Figure 3
Progestin induces miR-16 downregulation via c-Myc and Stat3. A, C4HD cells were treated with MPA or transfected with PR siRNAs or CTRL siRNAs before MPA stimulation. Western blot (WB) was performed with anti-c-Myc or anti-PR antibodies and filters were reprobed with an anti-β-actin antibody. The experiment shown was performed with PR siRNA #1, but the same results were obtained with PR siRNA #2. B, C4HD cells were transfected with c-Myc and CTRL siRNAs and were then treated with MPA. miR-16 levels were studied by RT-qPCR, and data analysis was performed as described in Figure 2. The WB in the right side of the figure shows the effects of siRNAs on c-Myc expression in C4HD cells. The experiment shown was performed with c-Myc siRNA #5, but the same results were obtained with c-Myc siRNA #6. C, C4HD cells were transfected with Stat3 siRNAs or CTRL siRNAs and then treated with MPA for 24 hours. miR-16 levels were studied by RT-qPCR, and data analysis was performed as described in Figure 2. The experiment shown was performed with Stat3 siRNA #1, but the same results were obtained with PR siRNA #3. D, C4HD and T47D cells were transfected with Stat3 siRNAs or CTRL siRNAs before MPA stimulation and WBs were performed with anti-c-Myc antibodies. Filters were reprobed with an anti-β-tubulin antibody. The experiment shown was performed with Stat3 siRNA #1, but the same results were obtained with PR siRNA #3. E, Recruitment of c-Myc, and H4 acetylation (AcH4) or trimethylation of lysine 9 histone H3 (H3K9me3) levels at the promoter of the DLEU2 gene was studied by ChIP. Amounts of immunoprecipitated DNA were normalized to inputs and are reported relative to the untreated control group, which was set to 1 (P < 0.001 for b versus a). Experiments shown in A to E were repeated in triplicate with similar results. Data shown represent the means of three independent experiments ± SEM (P < 0.001 for b versus a and c versus b). ChIP, chromatin immunoprecipitation; MPA, medroxyprogesterone acetate; SEM, standard error of the mean.