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EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines.

ABSTRACT: 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/?-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/?-catenin signaling pathway activities.

SUBMITTER: Liu Y

PROVIDER: S-EPMC7480130 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines.

Liu Yuting Y Yu Jun J Xie Yang Y Li Mengying M Wang Feng F Zhang Jing J Qi Jian J

Experimental and therapeutic medicine 20200901 5

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 o ...[more]

PMID: 32952624

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Project description:Secreted frizzled-related protein 1 (SFRP1), which is an extracellular inhibitor involved in Wnt signalling, is downregulated by promoter hypermethylation in the early stages of colorectal tumorigenesis. Polycomb (PCG) and methyl-CpG-binding domain (MBD) proteins that serve a role in epigenetic gene regulation. The aim of the present study was to determine the role of PCG and MBD proteins in the regulation of SFRP1 gene expression in colorectal cancer (CRC), specifically in CRC cell lines and the human embryo intestinal mucosa cell line CCC-HIE-2. The methylation status of the SFRP1 gene promoter were analysed using methylation-specific PCR (MSP), whereas SFRP1 mRNA expression was analysed using reverse transcription-quantitative PCR. The association between PCG and MBD proteins and the SFRP1 gene was assessed, where associated proteins were screened by chromatin immunoprecipitation and their expression were subsequently knocked down using RNA interference to determine their role in the regulation of SFRP1 gene expression. The SFRP1 promoter was demonstrated to be hypermethylated in CRC cell lines and partially methylated in the non-cancerous cell line CCC-HIE-2. SFRP1 mRNA expression was significantly lower in CRC cell lines compared with that of CCC-HIE-2 cells. The expression of PCGs enhancer of zeste homolog 2 (EZH2) and BMI1, along with MBD2, was indicated to be upregulated with SFRP1 methylation in HCT116 and SW480 cells. The SFRP1 promoter region was enriched with EZH2 in CCC-HIE-2 cells and enriched with EZH2 and MBD2 in SW480 cells, whereas none of the proteins examined were indicated on the SFRP1 promoter in HCT116 cells. The expression of SFRP1 was reactivated by MBD2 small interfering (si)RNA but not by EZH2 siRNA in SW480 cells, but combined MBD2 and EZH2 knockdown effectively restored SFRP1 gene expression without affecting the methylation status of the SFRP1 promoter. In conclusion, data from the present study revealed that MBD2 and EZH2 regulated SFRP1 expression without affecting the hypermethylation of SFRP1 in CRC cell lines. Instead, the regulation of SFRP1 expression may be through a distinct mechanism, which warrants further investigation.

Project description:The polycomb repressor complex 2 molecule EZH2 is now known to play a role in essential cellular processes, namely, cell fate decisions, cell cycle regulation, senescence, cell differentiation, and cancer development/progression. EZH2 inhibitors have recently been developed; however, their effectiveness and underlying molecular mechanisms in many malignancies have not yet been elucidated in detail. Although the functional role of EZH2 in tumorigenesis in neuroblastoma (NB) has been investigated, mutations of EZH2 have not been reported. A Kaplan-Meier analysis on the event free survival and overall survival of NB patients indicated that the high expression of EZH2 correlated with an unfavorable prognosis. In order to elucidate the functional roles of EZH2 in NB tumorigenesis and its aggressiveness, we knocked down EZH2 in NB cell lines using lentivirus systems. The knockdown of EZH2 significantly induced NB cell differentiation, e.g., neurite extension, and the neuronal differentiation markers, NF68 and GAP43. EZH2 inhibitors also induced NB cell differentiation. We performed a comprehensive transcriptome analysis using Human Gene Expression Microarrays and found that NTRK1 (TrkA) is one of the EZH2-related suppression targets. The depletion of NTRK1 canceled EZH2 knockdown-induced NB cell differentiation. Our integrative methylome, transcriptome, and chromatin immunoprecipitation assays using NB cell lines and clinical samples clarified that the NTRK1 P1 and P2 promoter regions were regulated differently by DNA methylation and EZH2-related histone modifications. The NTRK1 transcript variants 1/2, which were regulated by EZH2-related H3K27me3 modifications at the P1 promoter region, were strongly expressed in favorable, but not unfavorable NB. The depletion and inhibition of EZH2 successfully induced NTRK1 transcripts and functional proteins. Collectively, these results indicate that EZH2 plays important roles in preventing the differentiation of NB cells and also that EZH2-related NTRK1 transcriptional regulation may be the key pathway for NB cell differentiation.

Project description:BackgroundEmerging evidence has identified miR-138 as a tumor suppressor that can suppress the proliferation of various cancers. Meanwhile, the cause of abnormal miR-138 expression in cervical cancer remains uncertain. This study clarified the mechanism by which miR-138 regulates proliferation, invasion, metastasis, and EMT in cervical cancer cells.ResultsmiR-138 expression in human cervical cancer and adjacent normal tissue was measured using qPCR. SiHa and C33A cells were used to determine the function of miR-138 via miR-138 mimic or inhibitor transfection, followed by wound healing, Cell Counting Kit-8, flow cytometry, and Transwell assays. Epithelial and mesenchymal marker expression was analyzed using Western blotting. DNA methylation in the miR-138 promoter was examined using bisulfite sequencing PCR. The downstream target genes of miR-138 were identified via bioinformatics analysis and luciferase reporter assays. A tumor xenograft model was employed to validate DNA methylation-induced miR-138 downregulation and tumor growth inhibition in cervical cancer in vivo. miR-138 levels were significantly lower in cervical cancer tissues than in adjacent control tissues. Furthermore, lower miR-138 expression and higher CpG methylation in the miR-138 promoter were identified in lymph node-positive metastatic cervical cancer tumors versus that in non-metastatic tumor tissues. Upon miR-138 overexpression, cell proliferation, metastasis, invasion, and EMT were suppressed. miR-138 agomir transfection and demethylating drug treatment significantly inhibited cervical tumor growth and EMT in tumor xenograft models. DNA methylation inhibited miR-138 transcription, and enhancer of zeste homolog 2 (EZH2) downregulation mediated the tumor suppressor function of miR-138 in cervical cancer.ConclusionWe demonstrated that miR-138 suppresses tumor progression by targeting EZH2 in cervical cancer and uncovered the role of DNA methylation in the miR-138 promoter in its downregulation. These findings demonstrated the potential of miR-138 to predict disease metastasis and/or function as a therapeutic target in cervical cancer.

Dataset Information

EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines.

Publications

EZH2 regulates sFRP4 expression without affecting the methylation of sFRP4 promoter DNA in colorectal cancer cell lines.

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