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Published on: March 13, 2018
Hypermethylation of a New Distal Sodium/Iodide Symporter (NIS) enhancer (NDE) is associated with reduced NIS
Ana Luiza Galrão1, Rosalinda Y Camargo, Celso U Friguglietti
1Thyroid 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.
Context:
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.
Objectives:
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.
Design:
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.
Main Outcome Measures:
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.
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