title: miR-99a directly targets the mTOR signalling pathway in breast cancer side population cells.
abstract: OBJECTIVE
  miR-99a has been reported to function as a tumour suppressor in breast cancer. However, its role in the regulation of breast cancer stem cell (CSC) phenotype has up to now remained unknown.
  
  
  METHODS
  In this study, we isolated the side population (SP) cells by staining cultured MCF-7 and MDA-MB-231 cells with fluorescent DNA-binding dye Hoechst 33342, then by flow cytometric sorting. Next, we detected expression of miR-99a in the SP cells compared to non-SP cells using real-time PCR, and explored effects of miR-99a on the CSC phenotype of the breast cancer cells, including sphere formation, self-renewal, tumourigenicity and cell migratory capability.
  
  
  RESULTS
  We found that expression of miR-99a was down-regulated in the SP cells compared to non-SP cells. Restoration of expression of miR-99a inhibited cell migration and invasion, reduced sphere formation of breast SP cells in vitro, and suppressed tumour growth in vivo. Finally, bioinformatic prediction suggested the oncogene, mammalian target of rapamycin (mTOR) - a downstream effector of the PI3K/AKT signalling pathway, was a target gene of miR-99a in SP cells. Further, quantitative RT-PCR and western blot assays identified that overexpression of miR-99a suppressed expression of mTOR and its downstream gene, HIF-1α.
  
  
  CONCLUSIONS
  Collectively, these data suggest that miR-99a reversed the breast cancer malignant CSC phenotype, probably by targeting the mTOR signalling pathway.
authors.affiliation: Department of Breast and Thyroid Surgery, Affiliated Hospital of Binzhou Medical University, Binzhou, Shandong, 256603, China.
authors.forename: Z
authors.initials: Z
authors.lastname: Yang
authors.forename: Y
authors.initials: Y
authors.lastname: Han
authors.forename: K
authors.initials: K
authors.lastname: Cheng
authors.forename: G
authors.initials: G
authors.lastname: Zhang
authors.forename: X
authors.initials: X
authors.lastname: Wang
mesh_terms: D000818:Animals; D001483:Base Sequence; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D005260:Female; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D061986:MCF-7 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D035683:MicroRNAs / Q000737:chemistry / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016415:Sequence Alignment; D058985:Side-Population Cells / Q000166:cytology / Q000378:metabolism*; D015398:Signal Transduction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism*; D014183:Transplantation, Heterologous
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D035683:MicroRNAs; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; C497442:HIF1A protein, human; C561679:MIRN99 microRNA, human; C546842:MTOR protein, human