Yi Jin Wook, Ha Seong Yun, Jee Hyeon-Gun, Kim Kwangsoo, Kim Su-Jin, Chai Young Jun, Choi June Young, Lee Kyu Eun
Department of Surgery, Inha University Hospital, Inha University College of Medicine, Incheon, Korea.
Department of Surgery, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Korea.
Clin Exp Otorhinolaryngol. 2022 Aug;15(3):273-282. doi: 10.21053/ceo.2022.00206. Epub 2022 May 4.
The BRAFV600E 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 BRAFV600E mutation in thyroid cells.
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).
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.
Induction of the BRAFV600E 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 BRAFV600E mutation in thyroid cells modulates DNA methylation and results in cancer-related gene expression.
BRAFV600E突变是甲状腺乳头状癌的主要驱动突变。本研究旨在阐明甲状腺细胞中BRAFV600E突变诱导的DNA甲基化与基因表达变化之间的相关性。
我们使用通过将野生型BRAF基因转染Nthy/ori细胞（Nthy/WT细胞）和V600E突变型BRAF基因（Nthy/V600E细胞）产生的Nthy/BRAF细胞系。我们对Nthy/WT和Nthy/V600E细胞进行了基因表达微阵列和DNA甲基化阵列分析。整合两种类型的阵列数据以识别甲基化与基因表达之间的负相关。使用来自癌症基因组图谱（TCGA）的数据在计算机上验证结果，并通过焦磷酸测序和定量实时聚合酶链反应（qRT-PCR）在体内验证结果。
在Nthy/V600E细胞中，199,821个探针显著高甲基化，697个基因在Nthy/V600E中呈现“高甲基化-下调”模式。其中包括肿瘤抑制基因和凋亡相关基因。总共66,446个探针显著低甲基化，227个基因在Nthy/V600E细胞中呈现“低甲基化-上调”模式。其中包括原癌基因和发育蛋白编码基因。在TCGA分析中，491/697（70.44%）个基因呈现高甲基化-下调模式，153/227（67.40%）个基因呈现低甲基化-上调模式。十个选定基因在焦磷酸测序和qRT-PCR中呈现相似的甲基化-基因表达模式。
甲状腺细胞中BRAFV600E突变的诱导导致频繁的高甲基化。抗癌基因，如参与肿瘤抑制或凋亡的基因，因上游高甲基化而下调，而致癌基因，如原癌基因，则因低甲基化而上调。我们的结果表明，甲状腺细胞中的BRAFV600E突变调节DNA甲基化并导致癌症相关基因表达。
