Epigenome analysis in HPV(-) and HPV(+) squamous cell carcinoma cell lines
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Jung Kim
PROVIDER: E-GEOD-24087 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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2012-04-18 | E-GEOD-29895 | biostudies-arrayexpress
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2018-07-09 | E-MTAB-6430 | biostudies-arrayexpress
Project description:DNA methylation analysis using the Infinium HumanMethylation450 BeadChip platform (Illumina) of 96 Caucasian American, 96 Han Chinese American and 96 African American LCL samples determined differences in terms of differentially methylated sites. Importantly, the observed differences were confirmed in primary blood samples of 10 healthy Caucasian, 10 African American (GSE36064) and 10 Asian individuals. Genes associated to differentially methylated site suggest an influence of DNA methylation on phenotype differences. Interestingly, methylation differences could be partially traced back to genetic polymorphisms. 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 (2010.3) Methylation module (1.8.5) software. Methylation score of each CpG is represented as beta value.
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Project description:We used deep sequencinge technology to profile the transcriptome, gene copy number, and CpG island methylation status simultaneously in eight commonly used breast cell lines to develop a model for how these genomic features are integrated in estrogen receptor positive (ER+) and negative breast cancer. Total mRNA sequence, gene copy number, and genomic CpG island methylation were carried out using the Illumina Genome Analyzer. Sequences were mapped to the human genome to obtain digitized gene expression data, DNA copy number in reference to the non-tumor cell line (MCF10A), and methylation status of 21,570 CpG islands to identify differentially expressed genes that were correlated with methylation or copy number changes. These were evaluated in a dataset from 129 primary breast tumors. Gene expression in cell lines was dominated by ER-associated genes. ER+ and ER- cell lines formed two distinct, stable clusters, and 1,873 genes were differentially expressed in the two groups. Part of chromosome 8 was deleted in all ER- cells and part of chromosome 17 amplified in all ER+ cells. These loci encoded 30 genes that were overexpressed in ER+ cells; 9 of these genes were overexpressed in ER+ tumors. We identified 149 differentially expressed genes that exhibited differential methylation of one or more CpG islands within 5kb of the 5' end of the gene and for which mRNA abundance was inversely correlated with CpG island methylation status. In primary tumors we identified 84 genes that appear to be robust components of the methylation signature that we identified in ER+ cell lines. Our analyses reveal a global pattern of differential CpG island methylation that contributes to the transcriptome landscape of ER+ and ER- breast cancer cells and tumors. The role of gene amplification/deletion appears to more modest, although several potentially significant genes appear to be regulated by copy number aberrations. 8 commonly used breast cancer cell lines were sequenced for mRNA expression, CpG methylation and DNA copy number
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Project description:Bilateral retinoblastoma has served as a critical model for studying genetic predisposition to cancer, however international incidence of unilateral retinoblastoma is variable across populations suggesting the involvement of environmental factors. DNA from Human Papilloma Virus (HPV) has been found in human retinoblastoma tissue through polymerase chain reaction based methods in several independent reports. To search for HPV oncoproteins and characterize transcriptional profiles in retinoblastoma, we analyzed tumor tissue obtained after enucleation and cultured for one week from 13 previously untreated Mexican children with retinoblastoma. We identified the presence of E7 HPV oncoproteins using western blot analysis in HPV positive tumors, and using non-supervised methods of classification on gene expression microarrays data, tumors clustered according to the presence or absence of HPV DNA identified by PCR. These methods were unable to discriminate retinoblastoma tissues by laterality. 2,404 genes were differentially expressed between HPV positive and HPV negative retinoblastoma, including a group of genes involved in a transcriptional response to viral infection. This molecular evidence supports the hypothesis that HPV is involved in the molecular pathogenesis of these tumors. The 13 children and tumors examined here, are incident cases participating in a larger case-control study at two Pediatric Hospitals of third level of medical attention in Mexico City, that form part of the public health system. Nine different tumors positive and four negative to HPV by PCR were included in the experimental design. From these, seven are unilateral and six are bilateral and are considered biological replicas. Two unilateral and two bilateral cases were chosen to make technical replicas. From the surgical specimens, two tumor portions were obtained, one was frozen, DNA was extracted from it, and HPV status was assigned using MY09 and MY11 primers by PCR. Another portion of tumor tissue was used to grow in culture, separating viable cells from non viable cells through a ficoll gradient. In the eye, retinoblastoma usually grows into the vitreous which has no cells, and thus the tissue obtained is predominantly formed by tumor cells and few endothelial cells from capillaries in the tumor. Retinoblastoma grow in culture as non adherent cells, and when adherent cells were also present, cell suspension was moved to a new dish to keep retinoblastoma cells as the main or only cellular type as possible. We used a two channel platform with in house spotted microarrays. Since retinoblastoma has known classes: uni and bilateral, we sought to verify these classes or discover new, we used a class discovery approach and a reference experimental design. As the reference sample we used RNA form exponentially growing Saccharomyces cerevisiae because major signaling pathways and basic cellular processes among Saccharomyces cerevisiae and mammalian cells including cell cycle control and DNA repair mechanisms have a high degree of conservation. Also because yeast is easy to grow, and thus a reliable and robust source of reference RNA.
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