title: microRNA-29 negatively regulates EMT regulator N-myc interactor in breast cancer.
abstract: BACKGROUND
  N-Myc Interactor is an inducible protein whose expression is compromised in advanced stage breast cancer. Downregulation of NMI, a gatekeeper of epithelial phenotype, in breast tumors promotes mesenchymal, invasive and metastatic phenotype of the cancer cells. Thus the mechanisms that regulate expression of NMI are of potential interest for understanding the etiology of breast tumor progression and metastasis.
  
  
  METHODS
  Web based prediction algorithms were used to identify miRNAs that potentially target the NMI transcript. Luciferase reporter assays and western blot analysis were used to confirm the ability of miR-29 to target NMI. Quantitive-RT-PCRs were used to examine levels of miR29 and NMI from cell line and patient specimen derived RNA. The functional impact of miR-29 on EMT phenotype was evaluated using transwell migration as well as monitoring 3D matrigel growth morphology. Anti-miRs were used to examine effects of reducing miR-29 levels from cells. Western blots were used to examine changes in GSK3β phosphorylation status. The impact on molecular attributes of EMT was evaluated using immunocytochemistry, qRT-PCRs as well as Western blot analyses.
  
  
  RESULTS
  Invasive, mesenchymal-like breast cancer cell lines showed increased levels of miR-29. Introduction of miR-29 into breast cancer cells (with robust level of NMI) resulted in decreased NMI expression and increased invasion, whereas treatment of cells with high miR-29 and low NMI levels with miR-29 antagonists increased NMI expression and decreased invasion. Assessment of 2D and 3D growth morphologies revealed an EMT promoting effect of miR-29. Analysis of mRNA of NMI and miR-29 from patient derived breast cancer tumors showed a strong, inverse relationship between the expression of NMI and the miR-29. Our studies also revealed that in the absence of NMI, miR-29 expression is upregulated due to unrestricted Wnt/β-catenin signaling resulting from inactivation of GSK3β.
  
  
  CONCLUSIONS
  Aberrant miR-29 expression may account for reduced NMI expression in breast tumors and mesenchymal phenotype of cancer cells that promotes invasive growth. Reduction in NMI levels has a feed-forward impact on miR-29 levels.
authors.forename: Jack W
authors.initials: JW
authors.lastname: Rostas
authors.forename: Hawley C
authors.initials: HC
authors.lastname: Pruitt
authors.forename: Brandon J
authors.initials: BJ
authors.lastname: Metge
authors.forename: Aparna
authors.initials: A
authors.lastname: Mitra
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Bailey
authors.forename: Sejong
authors.initials: S
authors.lastname: Bae
authors.forename: Karan P
authors.initials: KP
authors.lastname: Singh
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Devine
authors.forename: Donna L
authors.initials: DL
authors.lastname: Dyess
authors.forename: William O
authors.initials: WO
authors.lastname: Richards
authors.forename: J Allan
authors.initials: JA
authors.lastname: Tucker
authors.forename: Lalita A
authors.initials: LA
authors.lastname: Shevde
authors.affiliation: Department of Pathology, University of Alabama at Birmingham, WTI-320E, 1824 6th avenue South, Birmingham, AL 35294, USA. rsamant@uab.edu.
authors.forename: Rajeev S
authors.initials: RS
authors.lastname: Samant
mesh_terms: D000465:Algorithms; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D019295:Computational Biology / Q000379:methods; D058750:Epithelial-Mesenchymal Transition; D005260:Female; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D061986:MCF-7 Cells; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D009361:Neoplasm Invasiveness / Q000235:genetics / Q000473:pathology; D015398:Signal Transduction
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C532045:MIRN29a microRNA, human; D035683:MicroRNAs; C099927:NMI protein, human; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3