Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Cell transcriptional state alters genomic patterns of DNA double-strand break repair in human astrocytes

ABSTRACT: The misrepair of DNA double-strand breaks in close spatial proximity within the nucleus can result in chromosomal rearrangements that are important in the pathogenesis of hematopoietic and solid malignancies. It is unknown why certain epigenetic states, such as those found in stem or progenitor cells, appear to facilitate neoplastic transformation. Here we show that altering the transcriptional state of human astrocytes alters patterns of DNA damage repair from ionizing radiation at a gene locus-specific and genome-wide level. Astrocytes induced into a reactive state exhibited increased DNA repair, compared to non-reactive cells, in actively transcribed chromatin domains after irradiation. In mapping these sites of repair, we identified misrepair events and repair hotspots that were unique to each state. The precise characterization of genomic regions susceptible to mutation in specific epigenetic transcriptional states provides new opportunities for addressing clonal evolution in solid cancers, particularly those where double-strand break induction is a cornerstone of management. Primary astrocyte cells were cultured to confluency and went through a linear monolyer scratch or no scratch.

ORGANISM(S): Homo sapiens

SUBMITTER: abdel elkahloun

PROVIDER: E-GEOD-45994 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:MicroRNAs (miRNAs) are an evolutionarily conserved large class of small non-coding RNAs that mediate post-transcriptional silencing of genes and influence a broad spectrum of biological processes ranging from embryonic development to organismal death. Our previous study identified the miR-29 family, three paralogous species of miR-29a/b/c, as the most predominantly expressed small RNA in aged mouse brain compared to neonate one. The mouse brain miR-29 is highly astrocytic. Its expression is quiescent during early brain development, and thereafter steadily increases to a saturating level around the end of the reproductive phase. To explore the functional relevance of miR-29 expression to the neural physiology of mouse brain from the mechanistic perspective of mammalian species-specific lifespan, we here designed a gain-of-function approach through forced expression of miR-29 in astrocyte from mouse fetus and surveyed the resulting alteration in the transcriptional and translational levels. DNA microarray analyses retrieved a total of 5,589 genes showing a temporal significant expression changes in the miR-29-transfected fetal astrocytes, and classified them into two gene groups positively or negatively regulated by miR-29. Mass spectrometry (MS)-based quantification of translational products of miR-29-responsive genes identified 18 species of miR-29 target candidates. We performed functional enrichment analyses using bioinformatics resources to elucidate the biological roles of the gene sets thus identified, and obtained their expression trend that favor the processes for facilitating cell differentiation while supporting normal cell proliferation /survival, which was different from the functional signatures of miR-29 at adult stages, implying the bifunctional property of miR-29 depending on the developmental context. Our present results strongly suggest that miR-29 serves as the central coordinator to shift the global gene expression in the developing mouse brain toward matured phenotype, and through which ensure the programmed transition to the post-developmental stage that is inherently set for the mouse lifespan. DNA microarray experiment: Sample labeling, hybridization and washing were performed following the standard protocol detailed in the Agilent One-Color Microarray-Based Gene Expression Analysis ver.5.7. Briefly, a One-color Spike-Mix was diluted 1000-fold and a 5-μl aliquot of the diluted mix were added to every 0.5 μg of total RNA samples (three each of extraction replicates of miR-29-treated sample and control sample named elsewhere) prior to labeling reaction. The labeling reaction was carried out separately for the RNA samples using a Quick Amp Labeling Kit one-color, in the presence of cyanine 3-CTP. The dye-labeled target (1.6 μg as cRNA) was fragmented and hybridized on an Agilent Whole Mouse Genome 4X44K microarray at 65°C for 17 hr with a Gene Expression Hybridization Kit. The hybridized slide was washed in Gene Expression Wash Buffer 1 at room temperature for 1 min, which was followed by a wash for 1 min in Gene Expression Wash Buffer 2. The processed microarrays were scanned using an Agilent DNA Microarray Scanner. Data extraction from raw image files was done with Agilent Feature Extraction software ver.10.7.

Project description:mRNA and microRNA expression was examined in global cellular fractions and in RNA-induced silencing complex (RISC)-immunoprecipitated cell fractions in cultured primary human astrocytes (ScienCell) and in cultured human U-87 MG astrocytoma cells (ATCC). ABSTRACT: Background: GW/P bodies are cytoplasmic ribonucleoprotein-rich foci that are involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. These mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 when bound to miRNA within the RNA-induced silencing complex (RISC). Although miRNAs and mRNAs are known to be localized to RISC in a variety of cells, to date no published study has examined the profile of specific miRNA and mRNA targeted to the RISC. Methodology/Principle Findings: In this study, RISC mRNA and miRNA components were profiled by microarray analysis of human U-87 astrocytoma cells and primary human astrocytes with total RNA extracted from the RISC as well as the global cellular fractions. The novel findings of this study were fourfold: (1) miRNAs are highly enriched in primary astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma cells and primary astrocytes each contain unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p were upregulated and miR-181b was downregulated in U-87 astrocytoma RISC as compared to primary astrocyte RISC, and (4) RISC contain mostly downregulated mRNAs in primary astrocytes and U-87 astrocytoma cells. Conclusions/Significance: We show that in U-87 astrocytoma cells, miR-34a and miR-195 were upregulated in RISC suggesting an oncogenic role for these miRNAs. Three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated. One miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in cancer, inflammatory disease, immunological disease, the cell cycle, cellular movement and numerous cell signaling pathways. This study points to the importance of the RISC and ultimately GW/P body composition and function and in miRNA and mRNA deregulation in astrocytoma cells and possibly for other brain tumors. Experiments outsourced to LC Sciences who are partnered with an AffymetrixM-BM-. Authorized Service Provider, SeqWright DNA Technology Services (Houston, TX). Using Affymetrix Human Genome U133A 2.0 Array, they analyzed 14,500 well-characterized human genes across 4 replicates in each of the 4 sample groups: (1) global cellular U-87 astrocytoma, (2) global cellular primary astrocyte, (3) RISC-IP U-87 astrocytoma, and (4) RISC-IP primary astrocyte. Experiments outsourced to LC Sciences (Houston, TX). Using M-BM-5Paraflo microfluidic chips, they analyzed 6211 target human miRNAs (Sanger miRBase version 11, http://microrna.sanger.ac.uk/sequences/) or control RNAs (array hybridization controls and single-base mismatch targets) and a polyethylene glycol spacer segment to extend the coding segment away from the substrate across 4 replicates in each of the 4 sample groups: (1) global cellular U-87 astrocytoma, (2) global cellular primary astrocyte, (3) RISC-IP U-87 astrocytoma, and (4) RISC-IP primary astrocyte.

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Cell transcriptional state alters genomic patterns of DNA double-strand break repair in human astrocytes

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