Abstract 1992: <i>In silico</i> transciptome analysis and experimental validation of epigenetically regulated genes in gastric carcinoma
Authors: Alejandro H. Corvalan, Eudocia Santibanez, Cynthia Villarroel, Francisco Aguayo, Pamela Gonzalez, Gonzalo Carrasco, Jose Valbuena, Francisco J. Ossandon, Jose I. Diaz, Maria J. Maturana
Journal: Cancer Research (2010)

Abstract Gastric Cancer is one of the most common cancers worldwide and no biomarkers are available for early detection. To search for potential biomarkers we analysed in silico transcriptome followed by experimental validation of epigenetically regulated genes. Methods. In silico transcriptome analysis was performed after download 4 normal and 10 tumor gastric Serial Gene Expression (SAGE) libraries from CGAP/GEO domains to create a single file containing 121,409 tags. Clustering libraries were identified by PCA, COA and Support Clustering. Significance Analysis for Microarray (SAM) was performed to select differentially expressed tags and SAGE Genie/TAGmapper was applied for tag-gene association. CpG island in the promoter region of selected genes was interrogated by UCSC or EMBOSS CpGPlot browsers. Functional annotation was performed by Gene Ontology (GO) using Babelomics. Experimental validation of epigenetically regulated genes was performed by RT-PCR in five gastric cancer cell lines (NCI-N87, MKN-45, KATO-III, AGS, HSC-38) and other cell lines (MCF7, ZR-75-1, A549, Hep2, JURKAT, CASKI, FS and Hek293T]) after 5 uM of 5-azaC incubation for 72 hrs. Genes with restored expression were further analyzed by bisulfite sequencing and clinico-pathological significance was evaluated by in 91 early gastric cancers and 148 chronic gastritis cases by Tissue Microarray (TMA) methodology. Results. Two thousand and four hundred and thirty seven (2%) tags were expressed in all normal libraries and all tumor libraries. PCA, COA and Support Cluster analyses clearly identified normal and tumor libraries and were confirmed by Support clustering analysis. SAM revealed 90 tags differentially expressed between these two libraries with 78 down-regulated and 12 up-regulated tags in tumor libraries. Tag to gene association identified 59 genes. Functional annotation revealed that most of downregulated genes were negative regulators of apoptosis (GO:0016265) and overexpressed genes were cell cycle regulators (GO:0007049) or angiogenesis inducers (GO:0001525). Interestingly, 16 genes did not have any prior investigation in gastric carcinoma. Finally by bona-fide CpG island analysis, 38 genes contained CpG in their promoter regions, and 12 demonstrated epigenetic regulation after 5-azaC incubation including FLT1 and STAT1. Bisulfite sequence confirmed CpG methylation changes for FLT1 gene and IHC staining of STAT1 on TMAs revealed almost significant overexpression in cancer-associated gastritis vs chronic gastritis control cases (p=0.057). Discussion. Experimental validation of in silico analysis of gastric carcinoma transcriptomes reveal epigenetic regulation of several genes. Among these, STAT1 is a promissory candidate to distinguish chronic gastritis from cancer-associated gastritis. Supported by FONDECYT 1080563 from Government of Chile. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1992.