ABSTRACT: This SuperSeries is composed of the following subset Series: GSE29359: Analysis of p53 target genes in melanoma part 1 GSE29361: Analysis of p53 target genes in melanoma part 2 Refer to individual Series
Project description:Although mutations in p53 are infrequent in human melanoma, its function is abnormal. In this study, whole genome bead arrays were used to examine the expression of p53 target genes in extracts from 82 metastatic melanoma samples, compared to extracts derived from diploid human melanocytes, to provide a global assessment of aberrant p53 function. Total RNA extracted from 82 tumour samples and 8 melanocyte cell lines was analysed. Metastatic melanomas were compared to melanocyte cell lines.
Project description:Although mutations in p53 are infrequent in human melanoma, its function is abnormal. In this study, whole genome bead arrays were used to examine the expression of p53 target genes in extracts from 6 melanoma cell lines, compared to extracts derived from diploid human melanocytes and fibroblasts, to provide a global assessment of aberrant p53 function. The expression of these genes was also examined in extracts derived from melanocytes and melanoma cell lines in which p53 expression had been inhibited using shRNA and compared to cells that had been transduced with a control shRNA. Total RNA extracted from 18 samples was analysed representing duplicates of 6 melanoma cell lines, 1 melanocyte cell line and 2 fibroblast cell lines. Melanoma cell lines were compared to normal cell lines. In addition, IgR3, Mel-RM and melanocyte cell lines were transduced with either control shRNA or p53 shRNA to evaluate the effect of p53 on its target genes. Cell lines transduced with control shRNA were compared to cell lines transduced with p53shRNA. Duplicates were analysed.
Project description:Malignant melanoma is a common and frequently lethal disease. Current therapeutic interventions have little effect on survival, emphasizing the need for a better understanding of the genetic, epigenetic, and phenotypic changes in melanoma formation and progression. We identified genes that were not previously known to be silenced by methylation in melanoma using a microarray-based screen following treatment of melanoma cell lines with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Experiment Overall Design: RNA was isolated following 0 and 48 hours of 5AzadC treatment of melanocytes and six melanoma cell lines (MelJuSo, UACC 903, C8161, Neo6 C8161, WM1205, and WM35) and used for the reexpression microarray analysis.
Project description:MiR-31 is one of the most highly overexpressed miRNAs in psoriasis skin; however, its biological role in the disease has not been studied. Here we show that miR-31 is markedly overexpressed in psoriasis keratinocytes. To study the biological role of miR-31 in keratinocytes, we transfected miR-31 hairpin inhibitor (anti-miR-31) into primary human keratinocytes to inhibit endogenous miR-31. We performed a global transcriptome analysis of keratinocytes upon suppression of endogenous miR-31 using Affymetrix arrays. Expression profiling of primary human keratinocytes transfected with 10nM miR-31 hairpin inhibitor (anti-miR-31) or control hairpin RNA (anti-miR-Ctrl) for 48 hours (biological triplicates in each group) was performed using the Affymetrix GeneTitan system.
Project description:Genetic variation is known to influence the amount of mRNA produced by a gene. Because molecular machines control mRNA levels of multiple genes, we expect genetic variation in components of these machines would influence multiple genes in a similar fashion. We show that this assumption is correct by using correlation of mRNA levels measured from multiple tissues in mouse strain panels to detect shared genetic influences. These correlating groups of genes (CGGs) have collective properties that on average account for 52â79% of the variability of their constituent genes and can contain genes that encode functionally related proteins. We show that the genetic influences are essentially tissue-specific and, consequently, the same genetic variations in one animal may upregulate a CGG in one tissue but downregulate the CGG in a second tissue. We further show similarly paradoxical behaviour of CGGs within the same tissues of different individuals. Thus, this class of genetic variation can result in complex inter- and intra-individual differences. This will create substantial challenges in humans, where multiple tissues are not readily available. Each sample is a single hybridisation from a given BxD strain to a C57BL/6J reference sample. Two-colour glass arrays were used. No dye swaps were employed. Data from whole brain.
Project description:Genetic variation is known to influence the amount of mRNA produced by a gene. Because molecular machines control mRNA levels of multiple genes, we expect genetic variation in components of these machines would influence multiple genes in a similar fashion. We show that this assumption is correct by using correlation of mRNA levels measured from multiple tissues in mouse strain panels to detect shared genetic influences. These correlating groups of genes (CGGs) have collective properties that on average account for 52â79% of the variability of their constituent genes and can contain genes that encode functionally related proteins. We show that the genetic influences are essentially tissue-specific and, consequently, the same genetic variations in one animal may upregulate a CGG in one tissue but downregulate the CGG in a second tissue. We further show similarly paradoxical behaviour of CGGs within the same tissues of different individuals. Thus, this class of genetic variation can result in complex inter- and intra-individual differences. This will create substantial challenges in humans, where multiple tissues are not readily available. Common reference design where each hybridisation compares a single BxD strain to a common reference of C57BL/6J in the green channel. Two-colour, spotted glass arrays were used. No dye swaps were employed.
Project description:In the early stages of wound healing, keratinocytes become “activated” and release inflammatory molecules such as interleukin-1 and interleukin-8 that are linked to innate immune responses and neutrophil recruitment. It is unclear, however, whether keratinocytes release molecules linked to adaptive immune responses, e.g. CCL20, in their early state of activation without signals from infiltrating T cells. This study aims to isolate the immediate alterations in protective and inflammatory gene expression that occur in epidermal keratinocytes, with a particular focus on molecules associated with cell-mediated immunity. We used dispase-separated epidermis, followed by intercellular disassociation by trypsinization, as a model for epidermal injury. We obtained a pure population of keratinocytes using flow cytometry. As a control for uninjured epidermis, we performed laser capture microdissection on normal human skin. Sorted keratinocytes had an early burst of upregulated gene expression, which included CCL20, IL-15, IL-23A, IFN-κ, and several antimicrobial peptides. Our results provide insight into the potential role of keratinocytes as contributors to cell-mediated inflammation, and expand knowledge about gene modulation that occurs during early wound healing. Our findings may be relevant to cutaneous diseases such as psoriasis, where micro-injury can trigger the formation of psoriatic plaques at the site of trauma. Compare keratinocyte response to cell disassociation with (1) epidermal samples isolated by laser caputre microscopy and (2) cultured KCs
Project description:In order to explain the molecular mechanisms of capsaicin-induced proliferation of gingival epithelial cells (GECs), a microarray analysis was performed. Several cell proliferation related genes were significantly up-regulated. These data suggest that TRPV1 signaling in GEC may induce transcriptional upregulation of growth factors, which results in an increased proliferation rate. Simian virus 40 (SV40) T-antigen-immortalized human gingival epithelial cell line, epi4, were stimulated with 1 M-NM-<M capsaicin for 4 hours. Total RNA was isolated and subjected to gene expression analysis using Agilent whole human genome oligo microarray.
Project description:Each expressed gene was tested for differential expression in three mouse tissues (Brain, Kidney, Liver) in a direct comparison of SJL/J vs. C57BL/6J Keywords: direct comparison, multiple tissues Whole RNA from a pool of 5 SJL/J mice was compared to a pool of RNA from C57BL/6J mice with three technical replicates per tissue, no dye swaps.
Project description:We investigated whether levels of serum microRNAs (miRNAs) could discriminate women with high-grade serous ovarian epithelial cancer (SEOC) from age matched healthy volunteers. miRNA expression profiling was performed on 4 SEOC cell lines and normal human ovarian surface epithelial cells (HOSE). miR-182, miR-200a, b and c were highly overexpressed in the SEOC cell lines. miR-103, miR-92a and miR-638 displayed relatively invariant expression and with RNU48, were assessed, as putative serum miRNA normalizers. The 7 miRNA and RNU48 were assessed in serum from SEOC patients (n = 30) and age-matched healthy volunteers (n = 32) by qRT-PCR following pre-amplification. No correlation between age and miRNA or RNU48 levels was observed (age range 30-79 years). miR-103 demonstrated the least variance and was chosen to normalize serum volume adjusted results. miR-200a, b and c were significantly elevated in SEOC serum (P = 0.002; 0.003; 0.0003 respectively) and a multivariate model was the best predictive classifier of SEOC (ROC-AUC = 0.806). A correlation between high miR-200b levels and residual macroscopic disease following cytoreductive surgery was identified (P = 0.006). Our results suggest that serum miR-200a, b and c may have utility as diagnostic biomarkers for SEOC. miRNA expression was arrayed in 1 human surface epithelial cells (HOSE) and 4 ovarian cancer cell lines (OV167, OV202, OVCAR-3, PE01). HOSE cells were run in triplicate (including 1 dye-swap). Ovarian cancer cell lines were run in duplicate (including 1 dye-swap).