Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression analysis of human seminoma samples

ABSTRACT: Purpose: The aim of the present study was to examine the biological differences between seminomas with occult and clinically apparent metastases at the time of diagnosis of the primary tumor to gain insight into the biology of these tumors and facilitate the identification of novel predictors of seminoma metastasis. Materials and Methods: Total RNA including small RNAs was isolated from testicular tumors of patients with pure seminoma presenting with lymphogenic metastasis (n=5, clinical stage IIb/c) and occult metastasis (n=5, clinical stage I). The regulation of biological processes was examined (1) throughout the mRNA transcriptome (whole genome microarrays, 8x60K Array, Agilent and 4 samples per group) and (2) the miRNA transcriptome employing small RNA next generation sequencing (SOLID, Life Technologies). Protein coding genes (mRNAs) and small RNAs showing a significant (> 2-fold) difference between the groups were identified. Finally (3), we examined 95 candidate miRNAs in 36 apparent metastasized and another 5 occult metastasized seminoma using logistic regression analysis. Results: Among 19,596 genes, on average 12,894 mRNAs appeared expressed (65.8%, SD +/- 2.4; range, 62.0M-bM-^@M-^S69.3%) and 16.99M-CM-^W106/13.94M-CM-^W106 small RNA reads were identified for apparent/occult metastasized seminoma. These reads on average convert into 9,901/9,675 small RNAs including 422/404 mature microRNAs. None of these mRNAs/small RNAs met our selection criteria for candidate genes. From 95 candidate miRNAs 44 appeared expressed, with 3 of them showing weak but significant (p=0.047-0.049) differences among both groups. Conclusions: Occult and apparent metastasized seminomas are biologically almost indistinguishable and probably represent no separate tumor entities. These findings may simplify future research on seminoma metastasis. Regarding the genome-wide expression profiling we analyzed gene expression changes in human seminoma samples with occult or lymphatic metastasis using Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays in 8x60K format in combination with a one-color based hybridization protocol. Microarray signals were detected using the Agilent DNA Microarray Scanner. Differential RNA expression was assessed by applying appropriate biostatistics to the data set. GeneSpring GX12.6 analysis software was used to normalize and analyze the raw data. Different filtering approaches were applied to identify up- and downregulated transcripts in the comparisons of interest. Identified differentially expressed genes were further characterized for their involvement in biological pathways and processes based on the Panther database. For mRNA transcriptome analysis we used 4 samples per group. Only significant and > 2-fold differences in gene expression between the two groups were considered for comparisons, and unadjusted p-values and those adjusted for multiple comparisons (false discovery rate) were calculated. All gene candidates underwent gene set enrichment analyses using PANTHER pathway software (http://www.pantherdb.org/), which groups genes with similar biological functions based on their annotation.

ORGANISM(S): Homo sapiens

SUBMITTER: Michael Abend

PROVIDER: E-GEOD-55198 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Acyl-coA synthases (ACSs) produce fatty acyl-CoAs that are used in metabolic and signaling pathways. Metazoans have a large number of ACS genes with differing expression patterns and substrate preferences, but the physiological roles of most ACS genes are unknown. Here, we focused on the C. elegans acyl CoA synthase, ACS-3, which is known to regulate fat uptake and de novo fat synthesis through the conserved nuclear hormone receptor, nhr-25. We performed microarray analysis of acs-3 mutants to elucidate the acs-3-regulated transcription program. This analysis revealed an enrichment among differentially regulated genes of those involved in lipid metabolism, pathogen and wounding responses, and sterol binding genes, among others. As the immunity genes were the most represented gene class, we performed pathogen sensitivity assays to test the phenotypic consequences of this immune gene regulation. Interestingly, acs-3 mutants were hypersensitive to the fungal pathogen D. coniospora, but only mildly sensitive to the bacterial pathogen P. aeruginosa. acs-3 mutation suppressed nhr-25 mutant sensitivity to P. aeruginosa, yet surprisingly microarray analysis of nhr-25(RNAi) animals revealed significant overlap with the acs-3 mutant transcriptome, with an enrichment of pathogen response genes. The upregulation of pathogen response genes in acs-3(ft5) mutants and following nhr-25 reduction-of-function (rf) does not appear to be due to a constitutive osmotic response or defective cuticle barrier, two potential explanations for the acs-3(ft5) and nhr-25(rf) expression of innate immunity genes in the absence of pathogen exposure. Together, these data indicate that ACS-3 promotes resistance to the fungal pathogen, D. coniospora and regulates innate immunity genes through an unknown mechanism. Potential roles for ACS-3 in innate immunity are discussed. We used two-color expression microarrays to compare the transcriptional profiles in two experimental conditions: 1) comparing wild-type (N2) L4 stage larval worms to acs-3(ft5) L4 larval mutant animals; and 2) animals grown to L4 larval stage on bacteria harboring vectors for either control or nhr-25 RNA-interference (RNAi). L4 stage was determined by morphology of the developing vulva. Three biological replicates were used for each experimental condition. Statistically significant changes in gene expression in each experimental were determined using M-bM-^@M-^\linear models for microarray dataM-bM-^@M-^] (limma).

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Gene expression analysis of human seminoma samples

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