title: Induction of cell proliferation and survival genes by estradiol-repressed microRNAs in breast cancer cells.
abstract: BACKGROUND
  In estrogen responsive MCF-7 cells, estradiol (E₂) binding to ERα leads to transcriptional regulation of genes involved in the control of cell proliferation and survival. MicroRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression. The aim of this study was to explore whether miRNAs were involved in hormonally regulated expression of estrogen responsive genes.
  
  
  METHODS
  Western blot and QPCR were used to determine the expression of estrogen responsive genes and miRNAs respectively. Target gene expression regulated by miRNAs was validated by luciferase reporter assays and transfection of miRNA mimics or inhibitors. Cell proliferation was evaluated by MTS assay.
  
  
  RESULTS
  E₂ significantly induced bcl-2, cyclin D1 and survivin expression by suppressing the levels of a panel of miRNAs (miR-16, miR-143, miR-203) in MCF-7 cells. MiRNA transfection and luciferase assay confirmed that bcl-2 was regulated by miR-16 and miR-143, cyclinD1 was modulated by miR-16. Importantly, survivin was found to be targeted by miR-16, miR-143, miR-203. The regulatory effect of E₂ can be either abrogated by anti-estrogen ICI 182, 780 and raloxifene pretreatment, or impaired by ERα siRNA, indicating the regulation is dependent on ERα. In order to investigate the functional significance of these miRNAs in estrogen responsive cells, miRNAs mimics were transfected into MCF-7 cells. It revealed that overexpression of these miRNAs significantly inhibited E₂-induced cell proliferation. Further study of the expression of the miRNAs indicated that miR-16, miR-143 and miR-203 were highly expressed in triple positive breast cancer tissues, suggesting a potential tumor suppressing effect of these miRNAs in ER positive breast cancer.
  
  
  CONCLUSIONS
  These results demonstrate that E₂ induces bcl-2, cyclin D1 and survivin by orchestrating the coordinate downregulation of a panel of miRNAs. In turn, the miRNAs manifest growth suppressive effects and control cell proliferation in response to E₂. This sheds a new insight into the integral post-transcriptional regulation of cell proliferation and survival genes by miRNAs, a potential therapeutic option for breast cancer.
authors.affiliation: Department of Pharmacology, School of Chemical Biology & Pharmaceutical Sciences, Capital Medical University, 100069 Beijing, China. xinfengyu@hotmail.com
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Yu
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zhang
authors.forename: Ishwori B
authors.initials: IB
authors.lastname: Dhakal
authors.forename: Marjorie
authors.initials: M
authors.lastname: Beggs
authors.forename: Susan
authors.initials: S
authors.lastname: Kadlubar
authors.forename: Dali
authors.initials: D
authors.lastname: Luo
mesh_terms: D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D056741:Cyclin D / Q000378:metabolism; D004958:Estradiol / Q000494:pharmacology*; D004967:Estrogens / Q000494:pharmacology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D019254:Genes, bcl-2 / Q000187:drug effects; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D000077022:Survivin
chemical_list: C107489:BIRC5 protein, human; D056741:Cyclin D; D004967:Estrogens; D051034:Inhibitor of Apoptosis Proteins; D035683:MicroRNAs; D000077022:Survivin; D004958:Estradiol