INTRODUCTION: Experimental and clinical evidence points to a critical role of 
progesterone and the nuclear progesterone receptor (PR) in controlling mammary 
gland tumorigenesis. However, the molecular mechanisms of progesterone action in 
breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are 
short ribonucleic acids which have also been found to play a pivotal role in 
cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is 
poorly explored. In this study we explored progestin modulation of miRNA 
expression in mammary tumorigenesis.
METHODS: We performed a genome-wide study to explore progestin-mediated 
regulation of miRNA expression in breast cancer. miR-16 expression was studied 
by RT-qPCR in cancer cell lines with silenced PR, signal transducer and 
activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. 
Breast cancer cells were transfected with the precursor of miR-16 and 
proliferation assays, Western blots or in vivo experiments were performed. 
Target genes of miR-16 were searched through a bioinformatical approach, and the 
study was focused on cyclin E. Reporter gene assays were performed to confirm 
that cyclin E 3'UTR is a direct target of miR-16.
RESULTS: We found that nine miRNAs were upregulated and seven were downregulated 
by progestin in mammary tumor cells. miR-16, whose function as a tumor 
suppressor in leukemia has already been shown, was identified as one of the 
downregulated miRNAs in murine and human breast cancer cells. Progestin induced 
a decrease in miR-16 levels via the classical PR and through a hierarchical 
interplay between Stat3 and the oncogenic transcription factor c-Myc. A search 
for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator 
cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR 
region. We found that, similar to the molecular mechanism underlying 
progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the 
induction of cyclin E expression by progestin. Moreover, overexpression of 
miR-16 abrogated the ability of progestin to induce cyclin E upregulation, 
revealing that cyclin E is a novel target of miR-16 in breast cancer. 
Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in 
vitro and in vivo, demonstrating for the first time, a role for miR-16 as a 
tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand 
heregulin (HRG) downregulated the expression of miR-16, which then participates 
in the proliferative activity of HRG in breast tumor cells.
CONCLUSIONS: In this study, we reveal the first progestin-regulated miRNA 
expression profile and identify a novel role for miR-16 as a tumor suppressor in 
progestin- and growth factor-induced growth in breast cancer.