Identification Of Differentially Expressed Genes In Stroma Of Colorectal Cancer Patients With Liver Metastasis

Objective Liver metastases of colorectal cancer(CRC) causes high morbidity andmortality worldwide.Nowdays more and more researches focus on the role which stromaplays in tumour progression. A few important markers of therapeutic targets and prognosticappear in CRC stroma.The research of molecular markers in CRC stroma will exactlyforecast liver metastases of CRC and lay the foundation of best targeted treatment.Lasercapture microdissection(LCM) and gene chip are two immerging techniques that havebecome important methods in cancer research.LCM under direct microscopic visualizationpermits defined cells or groups of cells from heterogeneous tissues via laser capture andmicrodissection,and thus allows the homogenous of tissure.Gene chip can analysethousands of DNA with high efficiency simultaneously,and then provides a powerful toolof identification of differentially expressed genes.The aim of this study is to identify the hepatic metastasis-associated genes in CRC stroma by theabove two kinds of technology.First, we used LCM to separate pure stroma from CRC(with or withoutliver metastases).Then,we studied the gene expression profiles of CRC with hepatic metastasis andCRC without hepatic metastasis stroma tissues by using the Agilent Human4*44K2.0chips,andanalyzed the differentially expressed genes of CRC stroma tissues screened by microarray by usingseveral bioinformatics tools.Methods Total RNA was extracted from human carcinoma tissue samples of4CRCwith liver metastases and4CRC without liver metastases. We used LCM to separate purestroma from CRC(with or without liver metastases),total RNA was isolated from thesetissues. Denatured gel electrophoresis was used to determine their quality.After linear amplification and one-color fluorescent-labeled, Agilent Nanodrop ND-1000was used todetermine their concentrations,quality and fluorescence labeling efficiency.The qualifiedcRNA was hybridized with Agilent Human4*44K2.0whole genome microarry,then waswashed and scanned. The GeneSpring GX v11.51Software was used to normalize thesignals.The siganal data acquired by further software analysis and comparism,selected outgenes different in expression.Using qRT-PCR verify the two top ranked up-and down-regulated genes.Result In4cases of fresh specimens hybridized with gene chip,a total of1948differentin gene expression was found,including1266up-regulation,682down-regulation.Thesegenes were divided by molecular function according to GO(gene ontology) Category,themost of these genes are relevant to cell differention,enzyme activity regulation,signaltransduction,as well as gene transcription and translation.According to Pathwayanalysis,the most of these genes are relevant to ECM-receptor interaction,focaladhesion,cell adhesion molecules,antigen processing and presentation,cytokine-cytokinereceptor interaction,TGF-β sigaling pathway et al.Test and verify4genes of all by qRT-PCR,which expression direction consistent withthe results detected by gene chip,according with expected results.Conclusion1.Combining laser capture microdissection and gene chip is an effectivemethod for identification of differential gene expression profiles in CRC(with or withoutliver metastases) stroma.2.Further research on these differentially expressed genes（IGF2、Periostin、CCL21、PGC-1）may provide a novel method for the diagnosis and molecular targetedtherapy of hepatic metastasis in colorectal cancer.