title: Aryl hydrocarbon receptor-microRNA-212/132 axis in human breast cancer suppresses metastasis by targeting SOX4.
abstract: BACKGROUND
  MicroRNAs (miRNAs) are a class of short non-coding RNAs that pave a new avenue for understanding immune responses and cancer progression. Although the miRNAs are involved in breast cancer development, their axis with the transcription factors that show therapeutic potential in breast cancer is largely unknown. Previous studies showed anti-metastatic roles of agonist-activated aryl hydrocarbon receptor (Ahr) in various breast cancer cell lines. Recently, we demonstrated that agonist-activated Ahr induced a highly conserved miRNA cluster, named miR-212/132, in murine cellular immune compartment. Therefore, current study was performed to examine if this miRNA cluster mediates the anti-metastatic properties of Ahr agonists.
  
  
  METHODS
  The expression of miR-212/132 cluster and coding genes were examined by real-time PCR, and the protein levels were detected by western blot. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3'-diindolylmethane (DIM) were used to activate Ahr in MDA-MB-231 and T47D breast cancer cells. Chromatin immunoprecipitation (ChIP) assay was used to identify the binding site(s) for Ahr on miR-212/132 promoter. For prediction of potentially target gene of the miRNA cluster, bioinformatics analysis was carried out, and to test targeting, luciferase activity was quantified. Besides, biological effects of Ahr-miR-212/132 axis were examined in vitro by cell migration, expansion and invasion, and examined in vivo by orthotopic model of spontaneous metastasis.
  
  
  RESULTS
  The miR-212/132 cluster was transcriptionally activated in MDA-MB-231 and T47D cells by TCDD and DIM, and this activation was regulated by Ahr. A reciprocal correlation was identified between Ahr agonists-induced miR-212/132 and the pro-metastatic SRY-related HMG-box4 (SOX4), and a new specific binding sites for miR-212/132 were identified on the untranslated region (3'UTR) of SOX4. Interestingly, miR-212/132 over-expression showed direct anti-migration, anti-expansion and anti-invasion properties, and an inhibition of the miRNA cluster mitigated the anti-invasive properties of TCDD and DIM. Further in vivo studies demonstrated that the Ahr-miR-212/132-SOX4 module was induced by Ahr activation.
  
  
  CONCLUSIONS
  Taken together, the findings provide the first evidences of the synergistic anti-metastatic properties of miR-212/132 cluster through suppression of SOX4. Also, current study suggest a new miRNA-based mechanism elucidating the anti-metastatic properties of Ahr agonists, suggesting possibility of using miR-212/132 to control metastasis in breast cancer patients.
authors.affiliation: Laboratory of Physiology, Biological Sciences Department, College of Science, King Faisal University, Faisal Bin Fahd road, Hofuf, 31982, Ahsaa, Saudi Arabia. hhanieh@kfu.edu.sa.
authors.forename: Hamza
authors.initials: H
authors.lastname: Hanieh
mesh_terms: D020413:3' Untranslated Regions; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D002465:Cell Movement / Q000235:genetics; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000008:administration & dosage; D051379:Mice; D035683:MicroRNAs / Q000096:biosynthesis* / Q000235:genetics*; D009362:Neoplasm Metastasis; D000072317:Polychlorinated Dibenzodioxins / Q000008:administration & dosage; D018336:Receptors, Aryl Hydrocarbon / Q000096:biosynthesis* / Q000235:genetics; D055751:SOXC Transcription Factors / Q000235:genetics*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C512018:1,1-bis(3'-indolyl)methane; D020413:3' Untranslated Regions; D007211:Indoles; C547077:MIRN132 microRNA, human; C551411:MIRN212 microRNA, human; D035683:MicroRNAs; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; C083431:SOX4 protein, human; D055751:SOXC Transcription Factors