Progestins induce miR-16 downregulation via the classical PR. A, C4HD and T47D cells were treated with 10 nM MPA for the times shown. B, C4HD cells were either pretreated with 10 nM RU486 (left panel) or transfected with 100 nM PR and control (CTRL) siRNAs, and were then stimulated with MPA for 24 hours or left untreated (middle panel). The western blot (WB) in the right panel shows the effect of siRNAs on PR expression in C4HD cells. The experiment shown was performed with PR siRNA #1, but the same results were obtained with PR siRNA #2. C, T47D and T47D-Y cells were treated with MPA for the indicated time or T47D-Y cells were transiently transfected with the PR-B isoform (T47D-Y-PR-B), PR-BmPro mutant (T47D-Y-PR-BmPro cells) or the C587A-PR mutant (T47D-YC587A-PR cells) before MPA stimulation. In A to C, miR-16 expression levels were determined by RT-qPCR. The fold change of miR-16 expression levels upon MPA treatment was calculated by normalizing the absolute levels of miR-16 to those of U6 snRNA, which was used as internal control, and setting the value of untreated cells to 1. D, C4HD cells were treated with 10 nM MPA for 48 hours (left panel) or T47D cells were treated with 10 nM MPA for 24 hours (right panel) and the incorporation of [3H]-thymidine was used as a measure of DNA synthesis. The middle panel shows cell counts for C4HD cells that were treated with 10 nM MPA for 48 hours and then stained with Trypan blue dye. Experiments shown in A to D were repeated in triplicate with similar results. The data shown represent the means of three independent experiments ± SEM (P < 0.001 for b versus a and c versus b). MPA, medroxyprogesterone acetate; PR, progesterone receptor; SEM, standard error of the mean.