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EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines
Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influen...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
D.A. Spandidos
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480130/ https://www.ncbi.nlm.nih.gov/pubmed/32952624 http://dx.doi.org/10.3892/etm.2020.9160 |
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author | Liu, Yuting Yu, Jun Xie, Yang Li, Mengying Wang, Feng Zhang, Jing Qi, Jian |
author_facet | Liu, Yuting Yu, Jun Xie, Yang Li, Mengying Wang, Feng Zhang, Jing Qi, Jian |
author_sort | Liu, Yuting |
collection | PubMed |
description | Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/β-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/β-catenin signaling pathway activities. |
format | Online Article Text |
id | pubmed-7480130 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | D.A. Spandidos |
record_format | MEDLINE/PubMed |
spelling | pubmed-74801302020-09-17 EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines Liu, Yuting Yu, Jun Xie, Yang Li, Mengying Wang, Feng Zhang, Jing Qi, Jian Exp Ther Med Articles Abnormal activation of the Wnt signaling pathway is found in 90% of colorectal cancers (CRCs). Secreted frizzled-related protein 4 (sFRP4) serves as an antagonist of the canonical Wnt signaling pathway. Epigenetic alterations, including changes in DNA methylation and histone methylation, may influence the expression of sFRP4. Polycomb group (PcG) proteins are epigenetic transcriptional repressors that selectively repress gene expression by forming polycomb repressive complexes (PRCs). Enhancer of zeste homolog 2 (EZH2), the core component of PRC2, is a histone-lysine N-methyltransferase that interacts with DNA methyltransferases. In the present study, the promoter DNA methylation status of sFRP4 in CRC cell lines was analyzed and the underlying mechanisms of action governing this modification was investigated. Firstly, the DNA methylation status of the sFRP4 promoter in CRC cell lines was assessed using methylation-specific PCR. Subsequently, the mRNA and protein levels of sFRP4 were measured using real-time qPCR and western blot analysis, respectively, to determine whether the DNA methylation status of the sFRP4 promoter is correlated with its transcriptional levels. To screen for important epigenetic modifiers that may regulate the promoter DNA methylation status of sFRP4, the expression levels of PcG proteins were examined by gene array analysis. ChIP-qPCR was performed to test whether the selected PcG proteins directly bind the promoter region of sFRP4. Finally, the downregulated PcG proteins EZH2, chromobox 7 (CBX7) and jumonji and AT-rich interaction domain containing 2 (JARID2) were identified and their association with sFRP4 expression levels and Wnt/β-catenin signaling pathway activity were investigated. The present study revealed that sFRP4 was hypermethylated in the promoter region and downregulated during the progression of the CRC cell lines from Dukes A to Dukes C. Expression levels of PcG proteins EZH2, CBX7 and JARID2 were upregulated and positively associated with the aberrantly activated Wnt signaling pathway in the CRC cell lines. EZH2, CBX7 and JARID2 were all enriched in the sFRP4 promoter region in CRC cells. EZH2 downregulation did not affect the promoter DNA methylation status of sFRP4 but increased its expression levels and decreased CRC cell proliferation. DNA methylation controls the expression of sFRP4. EZH2 regulates sFRP4 expression without affecting the DNA hypermethylation of the sFRP4 promoter and influences CRC cell proliferation and Wnt/β-catenin signaling pathway activities. D.A. Spandidos 2020-11 2020-09-01 /pmc/articles/PMC7480130/ /pubmed/32952624 http://dx.doi.org/10.3892/etm.2020.9160 Text en Copyright: © Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. |
spellingShingle | Articles Liu, Yuting Yu, Jun Xie, Yang Li, Mengying Wang, Feng Zhang, Jing Qi, Jian EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines |
title | EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines |
title_full | EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines |
title_fullStr | EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines |
title_full_unstemmed | EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines |
title_short | EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines |
title_sort | ezh2 regulates sfrp4 expression without affecting the methylation of sfrp4 promoter dna in colorectal cancer cell lines |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480130/ https://www.ncbi.nlm.nih.gov/pubmed/32952624 http://dx.doi.org/10.3892/etm.2020.9160 |
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