 0.1 % ethanol (veh) or 10 nM E2 for the indicated times. Total RNA was harvested and the expression level of hsa-miR-196a2* was determined by quantitative RT-PCR. d List of E2 down-regulated genes that are potential targets of hsa-miR-196a2*. e Effect of ERα or ERK2 knockdown on expression of three potential target mRNAs of hsa-miR-196a2*, TP63, SPRY1, and TFAP2. MCF-7 cells were transfected with siCtrl, siERα, or siERK2 for 48 h and then treated with 0.1 % ethanol (veh) or 10 nM E2 for 24 h. f Effect of siERα or siERK2 treatment on the level of TP63 (ΔNp63α) protein, and on ERα and ERK2 and pERK1/2 protein levels monitored by Western blot analysis. Cells were treated as described in (e). β-Actin was used as a loading control