Project description:Epigenetic changes largely contribute to the regulation of gene expression in cancer cells. DNA methylation is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stage I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis using NimbleGenM-BM-4s 385K Human CpG Island plus Promoter arrays. By testing for differences in methylation between tumors and corresponding non-malignant lung tissues, we identified 298 tumor-specifically methylated genes. From many of these genes epigenetic regulation was unknown so far. Gene Ontology analysis revealed an over-representation of genes involved in regulation of gene expression and cell adhesion. Expression of 182 of 298 genes was found to be upregulated after 5-aza-2M-BM-4-deoxycytidine (Aza-dC) and/or trichostatin A (TSA) treatment of 3 NSCLC cell lines by Affymetrix microarray analysis. In addition, methylation of selected genes in primary NSCLCs and corresponding non-malignant lung tissue samples were analyzed by methylation-sensitive high resolution melting analysis (MS-HRM). Our results obtained by MS-HRM analysis confirmed our data obtained by MeDIP-chip analysis. Moreover, by comparing methylation results from MeDIP-chip analysis with clinico-pathological parameters of the patients we observed methylation of HOXA2 as potential parameter for shorter disease-free survival of NSCLC patients. In conclusion, using a genome-wide approach we identified a large number of tumor-specifically methylated genes in NSCLC patients. Our results stress the importance of DNA methylation for the pathogenesis of NSCLCs. Overall, samples of 3 untreated, with Aza-dC treated and with Aza-dC/TSA treated NSCLC cell lines were hybridized to Affymetrix HG-U133_plus_2.0 microarrays (18 in total).
Project description:Epigenetic changes largely contribute to the regulation of gene expression in cancer cells. DNA methylation is part of the epigenetic gene regulation complex which is relevant for the pathogenesis of cancer. We performed a genome-wide search for methylated CpG islands in tumors and corresponding non-malignant lung tissue samples of 101 stage I-III non-small cell lung cancer (NSCLC) patients by combining methylated DNA immunoprecipitation and microarray analysis using NimbleGen´s 385K Human CpG Island plus Promoter arrays. By testing for differences in methylation between tumors and corresponding non-malignant lung tissues, we identified 298 tumor-specifically methylated genes. From many of these genes epigenetic regulation was unknown so far. Gene Ontology analysis revealed an over-representation of genes involved in regulation of gene expression and cell adhesion. Expression of 182 of 298 genes was found to be upregulated after 5-aza-2´-deoxycytidine (Aza-dC) and/or trichostatin A (TSA) treatment of 3 NSCLC cell lines by Affymetrix microarray analysis. In addition, methylation of selected genes in primary NSCLCs and corresponding non-malignant lung tissue samples were analyzed by methylation-sensitive high resolution melting analysis (MS-HRM). Our results obtained by MS-HRM analysis confirmed our data obtained by MeDIP-chip analysis. Moreover, by comparing methylation results from MeDIP-chip analysis with clinico-pathological parameters of the patients we observed methylation of HOXA2 as potential parameter for shorter disease-free survival of NSCLC patients. In conclusion, using a genome-wide approach we identified a large number of tumor-specifically methylated genes in NSCLC patients. Our results stress the importance of DNA methylation for the pathogenesis of NSCLCs.
Project description:We used Illumina Infinium 27k Human DNA Methylation BeadChip v1.2 to assess genome-wide DNA methylation profiling of normal colon epithelial, adenomas and colorectal adenocarcinomas across approximately 27,000 CpGs in fresh frozen colorectal tissue samples. We found that cancer-associated methylation changes with impact on transcription occur nearly as frequent at non-CpG island as CpG island promoters in colorectal cancer (CRC).
Project description:Methylated genomic fragments from tumor and matching normal DNA were enriched with the methylated-CpG island recovery assay (MIRA); amplified, labeled and hybridized on Agilent CpG island tiling oligo arrays. Samples from tumor were labeled with Cy5 while the matching normal were labeled with Cy3 so that methylation differences can be detected. Keywords: Cancer Comparison of methylation patterns between tumor and matching normal; five patients with stage I lung squamous cell carcinoma
Project description:Genome-wide DNA methylation of colorectal cancer patients with lymph node metastases showed global loss of DNA methylation in CG-poor, non-CpG island (CGI) regions. Overall CGI methylation was increased in tumour samples. Differential methylation analysis of CGIs identified 60 putative biomarkers, with >20% increase in DNA methylation in both primary tumour and metastasis samples compared to normal adjacent tissue.
Project description:Genome-wide DNA-methylation profiles of human lung cancer cell lines and normal lung cells were generated by Infinium bead chip technology DNA methylation patterns of over 480,000 CpG sites were analyzed in normal human bronchial epithelial cells (NHBEC) and three non small cell lung cancer cell lines (NSCLC: A427, A549 and H322) using bisulfite-based Illumina 450K BeadChip arrays
Project description:Gene set analysis as it is typically applied to genome-wide methylation assays is severely biased as a result of differences in the numbers and sizes of CpG islands associated with different classes of genes. We demonstrate this bias using published data from a study of differential methylation in lung cancer and a data set we generated to study methylation changes in patients with long-standing ulcerative colitis and show that several of the gene sets that appear enriched would also be identified with randomized data. We also report a method to correct the bias. Application of the corrected method to the lung cancer and ulcerative colitis data sets provides novel and potentially interesting biological insights into the role of methylation in cancer development and chronic inflammation. We used Agilent Human CpG Island microarrays to compare methylation patterns in sigmoid colon tissue between five individuals suffering from ulcerative colitis and five healthy age-matched controls.
Project description:We used Illumina Infinium 27k Human DNA Methylation BeadChip v1.2 to assess genome-wide DNA methylation profiling of normal colon epithelial, adenomas and colorectal adenocarcinomas across approximately 27,000 CpGs in fresh frozen colorectal tissue samples. We found that cancer-associated methylation changes with impact on transcription occur nearly as frequent at non-CpG island as CpG island promoters in colorectal cancer (CRC). Samples included 6 normal colon epithelial tissue samples, 6 adenomas, and 30 colorectal adenocarcinomas. Bisulfite-converted DNA from the 42 samples were hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2.
Project description:Investiage global methylation changes between patients at diagnosis and after relapse by CpG island methylation microarray analysis and determine the role of gene-specific methylation associated with relapse of cancer patients. Two-condition experiment, Biological replicates: 2 biological replicates and 2 samples from the same methylated CpG island amplification (MCA).