Journal
JOURNAL OF CLINICAL ENDOCRINOLOGY & METABOLISM
Volume 99, Issue 6, Pages E944-E952Publisher
ENDOCRINE SOC
DOI: 10.1210/jc.2013-1450
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Funding
- Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Sao Paulo, Brazil [2007/51235-7, 2007/51236-3, 2009/52517-1]
- Instituto da Tiroide, Sao Paulo, Brazil
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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 I-125 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 I-125 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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