Dataset Information

Induction of the BRAFV600E Mutation in Thyroid Cells Leads to Frequent Hypermethylation.

ABSTRACT:

Objectives

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.

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 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.

SUBMITTER: Yi JW

PROVIDER: S-EPMC9441509 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Publications

Induction of the BRAFV600E Mutation in Thyroid Cells Leads to Frequent Hypermethylation.

Yi Jin Wook JW Ha Seong Yun SY Jee Hyeon-Gun HG Kim Kwangsoo K Kim Su-Jin SJ Chai Young Jun YJ Choi June Young JY Lee Kyu Eun KE

Clinical and experimental otorhinolaryngology 20220504 3

<h4>Objectives</h4>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.<h4>Methods</h4>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 methyl ...[more]

PMID: 35538718

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Project description:BACKGROUND:Intranuclear inclusions (NI) in normal and neoplastic tissues have been known for years, representing one of the diagnostic criteria for papillary thyroid carcinoma (PTC). BRAF activation is involved among others in autophagy. NI in hepatocellular carcinoma contain autophagy-associated proteins. Our aim was to clarify if NI in thyroid carcinoma (TC) have a biological function. METHODS:NI in 107 paraffin-embedded specimens of TC including all major subtypes were analyzed. We considered an inclusion as positive if it was delimited by a lamin AC (nuclear membrane marker) stained intact membrane and completely closed. Transmission electron microscopy (TEM), immunohistochemistry (IHC), immunofluorescence (IF) and 3D reconstruction were performed to investigate content and shape of NI; BRAFV600E mutation was analyzed by next generation sequencing. RESULTS:In 29% of the TCs at least one lamin AC positive intranuclear inclusion was detected; most frequently (76%) in PTCs. TEM analyses revealed degenerated organelles and heterolysosomes within such NI; 3D reconstruction of IF stained nuclei confirmed complete closure by the nuclear membrane without any contact to the cytoplasm. NI were positively stained for the autophagy-associated proteins LC3B, ubiquitin, cathepsin D, p62/sequestosome1 and cathepsin B in 14-29% of the cases. Double-IF revealed co-localization of LC3B & ubiquitin, p62 & ubiquitin and LC3B & p62 in the same NI. BRAFV600E mutation, exclusively detected in PTCs, was significantly associated with the number of NI/PTC (p = 0.042) and with immunoreactivity for autophagy-associated proteins in the NI (p≤0.035). BRAF-IHC revealed that some of these BRAF-positive thyrocytes contained mutant BRAF in their NI co-localized with autophagy-associated proteins. CONCLUSIONS:NI are completely delimited by nuclear membrane in TC. The presence of autophagy-associated proteins within the NI together with degenerated organelles and lysosomal proteases suggests their involvement in autophagy and proteolysis. Whether and how BRAFV600E protein is degraded in NI needs further investigation.

Project description:BackgroundConflicting evidence exists on the predictive value of ultrasound characteristics for BRAFV600E gene expression in thyroid cancer. This study aimed to determine the predictive value of ultrasound features for BRAFV600E gene expression status in thyroid cancer.MethodsA systematic review of studies published before December 31, 2023, was conducted in the PubMed, Web of Science, and Cochrane Library databases. Studies evaluating the ultrasonographic features for predicting BRAFV600E gene mutations in thyroid cancer were included. The relevant data were extracted, and the quality of eligible studies was independently assessed by two reviewers. Statistical analysis was performed using RevMan 5.4 and Stata 12.0 software.ResultsThe meta-analysis included 13 studies involving a total of 2,250 thyroid cancer patients. Ultrasound features significantly associated with BRAFV600E gene expression status in thyroid cancer (P<0.05) comprised hypoechogenicity, absence of halo, irregular borders, and vertical orientation. Contrastingly, no significant differences were observed in solid composition, irregular shape, and microcalcifications (P>0.05). Among the seven ultrasound features, the ones with superior combined sensitivity for nodules were hypoechogenicity, solid composition, absence of halo, and irregular borders, with sensitivities of 0.93 [95% confidence interval (CI): 0.87-0.96], 0.93 (95% CI: 0.86-0.97), 0.83 (95% CI: 0.72-0.91), and 0.74 (95% CI: 0.64-0.83), respectively. Finally, the areas under the summary receiver operating characteristic (SROC) curve with the highest diagnostic performance were the absence of halo and hypoechogenicity, with area under the curve (AUC) of 0.84 (95% CI: 0.80-0.87) and 0.81 (95% CI: 0.77-0.84), respectively.ConclusionsThe expression status of the BRAFV600E gene in thyroid cancer correlates with nodules exhibiting hypoechogenicity, absence of halo, irregular borders, and taller-than-wide shape. Notably, the absence of a halo and hypoechogenicity were identified as the most predictive ultrasonic features. However, due to the limited sample size, there may be bias in the meta-analysis results, and more extensive research is necessary.

Dataset Information

Induction of the BRAFV600E Mutation in Thyroid Cells Leads to Frequent Hypermethylation.

Objectives

Methods

Results

Conclusion

Publications

Induction of the BRAFV600E Mutation in Thyroid Cells Leads to Frequent Hypermethylation.

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