Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human epithelial cells laser captured FROM intestinal mucosa FROM 4 individuals undergoing surgery FOR colon cancer, bowel obstruction, OR other non-inflammatory conditions

ABSTRACT: The small intestinal epithelium mediates vital functions of nutrient absorption and host defense. The spatial organization of the epithelial cells along the crypt-villus axis segregates them into regions of specialized function. However, many of the mechanisms governing intestinal epithelial cell migration and the coordination of interactions with adjacent cells and the extracellular matrix are not fully understood. We have evaluated in vivo gene expression patterns of ileal epithelial cells in healthy human subjects, isolated by laser capture microdissection from either the villus epithelial or crypt cell regions of the small intestinal mucosa. Expression profiles in villus epithelium and Paneth cell lineages were determined by quantitative real-time PCR, DNA microarray, and immunohistochemistry based methods. Relative expression levels of selected epithelial biomarkers were compared between the ileum, jejunum, duodenum, colon, stomach, and esophagus. Previously established biomarkers as well as a novel and distinct set of genes believed to be linked to epithelial cell motility, adhesion, and differentiation were found to be enriched in each of the two corresponding cell populations. Additionally, high baseline expression levels of innate antimicrobials, alpha defensin 5 (HD5) and regenerating islet-derived 3 alpha (Reg3A), were detected exclusively within the small bowel, most notably in the ileum, in comparison to other sites along the gastrointestinal tract. Our findings provide new and important insights regarding the molecular machinery employed by small intestinal epithelial cells to mediate their function and spatial organization in vivo. Experiment Overall Design: Surgical specimens of human intestinal mucosa were obtained from 4 individuals undergoing surgery for colon cancer, bowel obstruction, or other non-inflammatory conditions. The samples were fixed overnight in 4% (w/v) paraformaldehyde, dehydrated in a graded alcohol series and paraffin-embedded. At least 500 epithelial cells from the crypt or upper villus regions were then captured by LCMD from unstained 6Âµm thick sections using a PALM MicroLaser System Total RNA was extracted for microarray-based gene expression analysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Michael George

PROVIDER: E-GEOD-10629 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Genes encoding transcription factors function as hubs in gene regulatory networks because they encode DNA-binding proteins, which bind to promoters that carry their binding sites. In the present work we have studied gene regulatory networks defined by genes with transcripts belonging to different mRNA abundance classes in the small intestinal epithelial cell. The focus is the rewiring that occurs in transcription factor hubs in these networks during the differentiation of the small intestinal epithelial cell while it migrates along the crypt-villus axis and during its development from a fetal endodermal cell to a mature adult villus epithelial cell. We have generated transcriptome data for mouse small intestinal villus, crypt and fetal intestinal epithelial cells. In addition we have generated metabolome data from crypt and villus cells. Our results show that the intestinal crypt transcription factor hubs that are rewired during differentiation are involved in the cell cycle process (E2F, NF-Y) and stem cell maintenance (c-Myc). In contrast the villi are dominated by a HNF-4 villus hub, which is rewired during differentiation by the addition of network genes with relevance for lipoprotein synthesis and lipid absorption. Moreover, we have identified a villus NF-kB hub, which was revealed by comparison of the villus and endoderm transcriptomes. The rewiring of the NF-kB villus hub during intestinal development reflects transcriptional activity established by host and microflora interactions. To aid in the mining of our results we have developed a web portal (http://gastro.imbg.ku.dk/mousecv/) allowing easy linkage between the transcriptomic data, biological processes and functions. Experiment Overall Design: Four different sample categories were analyzed. Experiment Overall Design: 1) Small intestinal crypts isolated form 12-weeks old C57BL/6 mice. These samples are in triplicates. Experiment Overall Design: 2) Small intestinal villi isolated form 12-weeks old C57BL/6 mice. These samples are in triplicates. Experiment Overall Design: 3) Embryonic day 12 mesenchyme. These samples are in quadruplicate. each sample is derived from a pool of mesenchymes (10-40) Experiment Overall Design: 4) Embryonic day 12 endoderm. These samples are in quadruplicate. each sample is derived from a pool of endoderms (10-40)

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Transcription profiling of human epithelial cells laser captured FROM intestinal mucosa FROM 4 individuals undergoing surgery FOR colon cancer, bowel obstruction, OR other non-inflammatory conditions

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