Profiling, Comparison and Validation of Gene Expression in Gastric Carcinoma and Normal Stomach

Oien, Karin A (2002) Profiling, Comparison and Validation of Gene Expression in Gastric Carcinoma and Normal Stomach. PhD thesis, University of Glasgow.

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Gastric carcinoma is the second most common cause of cancer death world-wide but its molecular biology is not well understood. My aims were to catalogue the genes expressed in gastric carcinoma and normal stomach and to identify differentially expressed and gastric-specific transcripts. Serial analysis of gene expression (SAGE) produces comprehensive, quantitative and reproducible expression profiles. The method of SAGE was established in this laboratory then used to study normal gastric antral mucosa and two gastric adenocarcinomas of distal, intestinal type. The libraries were compared on-line with other glandular epithelial tissues. Selected genes were validated in a panel of 19 normal and tumour gastro-intestinal tissues and cell lines by Northern blotting and immunohistochemistry. 29,480 transcripts, derived from 10,866 genes, were identified. The validation studies corroborated the SAGE profiles although tumour heterogeneity was noted. 1% of genes were differentially expressed (by over five-fold and with a p-value below 0.01) between the pooled gastric carcinomas and normal stomach. The most abundant transcripts included ribosomal and mitochondrial proteins, of which most were up- regulated in the tumours, as were other widely expressed genes including transcription factors (Id1), signalling molecules (fibroblast growth factor receptor and serine/threonine protein kinases), coatomer and proteasome components, thymosin beta 10 and collagenase I. In contrast, cytoskeletal proteins (alpha actinin and profilin) were down-regulated in the tumours. Many genes which were more highly expressed in normal stomach are important in normal gastric function, including gastrin, immunoglobulin alpha, lysozyme, mucin (MUC5), trefoil peptides (pS2 and spasmolytic polypeptide) and pepsinogens, which were amongst 55 gastric-specific transcripts. Some transcripts had previously been characterised only minimally (prostate stem cell antigen) or not at all (aquaporin 5) in the stomach. Some genes (intestinal trefoil factor) which were up-regulated in gastric carcinoma reflect the intestinal-type histology. Some genes abundant in normal gastric antrum had previously been regarded as markers of pancreatic carcinoma. Many differentially expressed species, some tumour-associated, were novel and await investigation. One new gene which was identified was highly expressed in normal stomach but absent from gastric carcinomas. This new gene was selected for further investigation. The SAGE expression profile was confirmed by Northern blotting and in situ hybridisation by which the mRNA was located in the superficial/foveolar (pit) epithelium of the gastric mucosa, so the gene was termed foveolin. The transcript was expressed outwith the stomach only in metaplastic gastric epithelium, in Barrett's oesophagus or the ulcer-associated cell lineage in the gut, and outwith the gut only in ovarian mucinous tumours. The mRNA was present in the stomach of mouse, rat and cow in the same location as in humans. The 5' and 3' ends of the mRNA were characterised by Rapid Amplification of cDNA Ends (RACE). Homologous mouse and cow mRNAs were identified, characterised and compared. The full-length genomic sequences for the human and mouse were obtained using on-line databases, characterised and compared. A partial human genomic clone was obtained from a PAC library, and used to map the gene by fluorescent in situ hybridisation (FISH) to human chromosome 2. The predicted protein product, like the mRNA and DNA sequences, is highly conserved between the human, mouse and cow species. The protein shows no homology to any known protein sequences or motifs, but bears an initial signal peptide and is therefore predicted to have an extracellular location, being either retained on the outer cell surface or secreted into the gastric lumen, much like gastric mucin (MUC5) and the trefoil peptide pS2 (TFF1), with which foveolin shares a similar location in the superficial and foveolar gastric epithelium. These are the first global profiles of gene expression in the stomach. The molecular anatomy correlated with the morphology. The gastric carcinoma profiles resembled other tumours, which supports the existence of common cancer-associated molecular targets. The normal gastric profile differed from other normal glandular tissues but agreed with existing literature. Many new transcripts were identified, of which one has been further characterised here in its first detailed description. These data increase our knowledge about the genes involved in normal gastric function and in malignant change in the stomach, and provide a catalogue of candidates from which to develop markers for better diagnosis and therapy of gastric carcinoma.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Nicol Keith
Keywords: Genetics, Oncology
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-75730
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 18:30
Last Modified: 19 Nov 2019 18:30

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