Homo sapiens
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PROVIDER: PRJNA102899 | ENA |
REPOSITORIES: ENA
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Project description:DNA hypomethylation is an important epigenetic modification found to occur in many different cancer types, leading to the upregulation of previously silenced genes and loss of genomic stability. We previously demonstrated that hypoxia and hypoglycaemia (ischemia), two common micro-environmental changes in solid tumors, decrease DNA methylation through the downregulation of DNMTs in human colorectal cancer cells. Here, we utilized a genome-wide cross-platform approach to identify genes hypomethylated and upregulated by ischemia. Following exposure to hypoxia or hypoglycaemia, methylated DNA from human colorectal cancer cells (HCT116) was immunoprecipitated and analysed with an Affymetrix promoter array. Additionally, RNA was isolated and analysed in parallel with an Affymetrix expression array. Ingenuity pathway analysis software revealed that a significant proportion of the genes hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. A Matrigel invasion assay revealed that indeed HCT116 cells grown in hypoxic or hypoglycaemic conditions have increased mobility capabilities. Confirmation of upregulated expression of cellular movement genes was performed with qPCR. The correlation between ischemia and metastasis is well established in cancer progression, but the molecular mechanisms responsible for this common observation have not been clearly identified. Our novel results suggest that hypoxia and hypoglycaemia may be driving changes in DNA methylation through downregulation of DNMTs. This is the first report to our knowledge that provides an explanation for the increased metastatic potential seen in ischemic cells; i.e. that ischemia could be driving DNA hypomethylation and increasing expression of cellular movement genes. Genomic DNA from the Human Colorectal cancer Cell line HCT116 cultured normally or in hypoxic or Hypoglycaemic conditions was enriched using Methylated DNA Immunoprecipitation, Amplified using whole genome amplification, and hybridized in triplicate (for each treatment) to Affymetrix Human promoter 1.0R arrays arrays.
2014-07-07 | E-GEOD-58050 | biostudies-arrayexpress
Project description:Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive cancer with a poor prognosis. Using methylated DNA immunoprecipitation (MeDIP)-chip analysis, we found that 161 genes that were specifically hypermethylated in PANC-1 cells. Among them, miR-615-5p was hypermethylated in its putative promoter region, which silenced its expression in PDAC cell lines.
2013-09-01 | GSE44738 | GEO
Project description:Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive cancer with a poor prognosis. Using methylated DNA immunoprecipitation (MeDIP)-chip analysis, we found that 161 genes that were specifically hypermethylated in PANC-1 cells. Among them, miR-615-5p was hypermethylated in its putative promoter region, which silenced its expression in PDAC cell lines. Comparison between PANC-1 cell lines and normal pancreas tissue
2013-09-01 | E-GEOD-44738 | biostudies-arrayexpress
Project description:The DNA methylation program is at the bottom layer of the epigenetic regulatory cascade of vertebrate development. While the methylation at C-5 position of the cytosine (C) residues on the vertebrate genomes is achieved through the catalytic activities of the DNA methyltransferases (DNMTs), the conversion of the methylated cytosine (5mC) could be accomplished by the combined actions of the TET enzyme and DNA repair. Interestingly, it has been found recently that the mouse and human DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox condition-dependent manner. We report here the study of tracking down the fate of the methyl group removed from 5mC on DNA during in vitro demethylation reaction by mouse de novo DNMTs, i.e. DNMT3A and DNMT3B. Remarkably, the methyl group becomes covalently linked to the catalytic cysteines utilized by the two de novo DNMTs in their DNA methylation reactions. Thus, the forward and reverse reactions of DNA methylations by the DNMTs may utilize the same cysteine residue(s) as the active site despite of their distinctive pathways. Secondly, we demonstrate that active DNA demethylation of a heavily methylated GFP reporter plasmid by ectopically expressed DNMT3A or DNMT3B occurs in vivo in transfected human HEK 293 cells in culture. Furthermore, the extent of DNA demethylation by the DNMTs in this cell-based system is affected by Ca2+ homeostasis as well as by mutation of their putative active cysteines. These findings substantiate the roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation in vitro as well as in a cellular context.
2022-02-24 | PXD003401 | Pride
Project description:Identification and characterization of epigenetically silenced genes is very important for cancer research. Particularly, information of hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses, and genes frequently methylated in a tumor-specific manner can be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2’-deoxycytidine were widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting such methylation-mediated silenced genes in cancer and successfully developed a new method termed “methyl-CpG targeted transcriptional activation (MeTA)” by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD) that specifically recognizes and binds to methylated DNAs. Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the present study, we applied this method to three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control). All of these cell lines have already been analyzed their expression profiles by 5-aza-2’-deoxycytidine. We first analyzed the expression of five genes by RT-PCR with Southern hybridization, NEFH, NPTX2, SFRP1, TIMP3, and UCHL1; these genes are known to be methylated in at least any one of these cancer cell lines. Upregulation by “MeTA” was confirmed in all of these genes. Then we searched for upregulated-genes, by two-folds or more, in all the three cancer cell lines after MeTA; nineteen such upregulated genes were identified. Among these, sixteen genes except NEFH, HOXA9, and CLDN5 have not been reported previously using the conventional DNA methylation inhibitors. Methylation status of two genes, SLC32A1 and CSMD2, were further analyzed by methylation-specific PCR and found that SLC32A1 and CSMD2 were methylated in 100% (21/21) and 83% (15/18) pancreatic cancer cell lines analyzed, respectively. Our results suggest that “MeTA” is a highly efficient method to isolate methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer.
2012-12-11 | GSE26215 | GEO
Project description:Identification and characterization of epigenetically silenced genes is very important for cancer research. Particularly, information of hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses, and genes frequently methylated in a tumor-specific manner can be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2M-bM-^@M-^Y-deoxycytidine were widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting such methylation-mediated silenced genes in cancer and successfully developed a new method termed M-bM-^@M-^\methyl-CpG targeted transcriptional activation (MeTA)M-bM-^@M-^] by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD) that specifically recognizes and binds to methylated DNAs. Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the present study, we applied this method to three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control). All of these cell lines have already been analyzed their expression profiles by 5-aza-2M-bM-^@M-^Y-deoxycytidine. We first analyzed the expression of five genes by RT-PCR with Southern hybridization, NEFH, NPTX2, SFRP1, TIMP3, and UCHL1; these genes are known to be methylated in at least any one of these cancer cell lines. Upregulation by M-bM-^@M-^\MeTAM-bM-^@M-^] was confirmed in all of these genes. Then we searched for upregulated-genes, by two-folds or more, in all the three cancer cell lines after MeTA; nineteen such upregulated genes were identified. Among these, sixteen genes except NEFH, HOXA9, and CLDN5 have not been reported previously using the conventional DNA methylation inhibitors. Methylation status of two genes, SLC32A1 and CSMD2, were further analyzed by methylation-specific PCR and found that SLC32A1 and CSMD2 were methylated in 100% (21/21) and 83% (15/18) pancreatic cancer cell lines analyzed, respectively. Our results suggest that M-bM-^@M-^\MeTAM-bM-^@M-^] is a highly efficient method to isolate methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control) were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection.
2012-12-11 | E-GEOD-26215 | biostudies-arrayexpress
Project description:Identification of epigenetically silenced genes in tumor endothelial cells
| PRJNA98197 | ENA
Project description:Global Reactivation of Epigenetically Silenced Genes in Prostate Cancer
| PRJNA120275 | ENA
Project description:The identification of genes transcriptionally silenced by DNA hypermethylation is important in understanding the molecular basis of epigenetically regulated biological processes such as X chromosome inactivation, genomic imprinting, and cancer development. Our previously developed methyl-CpG targeted transcriptional activation (MeTA) method reactivates epigenetically silenced genes by using a methyl-CpG binding domain from MBD2 with a transcriptional activation domain. We applied either MeTA or a conventional DNA demethylating agent, 5-aza-cytidine (Aza-CR), to a human embryonic kidney cell line 293T and analyzed gene expression profiles by microarray; 138 and 202 genes that are upregulated 5-fold or more were identified by MeTA and Aza-CR, respectively. The top ten upregulated genes detected by MeTA were further analyzed. We found associations between expressional restorations by MeTA, methylation status, and NFkB(AD)-MBD fusion protein bindings in CpG islands (CGIs) around the transcription start site of the genes. Importantly, MeTA can upregulate genes meeting the stringent criteria of CGIs defined by Takai and Jones at the promoter region at higher frequency; 109 of 138 (79.0%) genes in MeTA vs. 121 of 202 (59.9%) genes in Aza-CR. Interestingly, only 27 genes were upregulated by both methods; MeTA may identify methylated genes that show low levels of induction by the DNA demethylating agents; demethylating agents may also induce factors that help re-expression of genes that harbor less stringent or no CGIs. These results suggest that microarray coupled with MeTA (MeTA-array) is an efficient alternative way to identify transcriptionally silenced genes by DNA hypermethylation. 293T cells were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection. In contrast, 293T cells were treated with 5-aza-cytidine (Aza-CR, 25 uM) or the same volume of PBS for 96 h and medium replaced every 24 h.
2011-06-03 | E-GEOD-25981 | biostudies-arrayexpress
Project description:Aberrant CpG methylation is a universal trait of cancer cell genomes and can result in epigenetic modulation of gene activity; however, at which stages tumour-specific epigenetic patterns arise is unknown. Here, we analyse the methylome of APCM in mouse intestinal adenoma as a model of intestinal cancer initiation, and inventory a map of over 13,000 adenoma-specific recurrent differentially methylated regions (DMRs). We find that multiple genes coding for Polycomb proteins are upregulated in adenoma, and concomitantly, hypermethylated DMRs form preferentially at Polycomb target sites. We establish that DMRs are absent from proliferating intestinal epithelial cells or intestinal stem cells, and thus arise de novo after loss of APC. Importantly, a core set of DMRs is conserved in human colon cancer, defining a class of early epigenetic alterations that are distinct from known sets of epigenetically silenced tumour suppressors. The data presented suggests a sequence of events that leads to an altered methylome of colon cancer cells, and may allow more specific selection of clinical epigenetic biomarkers. Analysis of the methylome and RNA expression in adenoma of Apc-Min/+ mutant mice and of normal intestine in Apc-Min/+ and Apc-+/+ wild type mice.
2013-04-03 | E-GEOD-38983 | biostudies-arrayexpress