title: Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS expression in thyroid tumors.
abstract: BACKGROUND
  In thyroid tumors, reduced radioiodine uptake is associated with worse patient outcome concomitantly with low sodium/iodide symporter (NIS) mRNA expression. Previous studies showed that CpG-island methylation in the NIS proximal promoter does not correlate with mRNA expression.
  
  
  OBJECTIVE
  The aim of the study was to identify new CpG-islands within the NIS 5'region and investigate the involvement of their methylation in NIS expression.
  
  
  METHODS
  DNA was obtained from 30 pairs of thyroid samples: tumor (T) and surrounding nontumor (NT) samples. All T samples had reduced NIS mRNA expression compared to NT samples.
  
  
  METHODS
  Methylation degree was quantified by bisulfite sequencing, and NIS expression by real-time PCR and Western blot. Reporter gene assays were performed to determine CpG-island functionality. Tumor cell cultures were treated with 5-Aza demethylating agent to determine NIS expression, methylation status, and (125)I uptake.
  
  
  RESULTS
  We identified a new CpG-island2 with 14 CpG sites, located -2152/-1887 relative to ATG site. CpG-island2 was hypermethylated in T compared to NT samples, in both benign and malignant tumor groups. There was a significant inverse correlation between NIS mRNA expression and degree of CpG-island2 methylation in NT and T samples. This sequence increased the expression of a reporter gene; thus, it was considered a new enhancer. Cell culture treatments with 5-Aza reduced CpG-island2 methylation levels concomitantly with restoration of NIS mRNA and protein expression and (125)I uptake.
  
  
  CONCLUSIONS
  We identified a new distal enhancer, NIS distal enhancer, that regulates gene expression through DNA methylation. This enhancer is hypermethylated in T compared to NT samples and is associated with decreased NIS expression in tumors. This epigenetic deregulation may be an early event in tumorigenesis.
authors.affiliation: Thyroid Unit (A.L.G., R.Y.C., G.M.-N.), Cellular and Molecular Endocrine Laboratory, LIM-25, University of São Paulo Medical School (FM-USP), 01246-903 São Paulo, Brazil; Head and Neck Surgery of Santa Catarina Hospital (C.U.F.), 01310-000 São Paulo, Brazil; Genetic Bases of Thyroid Tumors Laboratory (L.M., J.M.C.), Division of Genetics, Department of Morphology and Genetics, Federal University of São Paulo (UNIFESP), 04039-032 São Paulo, Brazil; Department of Physiology and Biophysics (C.S.-N.), Institute of Biomedical Sciences, University of São Paulo, 05508-900 São Paulo, Brazil; Center of Biotechnology (M.F.S.), Instituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, 05508-000 São Paulo, Brazil; and Department of Biological Sciences (I.G.S.R.), UNIFESP, 09972-270 São Paulo, Brazil.
authors.forename: Ana Luiza
authors.initials: AL
authors.lastname: Galrão
authors.forename: Rosalinda Y
authors.initials: RY
authors.lastname: Camargo
authors.forename: Celso U
authors.initials: CU
authors.lastname: Friguglietti
authors.forename: Lais
authors.initials: L
authors.lastname: Moraes
authors.forename: Janete Maria
authors.initials: JM
authors.lastname: Cerutti
authors.forename: Caroline
authors.initials: C
authors.lastname: Serrano-Nascimento
authors.forename: Miriam F
authors.initials: MF
authors.lastname: Suzuki
authors.forename: Geraldo
authors.initials: G
authors.lastname: Medeiros-Neto
authors.forename: Ileana G S
authors.initials: IG
authors.lastname: Rubio
mesh_terms: D001374:Azacitidine / Q000494:pharmacology; D018265:Carcinoma, Papillary, Follicular / Q000235:genetics* / Q000473:pathology; D018899:CpG Islands; D019175:DNA Methylation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D057809:HEK293 Cells; D006801:Humans; D027981:Symporters / Q000235:genetics*; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000473:pathology; D000099209:Sodium-Iodide Symporters
chemical_list: D001374:Azacitidine; D027981:Symporters; D000099209:Sodium-Iodide Symporters