Project description:In order to improve our understanding of microRNA (miRNA) deregulation in melanoma development and possible consequences for patient survival, miRNA expression profiles were determined, using an array based approach, in melanoma tumors, melanoma cell lines and normal melanocytes. Differentially expressed miRNAs were evaluated in relation to clinical characteristics, patient prognosis in terms of melanoma-specific survival, and mutational status for BRAF and NRAS.
Project description:DNA methylation profiling of human melanocytes and melanoma cell lines. Goal was to identify hypermethylated gene promoters in melanoma Genomic DNA from 4 human melanoma cell lines and normal human epidermal melanocytes was subjected to methylated DNA immunoprecipitation (MeDIP) and hybridized to Agilent's G4489A Human Promoter ChIP-on-Chip Set 244K
Project description:In order to improve our understanding of microRNA (miRNA) deregulation in melanoma development and possible consequences for patient survival, miRNA expression profiles were determined, using an array based approach, in melanoma tumors, melanoma cell lines and normal melanocytes. Differentially expressed miRNAs were evaluated in relation to clinical characteristics, patient prognosis in terms of melanoma-specific survival, and mutational status for BRAF and NRAS. Agilent microarray platform containing 470 miRNAs was used to determine miRNA expression profiles in 3 normal melanocytes (as non-neoplastic control), 21 melanoma cell lines and 16 clinical samples from fresh frozen regional lymph node metastases. To validate the microarray platform, the expression levels of some miRNAs were evaluated using RT-PCR and the correlation between the two platforms was assessed using Pearson Correlation analysis. The results obtained were further verified and confirmed by RT-PCR in an independent set of melanoma samples. Association between deregulated miRNAs and survival was determined by Univariate Cox proportional hazards model and log rank test.
Project description:MicroRNAs (miRNAs) influence cancer development through post-transcriptional negative regulation of both tumor suppressors and oncogenes. We subjected melanoma cell lines, normal melanocytes, and keratinocytes to array based miRNA profiling, and identified several distinct miRNAs with differential expression. Specifically, miR-211 levels were depleted in all eight melanoma cell lines examined, and also in 23 of 30 distinct patient melanoma samples (graded as primary in situ, regional metastatic, distant metastatic and nodal metastatic). Putative target genes of miR-211 were identified, and their anticipated increased expression levels were confirmed in melanoma cell lines, which were reduced in two melanoma cell lines that artificially over-expressed miR-211. Four such target genes (TCF12, RAB22A, KCNMA1 and SLC37A3) were confirmed by a target cleavage assay. Stable over-expression of miR-211 in two melanoma cell lines caused significant growth inhibition and reduced invasiveness. The differential expression of miR-211 in a variety of melanoma cell lines and clinical samples, consistent inverse correlation between miR-211 and its target mRNA levels, and growth retardation and reduced invasiveness of melanoma cell lines by miR-211 are all consistent with the idea that the depletion of miR-211 is a key step in melanoma development and/or progression The 15 Samples in this submission represent gene-level expression profiling of isolated total RNA from WM1552C, WM1552+miRNA211, A375, A375+miRNA211 and melanocytes hybridized to Affymetrix exon ararys.
Project description:Aberrant DNA methylation and histone modifications both contribute to carcinogenesis, but how these two epigenetic factors interact to impact gene expression remain unclear. To address this issue, we studied gene expression profiles, DNA methylation and two key histone modifications (H3K4me3 and H3K27me3), in two types of normal melanocytes (HEMn and HEMa) and two melanoma cell lines SK-MEL-28 and LOXIMVI. Using these data, we analyzed the relationship between epigenetic factors and gene expression status in both normal and melanoma cells, and the impact of epigenetic switches on gene expression during melanomagenesis. ChIP-seq analysis of H3K4me3 and H3K27me3 in two types of normal melanocytes (HEMn and HEMa) and two melanoma cell lines (SK-MEL-28 and LOXIMVI).
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:Genome wide DNA methylation profiling of primary uveal melanoma cells, normal uveal melanocytes, neural crest stem cells, embryonic stem cells and uveal melanoma cell lines. The Illumina Infinium 27k Human DNA methylation Beadchip Rev B was used to obtain DNA methylation profiles across approximately 27,000 CpGs in the samples. Samples included 58 primary UM, 3 NUM and NCSC controls and 2 cell lines.
Project description:DNA methylation profiling of human melanocytes and melanoma cell lines. Goal was to identify hypermethylated gene promoters in melanoma