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Figure 5 | Breast Cancer Research

Figure 5

From: Downregulation of the tumor-suppressor miR-16 via progestin-mediated oncogenic signaling contributes to breast cancer development

Figure 5

miR-16 is a tumor suppressor in progestin-induced breast cancer growth in vitro and in vivo. A, miR-16 inhibits in vitro progestin-induced breast cancer growth. C4HD or T47D cells were transfected with pre-miR-CTRL or pre-miR-16. After 48 hours of transfection, cells were treated with 10 nM MPA or left untreated, and proliferation was measured by [3H]-thymidine incorporation as described in Figure 2D. B, C4HD cells were transfected with pre-miR-CTRL or pre-miR-16. After 48 hours, cells were treated with 10 nM MPA for 48 or 120 hours or left untreated, and proliferation was measured by cell count as described in Figure 2D. The experiments shown in A and B were repeated four times with similar results. The data shown represent the means of the data from three independent experiments ± SEM (P < 0.001 for b versus a and c versus b). C, miR-16 inhibits in vivo progestin-induced breast cancer growth. C4HD cells were transfected with pre-miR-CTRL or pre-miR-16 for 48 hours and then injected subcutaneously (s.c.) into BALB/c mice at 2 × 106 cells/mouse. Mice were simultaneously injected with a 40 mg MPA depot. Tumor volume was calculated as described in the Methods. Each point represents the mean volume ± SEM of six independent tumors for both experimental groups. The experiment shown in C was repeated twice with similar results. *P < 0.01 or **P < 0.001 versus control. Inset, levels of pre-miR-16 in pre-miR-CTRL-C4HD and pre-miR16-C4HD tumors were studied by RT-qPCR at day 14; data analysis was performed as described in Figure 2. D, Cyclin E is an in vivo target of miR-16. Immunohistochemistry (IHC) for cyclin E in pre-miR-CTRL-C4HD and pre-miR-16-C4HD tumors (400×). Representative images are shown. As control, IHC was performed using an irrelevant rabbit antibody. Scale bar, 50 μM. Inset, average H-score, used to quantify the levels of cyclin E in pre-miR-CTRL-C4HD and pre-miR-16-C4HD tumors. E, BT-474.m1 cells were injected s.c. into nude mice at 20 × 106 cells/mouse. Mice were simultaneously injected with a 0.72 mg E2 depot. Seven days after cell injection, half of the mice were injected with a 40 mg MPA depot (arrow). Tumor volume was calculated as described in Methods. Each point represents the mean volume ± SEM of six independent tumors for both experimental groups. *P < 0.01 or **P < 0.001 versus control. F, c-Myc WB was performed in whole protein extracts from BT-474 tumors growing into mice treated or not with MPA (upper panel). WB from two representative animals from each group is shown. miR-16 levels were measured in RNA from BT-474 tumors from mice treated or not with MPA (lower-left panel). Quantification of cyclin E from WB performed on whole protein extracts from BT-474 tumors from mice treated or not with MPA (lower-right panel). MPA, medroxyprogesterone acetate; SEM, standard error of the mean; WB, western blot.

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