Cyclin E is a target of miR-16 in breast cancer cells. A, Upper panel, C4HD cells were transfected with pre-miR-16 or pre-miR-control (CTRL) before MPA stimulation. WB was performed with an anti-cyclin D1 antibody, and filters were reprobed with an anti-β-tubulin antibody. Bottom panel, as a control of transfection efficiency, miR-16 levels are shown in pre-miR-16-C4HD and pre-miR-CTRL-C4HD cells. B, C4HD cells were treated with MPA or pretreated with 10 nM RU486 before MPA stimulation, and mRNA expression levels of candidate miR-16 target genes were determined by RT-qPCR. The fold change of mRNA expression levels was calculated by normalizing the absolute levels of the gene-of-interest (GOI) mRNA to GAPDH levels, which were used as an internal control, and setting the value of untreated cells to 1. RAP2C, member of RAS oncogene family; RAF1, v-raf-1 murine leukemia viral oncogene homolog 1; CCNT2, cyclin T2; TCFAP2D, transcription factor AP-2 delta; BCL2L2, BCL2-like 2; CCNE1, cyclin E; TRAF3, TNF receptor-associated factor 3; Akt3, v-akt murine thymoma viral oncogene homolog 3; DMTF1, cyclin D binding myb-like transcription factor 1; WNT3A, wingless-type MMTV integration site family, member 3A; RREB1, ras responsive element binding protein 1; BDNF, brain-derived neurotrophic factor. C, C4HD cells were transfected with PR and CTRL siRNAs and then treated with MPA for 24 hours. Left panel, cyclin E mRNA levels were studied by RT-qPCR and data analysis was performed as described in Figure 2B. Right panel, WB was performed with an anti-cyclin E antibody and filters were reprobed with an anti-β-actin antibody. Longer exposures showing the expression of the low molecular weight (LMW) cyclin E isoforms are shown in the middle panel. The experiment shown was performed with PR siRNA #1, but the same results were obtained with PR siRNA #2. D, C4HD cells were transfected with Stat3 or CTRL siRNAs and were then treated with MPA or remained untreated. WB was performed as described in C. As a control for siRNA efficiency, the membranes were probed with an anti-Stat3 antibody. The experiment shown was performed with Stat3 siRNA #1, but the same results were obtained with PR siRNA #3. E, C4HD cells were transfected with c-Myc or CTRL siRNAs and then treated with MPA. WB was performed as in Figure 4C. The experiment shown was performed with c-Myc siRNA #5, but the same results were obtained with c-Myc siRNA #6. F, C4HD cells were transfected with pre-miR-16 or pre-miR-CTRL before MPA stimulation and WB was performed as in C. G, A scheme depicting the different constructions used is shown in the left panel. C4HD cells were transfected with a construct carrying the CCNE1 3' UTR cloned downstream of the firefly luciferase reporter gene (luc-3'CCNE1), middle panel, or with a construct that carried a minimal region of CCNE1 3'UTR which comprised only one of the miR-16 responding sites either wild type (luc-3' 1×TS) or mutated (luc-3' mTS), right panel. As a control, cells were transfected with a construct that lacks the 3' UTR cloned downstream of the luciferase gene (luc-3'EMPTY). Cells were co-transfected with pre-miR-16 or pre-miR-CTRL (middle panel) or treated with 10 nM MPA for 24 hours (right panel). Firefly luciferase activity was measured as described in the Methods. Renilla luciferase was used for normalization. The experiments shown in A to G 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; WB, western blot.