DNA Hypermethylation Suppresses Thyroid Peroxidase Expression and May Be a Promising Diagnostic Marker for Thyroid Cancer.

Abstract

Background: Decreased expression of thyroid peroxidase (TPO) is frequently observed in thyroid cancer, but the underlying regulatory mechanism remains largely unknown. This study aimed to elucidate the epigenetic basis of TPO silencing and assess its potential value for the diagnosis of thyroid cancer.

Methods: DNA methylation and expression levels of TPO were analyzed using the Infinium HumanMethylation450 array and transcriptome data from The Cancer Genome Atlas thyroid cancer dataset and validated in clinical tissue samples by bisulfite sequencing polymerase chain reaction (PCR) and quantitative PCR (qPCR). Thyroid cancer cell lines MDA-T41, KTC-1, CAL62, and TTA1 were treated with the demethylating drug decitabine and subjected to DNA methylation and mRNA expression analyses. Potential regulatory proteins were identified through DNA pull-down coupled with LC-MS/MS and validated by chromatin immunoprecipitation, luciferase reporter assay, and qPCR. A pyrosequencing assay was designed to quantify the methylation level of TPO.

Results: TPO expression was markedly downregulated in thyroid cancer and showed a strong inverse correlation with DNA methylation level at TPO differentially methylated region (DMR). Treatment of thyroid cancer cells with decitabine induced significantly decreased methylation level of TPO DMR and increased expression of TPO. Mechanistic analyses demonstrated that DNA hypermethylation suppressed TPO expression by recruiting methyl-CpG binding domain protein 2 (MBD2). Moreover, a pyrosequencing-based method for quantifying TPO methylation level was developed, and a remarkable difference between malignant and benign thyroid nodules (BTNs) was observed. Two CpG sites within TPO DMR achieved diagnostic performance with areas under the receiver operating characteristic curves of 0.927 (95% confidence interval [CI]: 0.864-0.989) and 0.916 (95% CI: 0.846-0.987), respectively.

Conclusions: DNA hypermethylation suppresses TPO transcription by recruiting MBD2 in thyroid cancer. Quantitative assessment of TPO methylation by pyrosequencing offers a promising molecular diagnostic approach to distinguish malignant cancer from BTN. Further studies are warranted to ascertain the clinical diagnostic utility of these findings.