Unknown,Transcriptomics,Genomics,Proteomics

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A novel epigenetic regulated gene and involved in phenotypic switch classifies primary melanomas depending on the aggressiveness grade (Illumina)

ABSTRACT: Loss of methylation of a GTPase-activating protein in melanoma mediates higher proliferation, lower migration, and aggressiveness of primary melanomas. Sequencing of bisulfite converted DNA and array based analysis of melanoma, breast, and colon primary and metastatic related tumorigenic cell lines.

ORGANISM(S): Homo sapiens

SUBMITTER: Antonio Gómez

PROVIDER: E-GEOD-46419 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:One pending issue in cardiovascular epigenetics is whether cerebrovascular events, a major complication of atherosclerosis, are associated with any specific DNA methylation changes in the carotid plaque. To clarify that topic, we profiled the DNA methylomes of human symptomatic carotid plaques (SCPs) obtained from patients who suffered cerebrovascular events (n=19) and asymptomatic counterparts (ACP; n=19) with a high-density microarray (~485,000 CpG sites, Illumina), and crossed DNA methylation data with RNAseq-based expression data from an independent SCP set (n=8). Few (30) CpGs showed a significant (p<0.05; absolute Delta-Beta>0.20) differential methylation between the two groups. Within SCPs, methylation correlated significantly with post-cerebrovascular event time (PCET; range: 3-45 days; r-value range -0.926 to 0.857; p<0.05) for ~45,000 CpGs, the vast majority of which became hypomethylated with increasing PCET. Hypomethylation was not due to erasure of the gene-body and CG-poor region hypermethylation that accompany the progression of stable lesions, but rather targeted promoters and CpG islands. Noticeably, promoter hypomethylation and increased expression were observed for genes involved in the inhibition of the inflammatory response, defence against oxidative stress and active DNA demethylation. Our data show that only weak changes in the DNA methylome distinguish symptomatic from asymptomatic carotid plaques, but a widespread demethylation resulting in permissive transcriptional marks at atheroprotective gene promoters is established in plaques after a cerebrovascular event, thus mirroring previous observations that ruptured plaques tend to revert to a more stable structure. The identified loci are candidate targets to accelerate the pace of carotid plaque stabilization. DNA was quantified by Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and the integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of 600 ng of each sample was perform according to the manufacturer's recommendation for Illumina Infinium Assay. Effective bisulphite conversion was checked for three controls that were converted simultaneously with the samples. 4 ul of bisulfite converted DNA were used to hybridize on Infinium HumanMethylation 450 BeadChip, following Illumina Infinium HD Methylation protocol. Chip analysis was performed using Illumina HiScan SQ fluorescent scanner. The intensities of the images are extracted using GenomeStudio (2011.2) Methylation module (1.8.5) software. Methylation score of each CpG is represented as beta value.

Project description:Non-small cell lung cancer (NSCLC) is a very common solid tumor where only small advances in the reduction of relapse-free survival and overall survival have been accomplished. The issue is particularly critical for stage I patients where there are not available biomarkers, in the absence of detectable nodal or other metastatic involvement, that might indicate which high-risk patients should receive the beneficially proved adjuvant chemotherapy. We aimed to find DNA methylation markers with prognostic value that could be helpful in this regard and complement the conventional staging. A DNA methylation microarray that analyzes 450,000 CpG sites in the human genome was used to study primary tumoral DNA obtained from a multicenter cohort of 490 patients with NSCLC, corresponding to 339 adenocarcinomas, 133 squamous carcinomas and 18 large cell carcinomas, in addition to 25 normal lung epithelium samples. The obtained prognostic DNA methylation markers were validated by the development of a single methylation-pyrosequencing assay in an independent cohort of 143 patients with stage I NSCLC. The unsupervised clustering of the studied primary NSCLC distinguished two branches with distinct clinical outcomes. Those CpG sites that were the best predictors of recurrence in stage I NSCLC patients were further confirmed in the described independent cohort. Both the global DNA methylation classifier and the highly-ranked aberrantly methylated single genes improved prognostic accuracy beyond standard stagings. DNA was quantified by Quant-iT PicoGreen dsDNA Reagent (Invitrogen) and the integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of 600 ng of each sample was performed according to the manufacturer's recommendations for the Illumina Infinium Assay. Effective bisulfite conversion was checked for three controls that were converted simultaneously with the samples. 4 ul of bisulfite-converted DNA were used to hybridize on an Infinium HumanMethylation 450 BeadChip, following the Illumina Infinium HD Methylation protocol. Chip analysis was performed using the Illumina HiScan SQ fluorescent scanner. The intensities of the images were extracted using GenomeStudio (2011.2) Methylation module (1.8.5) software. The methylation score of each CpG is represented as a beta value. This dataset includes 444 patient samples.

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A novel epigenetic regulated gene and involved in phenotypic switch classifies primary melanomas depending on the aggressiveness grade (Illumina)

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