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Experimental Study on Promoter Methylation Status of CDX2 Gene and Its Significance in Intestinal Metasplasia and Gastric Cancer

Author: XiaoFa
Tutor: SongYuGang；YaoYongLi
School: Southern Medical University,
Course: Internal Medicine
Keywords: CDX2 Intestinal metaplasia Gastric cancer Immunohistochemistry Methylation specific PCR
CLC: R735.2
Type: Master's thesis
Year: 2009
Downloads: 177
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Abstract

The background and purpose of gastric cancer refers to a malignant tumor from gastric epithelial and glandular epithelium, gastric cancer accounts for about 95% of malignant tumors of the stomach. Gastric cancer is a common malignant tumors of the human, and the second largest in the incidence of all malignancies, is one of the serious harm to human life and health of malignant disease. With other tumors, the incidence of gastric cancer is a multifactorial, complex multi-step process, a variety of process from quantitative to qualitative change in the intracellular accumulation. Correa has summed up the results of the study of the etiology of gastric cancer epidemic proposed the mode of occurrence of intestinal type gastric cancer: normal gastric mucosa - chronic superficial gastritis - chronic atrophic gastritis - intestinal metaplasia - dysplasia - early cancer - advanced cancer . Gastric intestinal metaplasia (also known as intestinal metaplasia) into gastric epithelial and glandular epithelium in pathological cases of intestinal epithelial and glandular epithelium. Gastric intestinal metaplasia is an important step in the process of gastric cancer. Gastric intestinal metaplasia process with a variety of genes related to gastric cancer progression. Tail type homeobox gene of CDX2 (caudal type homeobox gene 2) belong to a family of the homeobox gene (homeobox genes, Hox genes), was first isolated Drosophila by Mlodzik, it is a gut-specific transcription factor in embryonic development in their expression products are widely present in embryonic digestive tract glandular epithelium, the rear body section and the formation of the digestive tract, play an important role in the development and maturation, after birth, under normal circumstances, not in gastric epithelial expression, is widely expressed in the small intestine and colon mucosa, intestinal epithelial differentiation and the maintenance of the morphology and function of decisive significance. Some studies have shown that high expression of CDX2 in chronic atrophic gastritis, intestinal metaplasia and expressed in some gastric possible with the gastric mucosa and gastric cancer occurred related to the intestinal epithelium into. Of modern molecular biology, genetics, and epigenetics common regulation of the expression of the biological information, genetic information provides a \expression of these information. Epigenetics refers in the case does not affect the gene sequence of DNA methylation / de-methylation and histone acetylation / deacetylation of different states caused by changes in gene expression. DNA methylation mediated DNA methyltransferase enzyme (DNA methyltransferases, DNMTs), adding a methyl group in the the CpG dinucleotide cytosine five carbon atoms, into 5 - methylcytosine (5-mC), chemical reactions, biological functions mainly as unchanged in the sequence, the control of gene expression, and maintenance of the integrity of chromosomes and adjusting reorganization and certain genomic regions, transcriptional activity. Is different mutation genetics gene abnormalities, the methylation status of the tumor suppressor gene is reversible, application of methyl transferase inhibitors in the methylation status of gene activation demethylation, can restore some of the key the function of tumor suppressor genes play a role for the prevention and treatment of cancer, and provides a good basis for further study demethylating agent anti-tumor, tumor suppressor gene methylation cancer etiology, early diagnostic and prognostic evaluation of great value, and plays an irreplaceable role in tumor gene therapy research. Abnormal methylation of CDX2 in gastric cancer, colorectal cancer, esophageal cancer have been reported and speculated that treatment with tumor prognosis. The CDX2 gene CpG island methylation status in gastric intestinal metaplasia small. This study by detecting CDX2 gene expression and methylation status in different subtypes of intestinal metaplasia and gastric cancer, and its relationship with clinical features, explore CDX2 in intestinal metaplasia to gastric role in the transformation process and mechanism. Include: 1.CDX2 in intestinal metaplasia and gastric protein; 2.CDX2 promoter methylation status in intestinal metaplasia and gastric cancer; 3.5-aza-CdR on human gastric cancer cells KATO-Ⅲ AGS CDX2 mRNA expression and promoter methylation status. Materials and Methods (a) material 1.2008 from March to July 2008 during Digestive Endoscopy Center, Nanfang Hospital because of upper abdominal discomfort, early satiety, belching and other symptoms of upper gastrointestinal endoscopy to check and pathological diagnosis of 53 cases of patients with intestinal metaplasia, which 37 males and 16 females, aged 35-74 years, mean 57 years old. In another 18 patients without subjective symptoms and pathological examination to exclude IM gastric mucosa and gastric health check. Cases were in the 1-2cm at biopsy from the secluded entrance. Gastric cancer tissues from the same period Nanfang Hospital, surgical removal of tissue. All specimens were obtained from the body within 20min after part of making wax block, another part of the frozen in liquid nitrogen and stored at -80 ℃ and then placed in the. All patients had no known serious heart, liver and kidney dysfunction, as well as a history of mental illness who did not receive chemotherapy or radiation therapy in patients with gastric cancer before surgery. All patients were informed consent. KATO-Ⅲ of human gastric cancer cell lines AGS, from ATCC. (B) 1. Use of high-speed rail diammonium - love the first blue - PAS reaction (HID-AB-PAS) intestinal metaplasia divided into Ⅰ, Ⅱ, Ⅲ type gastric cancer, according to Lauren's parting divided into intestinal status of the CDX2 promoter hypermethylation in the detection of tissue type and diffuse type; 2. CDX2 protein expression in the use of immunohistochemistry to detect tissue; 3. methylation-specific polymerase chain reaction (MSP). 4.RT-PCR and MSP were detected before and after the treatment of 5-aza-CdR two gastric cancer cell CDX2mRNA expression and promoter methylation status. (C) Statistical analysis of experimental data using the SPSS13.0 package x ~ 2 test and analysis of variance test methods for statistical analysis. Results (a) Basic: 18 cases of normal gastric mucosa, intestinal metaplasia in 53 cases, including 22 cases of type Ⅰ, Ⅱ 18 cases, Ⅲ 13 cases. 36 cases of gastric cancer, including 21 cases of intestinal type gastric cancer, diffuse-type gastric cancer in 15 cases. (B) the expression of CDX2 expression in normal gastric mucosa in 1.CDX2. Different subtypes of intestinal metaplasia CDX2 expression positive rate (95.5% of the type I and type Ⅱ 83.3%, Ⅲ 61.5%) were significantly different (P = 0.036), and was gradually decreased. Gastric CDX2 positive expression rate (52.8%) was significantly lower than the intestinal metaplasia (83.0%) (P = 0.002), and histological type of gastric, intestinal type gastric CDX2 positive expression rate (71.4%) was significantly higher than the diffuse-type gastric cancer (26.7%) (P = 0.008). 2. CDX2 expression in intestinal metaplasia and gastric cancer cases are mainly concentrated in H.pylori infection negative intestinal metaplasia in both columns Contact number C = 0.267, P = 0.067, gastric cancer tissues both columns contact number C = 0.121, P = 0.463, H.pylori infection CDX2 expression had no effect. 3. CDX2 expression in gastric cancer, independent of age, gender; without lymph node metastasis CDX2 positive expression rate significantly higher than the lymph node metastasis group (43.3%) (100.0%) (column Contact number C = 0.390, P = 0.020), but both not a close relationship; of CDX2 and depth of invasion the column contact number C = 0.317, P = 0.106, prompted the depth of tumor invasion and CDX2 expression is not relevant. (C) CDX2 promoter methylation status of normal gastric mucosa all showed CDX2 promoter methylation, intestinal metaplasia CDX2 promoter methylation was 43.4% (type Ⅰ 22.7%, Ⅱ type 55.6%, Ⅲ 61.5%), significantly lower than the CDX2 promoter hypermethylation in gastric (69.4%) (P = 0.016), and different subtypes of intestinal metaplasia there are significant differences in CDX2 promoter methylation (P = 0.036). CDX2 promoter methylation rate of intestinal type gastric cancer (66.7%, 14/21) is less than the diffuse type (73.3%, 11/15), but there was no significant difference (P> 0.05). 2 gastric cancer without lymph node metastasis CDX2 promoter methylation (50.0%) lower than the lymph node metastasis group (73.3%) (column Contact number C = 0.185, P = 0.343), but the difference was not statistically significant; CDX2 start promoter methylation status and depth of invasion the column contact number C = 0.323, P = 0.041, prompt tumor infiltration depth and CDX2 promoter methylation status, and the deeper infiltration, the higher the rate of methylation, the relationship between the two Not close. 3 intestinal metaplasia in CDX2 promoter methylation-positive rate (36.4%) cases in CDX2 expression was significantly lower than the unmethylated cases listed contact number (C = 0.299, P = 0.031), gastric CDX2 promoter A CDX2 expression in the base of cases positive rate (47.4%) was significantly lower than unmethylated cases (94.1%) (column Contact number C = (0.452). At the optimum, P = 0.002) of CDX2 promoter methylation can lead to reduced CDX2 expression. (D) without 5-aza-CdR demethylation treatment of gastric cancer cells AGS expression promoter of CDX2mRNA unmethylated CDX2 mRNA expression was not found; KATO-Ⅲ cells, promoter A Residues. Demethylation after 5-aza-CdR drug line KATO-Ⅲ cells treated CDX2 mRNA re-expression of promoter transition unmethylated. Conclusion (a) CDX2 in the process of conversion to gastric intestinal metaplasia was gradually decreased, may play inhibit oncogene role; (b) CDX2 promoter methylation is the main mechanism of CDX2 expression missing epigenetic learning mechanism is an important molecular mechanism of gastric cancer. (C) 5-aza-CdR through CDX2 promoter demethylation leaving gastric cancer cells KATO-Ⅲ again CDX2 mRNA expression of CDX2 gene promoter methylation of the gene inactivation of important reasons, norepinephrine The base of the gene re-expression. CDX2 gene methylation status by drugs or other means of regulation to achieve inhibition of gastric cancer, which is expected to provide new ideas on the clinical treatment of gastric cancer.

Experimental Study on Promoter Methylation Status of CDX2 Gene and Its Significance in Intestinal Metasplasia and Gastric Cancer

Abstract

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