Backgroud and ObjictiveGastric cancer is one kind of malignant tumors derived from gastric mucosal epithelium and glandular epithelium,accounts for about 95%of malignant tumors in the stomach.Human gastric cancer is common,and accounted for the second place in the incidence of all malignant tumors.Therefore,it is one of the malignant disease which seriously endangering the health of human beings.Just like other tumors, gastric cancinogenesis is a multi-factor,multi-step and complex procedure,many changes are accumulated in cells,from quantitative change to qualitative change process.Correa summed up the epidemiological studies of gastric cancer,the modes of intestinal-type gastric cancer:normal gastric mucosa-chronic superficial gastritis-chronic atrophic gastritis-intestinal metaplasia-dysplasia-early cancer-advanced cancer.Gastric intestinal metaplasia refers to the metaplasia that the gastric epithelial and glandular epithelium changes into intestinal epithelium and glandular epithelium in pathological circumstances.The opinion that gastric intestinal metaplasia is precancerous lesion of gastric cancer has now been proved by many clinical trials and research.The procedure of gastric intestinal metaplasia progressing into gastric cancer relates with multiple genes.Caudal type homeobox gene 2(CDX2) belongs to homeobox gene(Hox) family and abstracted successfully from Drosophila by Mlodzik.CDX2 is intestinal-specific transcription factor.During the embryonic development,their products express extensively in the embryonic digestive tract epithelium,and play an important role for growth and maturity the rear body section and the formation of the digestive tract.After birth,CDX2 doesn\'t express in normal gastric epithelium but expresses widely in the small intestine and colon mucosa,and of decisive significance to intestinal epithelial differentiation and maintenance of its morphology and function.Studies have shown that CDX2 expresses highly in chronic atrophic gastritis-related intestinal metaplasia and some gastric carcinoma,indicating the possibility that CDX2 is related occurrence of gastric cancer.Modern molecular biology argues that genetics and epigenetics co-regulate the expression of biological information.While genetic information provides the "blueprint" necessary for "production" of proteins,epigenetics are also give advice to the cells how,when and where express of these information.Epigenetics refers to change of gene expression without change of gene sequences but with DNA methylation/demethylation or histone acetylation/deacetylation.DNA methylation refers to chemical reactions which add one methyl-group to cytosine 5 carbon atoms in CpG dinucleotide and make it into a 5-cytosine(5-mC),all of those was mediated by DNA methyltransferase(DNMTs).Biological functions are manifested in the maintaining gene sequence,controlling gene expression,maintaining the integrity of chromosomes and regulating the transcriptional activity of certain specific genomic region.Different from gene mutation,hypermethylation status of the tumor suppressor gene is reversible.Methyl transferase inhibitors will activate gene demethylation and restore function of some critical tumor suppressor gene.Therefor, it plays important role in prevention and treatment of tumor.All of those provided a good evidence for further study in demethylation reagents as anti-tumor.So study of tumor suppressor gene methylation status plays an irreplaceable role in definding cause of malignant,early diagnosis,tumor gene therapy research and prognosis evaluation.At present,methylation of CDX2 in gastric cancer,colorectal cancer,esophageal tumor and other tumors has been reported,and we predicted that it is related to treatment and prognosis of tumor.However,study on CDX2 promotor methylation status in gastric intestinal metaplasia is short.In this study,we detected the expression and promoter methylation status of CDX2 in different subtypes of intestinal metaplasia and gastric cancer and their relationship with clinical features,in order to find the role of CDX2 in the procedure of intestinal metaplasia to gastric cancer.Our study includes:1.CDX2 expression in intestinal metaplasia and gastric cancer;2.CDX2 methylation status in intestinal metaplasia and gastric cancer;3.Effect of 5-aza-CdR CDX2 mRNA expression and promoter methylation status on human gastric cancer cells KATO-Ⅲand AGS.Material and Method1.Material(1) 53 cases of intestinal metaplasia come from patients who undertook gastroscopy during March 2008 to July 2008 in Digestive Endoscopy Center of Southern Hospital,due to abdominal discomfort,early satiety,belching symptoms and pathologically diagnosed.Another 18 cases without subjective symptoms were also admited.Each cases biopsied from 1-2cm distance from pylorus.Gastric cancer tissues come from surgical resection cases in the Southern hospital in the same period. All specimens were obtained in 20min after vitro acquisition,some of them was paraffinum-embeded,while others was quick-frozen in liquid nitrogen and then placed in the -80℃fridge.All the patients had no known serious heart,liver and kidney dysfunction as well as the history of mental illness.Gastric cancer cases haven\'t received any chemotherapy or radiotherapy.All patients were informed consent.(2).Human gastric cancer cell line KATO-Ⅲand AGS,from ATCC.2.Method(1)Use High iron diamine-Alcin blue-Periodic acid Schiff reaction (HID-AB-PAS) to classified intestinal metaplasia toⅠ,Ⅱ,Ⅲtype,gastric cancer and according to Lauren\'s classification will be classified into intestinal type and diffuse type;(2) CDX2 expression was tested by immunohistochemical methods;(3) CDX2 methylation status was tested by methylation-specific polymerase chain reaction(MSP).(4) RT-PCR and MSP were used to detect CDX2 mRNA expression and methylation status before and after the 5-aza-CdR treatment of gastric cancer cells.3.Statistical analysisSPSS13.0 was used to analyse the experimental data.x~2 test,Speraman correlation analysis variance test,et al was adopted.Results1.Basic Information:all of our cases include 18 cases of normal gastric mucosa,53 intestinal metaplasia,which are classified into 22 cases of typeⅠ,18 typeⅡand 13 typeⅢ,36 gastric cancers,including 21 cases of intestinal type gastric cancers,15 cases of diffuse type of gastric cancer.2.CDX2 expression(1) Normal gastric mucosa did not express CDX2.CDX2-positive rate in different subtypes of intestinal metaplasia(95.5%,83.3%,61.5%,respectively) were significantly different(P=0.036),showed a gradually decrease tendency.Positive rate in gastric cancer(52.8%) was significantly lower than that in intestinal metaplasia(83.0%)(P=0.002).CDX2 expression positive rate was related to histology type of gastric cancer.Positive rate in intestinal type gastric(71.4%) was significantly higher than that in diffuse type(26.7%)(P=0.008).(2) Expression of CDX2 in both intestinal metaplasia and gastric cancer cases was focused mainly on case without H.pylori infection.Contingency coefficient between them in intestinal metaplasia is 0.267(P=0.067),while 0.121(P=0.463) in gastric cancers.H.pylori infection had no effect on the expression of CDX2.(3) CDX2 expression in gastric cancer cases has no relationship to age or gender.Positive expression rate of CDX2 in gastric cancer without lymph node metastasis(100.0%) was significantly higher than that with lymph node metastasis (43.3%)(Contingency coefficient C=0.390,P=0.020),but their relations were not close;Contingency coefficient between CDX2 pasitive expression and the depth of invasion was 0.317(P=0.106),there was a negtive correlation between them.3.CDX2 promoter methylation status(1) All of the normal gastric mucosa showed CDX2 promoter methylation, CDX2 methylation promoter rate in intestinal metaplasia was 43.4%(22.7%,55.6%, 61.5%,respectively in the three subtypes) was significantly lower than that in gastric cancer(69.4%)(P=0.016).CDX2 promoter methylation in different subtypes of intestinal metaplasia shows significant difference(P=0.036).CDX2 promoter methylation rate in the intestinal type gastric cancer(66.7%,14/21) was higher than that in diffuse type(73.3%,11/15),but there was no significant difference(P＞0.05).(2) CDX2 promoter methylation rate in gastric cancers without lymph node metastasis(50.0%) was significantly lower than in those with lymph node metastasis (73.3%)(Contingency coefficient C=0.185,P=0.002),but the difference was not significantly.Contingency coefficient between CDX2 promoter methylation and infiltration depth was 0.323(P=0.041),there was a positive correlation between them。However,the relationship between them was not close,(3) CDX2 positive expression in intestinal metaplasia with methylated CDX2 promoter(36.4%) was significantly lower than those with unmethylated CDX2 promoter(Contingency coefficient C=0.299,P=0.031),while that in gastric cancer with methylated CDX2 promoter(47.4%) was also significantly lower than those with unmethylated one(94.1%)(Contingency coefficient C=0.452,P=0.002),CDX2 promoter methylation could lead to the reduction of CDX2 expression.4.Human gastric cancer cells AGS showed expression of CDX2 mRNA before demethylation treatment by 5-aza-CdR for and CDX2 promoter showed unmethylation,while KATO-Ⅲshowed no expression of CDX2 mRNA and CDX2 promoter showed methylation.After demethylation treatment by 5-aza-CdR, KATO-Ⅲshowed reexpression of CDX2 mRNA and CDX2 promoter showed unmethylation.Conclusion1.Change of CDX2 expression during procedure of intestinal metaplasia to gastric cancer showed a gradually descending tendency.CDX2 probably played a important role in inhibiting oncogene;2.CDX2 promoter methylation is the main mechanism for loss of CDX2 expression in gastric cancer.Epigenetics mechanism probably is an important molecular mechanism of gastric carcinogenesis.3.5-aza-CdR can make CDX2 promoter demethylation so that gastric cancer cells KATO-Ⅲshowed CDX2 mRNA re-expression,suggesting that CDX2 promoter methylation are important reasons of the inactivation of CDX2 gene.CDX2 promotor methylation status can be regulated and controlled through drugs or other means to inhibit gastric cancer.