miR-9-5p as a Regulator of the Androgen Receptor Pathway in Breast Cancer Cell Lines

Abstract

Breast cancer (BC) is the most diagnosed carcinoma and the leading cause of cancer death in female over 100 countries. Thanks to the advance in therapeutic strategies, patients' survival has improved. However, the lack of response to treatments and drug resistance are still a main concern, demanding for new therapeutic approaches, in particular for the advanced stages of the disease. Androgen receptor (AR) is gaining increasing interest as a fourth targetable receptor in BC, however, its regulation in BC cells is still poorly understood. MicroRNAs (miRNAs) regulate gene expression post-transcriptionally. Here, we identified miR-9-5p as an inhibitor of AR expression, we validated the inverse correlation between miR-9-5p and AR in primary BC samples and we further identified a feedback loop in which androgen agonists of AR up-regulate miR-9-5p. We also provided evidence that miR-9-5p elicits anti-proliferative effects in BC cell lines regardless of their estrogen receptor status. Finally, we showed that miR-9-5p can revert AR-downstream signaling even in presence of AR-agonists, highlighting the role of this miR in the hormonal response of BC. In conclusion, this study supports the role of miR-9-5p as an anti-proliferative miR in BC and as a central modulator of AR-signaling response to circulating androgens in BC.

Keywords: androgen receptor; breast; cancer; miR-9-5p; microRNAs.

FIGURE 1

MiR-9-5p is downregulated in BC cell lines and down-regulates AR. (A) MiR-9-5p (mir-9) expression was measured by qRT-PCR in three BC cell lines and compared to Total Breast RNA. (B) Predicted miR-9-5p target sites in the AR 3′UTR mRNA, as shown by the Software TargetScan Human Release v6.2. in green, the seed region of miR-9::AR mRNA predicted interaction. (C) mRNA expression level of AR in MDA-MB-453, MCF-7, and T-47D cell lines, 48 h after transfection with pre-miR-9-5p (miR-9), or scrambled (SCR). (D) Protein expression level of AR in MDA-MB-453, MCF-7 and T-47D cell lines, 48 h after transfection with pre-miR-9-5p (miR-9), or scrambled (SCR). (E) AR levels after transfection of MDA-MB-453, MCF-7, and T-47D cell lines with AR siRNA (siAR) compared to scrambled (siSCR). (F) MiR-9-5p (miR-9) expression in MDA-MB-453, MCF-7, and T-47D cell lines after 48 h transfection with AR siRNA (siAR) or scrambled (siSCR). **p < 0.01, ***p < 0.001, n.s., not significant. Multiple t-test, corrected for multiple comparison using the Holm-Sidak method in (A–D).

FIGURE 2

Effects of miR-9-5p (mir-9) on proliferation of BC cell lines (A) MDA-MB-453 (B) MCF-7 (C) T-47D. **p < 0.01, ***p < 0.001, n.s., not significant. Multiple t-test, corrected for multiple comparison using the Holm-Sidak method in (A–C).

FIGURE 3

Cytofluorimetric evaluation of the effects of miR-9-5p (miR-9) on DNA replication at 72 h post-transfection. Cytofluorimetric dot plots of proliferating MDA-MB-453 (A), MCF-7 (B), and T-47D (C) BC cell lines, following BrdU incorporation, and anti-BrdU antibody incubation. The percentage of cells in S phase, which results in BrdU positive cells (BrdU+) was presented in the histograms on the right.

FIGURE 4

Effects of AR silencing (siAR) on proliferation of BC cell lines (A) MDA-MB-453 (B) MCF-7 (C) T-47D. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. Multiple t-test, corrected for multiple comparison using the Holm-Sidak method in (A–C).

FIGURE 5

Effect of DHT and DHEA on proliferation of BC cell lines at the indicated time points (A–C) and on DNA replication at 48 h post-transfection (D–F). **p < 0.01, ***p < 0.001, n.s., not significant. Multiple t-test, corrected for multiple comparison using the Holm-Sidak method in (A–F).

FIGURE 6

Effects of DHT and DHEA on miR-9-5p (miR-9) expression at 24 h post-transfection (A,B). **p < 0.01, ***p < 0.001. Multiple t-test, corrected for multiple comparison using the Holm-Sidak method in (A,B).

FIGURE 7

Effects of miR-9-5p (mir-9) on AR signaling. AR transcriptional activity as measured with the AR Cignal reporter assay at 48 h in MDA-MB-453 (A), MCF7 (B), and T47D (C) cells. The modulation of AR transcription factor was determined by comparing the normalized luciferase activities of the reporter in treated vs. untreated transfectants (SCR vs. miR-9, SCR vs. SCR + DHT, miR-9 vs. miR-9+DHT, and SCR+DHT vs. miR-9+DHT), in all the BC cell lines MDA-MB-453 (A), MCF-7 (B), and T-47D (C). Firefly: Renilla activity ratios were calculated from experimental and control transfections. Then, ratios from AR responsive reporter transfections were divided by ratios from negative controls to obtain relative luciferase units (RLU). *p < 0.05, ** p < 0.01, ***p < 0.001, n.s., not significant. Multiple t-test, corrected for multiple comparison using the Holm-Sidak method in (A–C).

FIGURE 8

MiR-9-5p is downregulated in BC FFPE samples. (A) Box-plot representation of the miR-9-5p (miR-9) qRT-PCR expression in adjacent normal (N) vs. tumoral (T) samples for all FFPE samples examined (n = 11). (B) Box-plot representation of the miR-9-5p (miR-9) and AR qRT-PCR expression in all FFPE samples examined (n = 11). The expression of AR and miR-9 have been normalized to GAPDH and RNU44, respectively. A non-parametric unpaired t-test (Kolmogorov-Smirnov test) method in (A,B). Box-plots have been presented on a ln-scale.