Clin Exp Otorhinolaryngol.  2022 Aug;15(3):273-282. 10.21053/ceo.2022.00206.

Induction of the BRAF^V600E Mutation in Thyroid Cells Leads to Frequent Hypermethylation

• Yi JW
• Ha SY
• Jee HG
• Kim K
• Kim Sj
• Chai YJ
• Choi JY
• Lee KE

• KMID: 2532991
• DOI: http://doi.org/10.21053/ceo.2022.00206

Abstract

Objectives
. The BRAF^V600E mutation is a major driver mutation in papillary thyroid cancer. The aim of this study was to elucidate the correlation between DNA methylation and gene expression changes induced by the BRAF^V600E mutation in thyroid cells.
Methods
. We used Nthy/BRAF cell lines generated by transfection of Nthy/ori cells with the wild-type BRAF gene (Nthy/WT cells) and the V600E mutant-type BRAF gene (Nthy/V600E cells). We performed gene expression microarray and DNA methylation array analyses for Nthy/WT and Nthy/V600E cells. Two types of array data were integrated to identify inverse correlations between methylation and gene expression. The results were verified in silico using data from The Cancer Genome Atlas (TCGA) and in vivo through pyrosequencing and quantitative real-time polymerase chain reaction (qRT-PCR).
Results
. In the Nthy/V600E cells, 199,821 probes were significantly hypermethylated, and 697 genes showed a “hypermethylation-downregulation” pattern in Nthy/V600E. Tumor suppressor genes and apoptosis-related genes were included. In total, 66,446 probes were significantly hypomethylated, and 227 genes showed a “hypomethylation-upregulation” pattern in Nthy/V600E cells. Protooncogenes and developmental protein-coding genes were included. In the TCGA analysis, 491/697 (70.44%) genes showed a hypermethylation-downregulation pattern, and 153/227 (67.40%) genes showed a hypomethylation-upregulation pattern. Ten selected genes showed a similar methylation-gene expression pattern in pyrosequencing and qRT-PCR.
Conclusion
. Induction of the BRAF^V600E mutation in thyroid cells led to frequent hypermethylation. Anticancer genes, such as those involved in tumor suppression or apoptosis, were downregulated by upstream hypermethylation, whereas carcinogenic genes, such as protooncogenes, were upregulated by hypomethylation. Our results suggest that the BRAF^V600E mutation in thyroid cells modulates DNA methylation and results in cancer-related gene expression.

Keyword

Thyroid Neoplasms; Methylation; Gene Expression

Figure

• 

  Fig. 1. Schematic diagram of the correlation analysis between differentially methylated probes (DMPs) in promoters and differentially expressed genes in exons. UTR, untranslated region.

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  Fig. 2. Distribution of differentially methylated probes according to genomic location and CpG site. (A) Number of hypermethylated probes in Nthy/V600E cells; genomic structure. (B) Number of hypermethylated probes in Nthy/V600E cells; geographic regions from the CpG area. (C) Number of hypomethylated probes in Nthy/V600E cells; genomic structure. (D) Number of hypomethylated probes in Nthy/V600E cells; geographic regions from the CpG area. UTR, untranslated region; IGR, intergenic region.

• 

  Fig. 3. Pyrosequencing and quantitative real-time polymerase chain reaction results for selected genes. (A) Methylation status of selected genes that showed hypermethylation and downregulation in Nthy/V600E cells. (B) mRNA expression status of selected genes that showed hypermethylation and downregulation in Nthy/V600E cells. (C) Methylation status of selected genes that showed hypomethylation and upregulation in Nthy/V600E cells. (D) mRNA expression status of selected genes that showed hypomethylation and upregulation in Nthy/V600E cells. Nthy/WT, Nthy/ori cells with the wild-type BRAF gene; Nthy/V600E, V600E mutant-type BRAF gene.

• 

  Fig. 4. Possible carcinogenic mechanisms of the BRAFV600E mutation.

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