Functional Study Of Cdx2 In Development Of Gastric Intestinal Metaplasia

Intestinal metaplasia(IM) of the gastric mucosa was regarded as theprecancerosis of gastric cancer.Varied expressions of some transcriptional factors andtheir controlled genes were intensively involved in the process of intestinalmetaplasia and further carcinogenesis of the gastric mucosa.Cdx2 is a specificintestinal transcription factor and plays a critical role in IM.Overexpressed Cdx2could cause intestinal metaplasia in vitro,and its function was reported largelymediated by LI-CD,another key regulator of IM.Objectives:To investigate the expression variation of Sox2,Cdx2 and LI-CD and investigatetheir relationships in the process of intestinal metaplasia.To verify whether Cdx2 isthe upstream regulator of LI-CD in IM and to elucidate the possible mechanism ofCdx2 induced intestinal metaplasia.Methods:1.Determined the relative level of Sox2,Cdx2 and LI-CD transcript by real-timequantitative PCR in grouped gastric mucosa samples which were collected bybiopsy with the pathological diagnosis.Immunohistochemically (IHC) evaluatedthe expression and distribution of Cdx2 and LI-CD protein in slided gastric mucosasamples with chronic gastritis,mild,moderate and severe IM.2.Constucted the recombinant eukaryotic expression plasmid of pcDNA3.1/wtCdx2which contains wild type human Cdx2 gene in plasmid pcDNA3.1.In vitro culturedgastric epithelial cell line GES-1 was transfected by pcDNA3.1/wtCdx2 in differentdose.The effect of wtCdx2 tranfection was determined by detecting the expressionof Sox2 and villin by Western Blot.3.Recombinant plasmid pcDNA3.1/mutCdx2 was also constructed by site directedmutagenensis,which contains a DNA binding domain mutated Cdx2 gene.GES-1celline was transfected by plasmid pcDNA3.1/mutCdx2,then the expressions ofCdx2,Sox2 and villin were also determined.Results 1.Compared to gastritis group,elevated expressions of Cdx2 and LI-CD in IMgroups were confirmed by Real-time PCR (P＜0.05).The expressions of Cdx2significant related with LI-CD and they are all in consistance with the progress ofIM.2.IHC assays of the clinical samples also showed Cdx2 and LI-CD expression wereup-regulated in different IM groups (P＜0.001).Their expressions were in positiverelationship with the status of IM (r=0.827,P＜0.01).3.Over expressed wtCdx2 could increase villin expression and decrease Sox2expression in transfected GES-1 cell line,while mutCdx2 did not have this kind offunction although increased expression of mutated Cdx2 protein was detected.Conclusions1.Cdx2,Sox2 and LI-CD are all intensively involved in the process of IM,whereCdx2 and LI-CD stimulate IM while Sox2,inhibits IM.There is good correlationbetween the expression of Cdx2 and LI-CD,they might exert their functionsthrough the same signaling pathway.On the contrary,it is showed inversecorrelation between Cdx2 and Sox2 in transcriptional level.2.Over-expressed Cdx2 by pcDNA3.1/wtCdx2 transfection in in vitro culturedGES-1 cell line cause increasing expression of villin implying intestinal phenotypeformed,and decreasing expression of Sox2 implying gastric phenotype disappeared.Cdx2 over-expression is critical to IM generation,and Sox2 should be adownstream target gene of Cdx2 at least in one pathway.3.IM cell model is successfully constructed by over-expression of wtCdx2.