Studies On Methylation Of RUNX3 And DAPK Gene In Human Gastric Cancer

IntroductionGastric cancer is one of the most common malignant tumor, the inactivation of tumor suppressor genes contributes to it's development and progression. Recent studies indicate that promoter methylation associated with loss of expression of some tumor suppressor genes in gastric cancer, with demethylation agents, many gastric cancer cell lines could reactivate these genes expression. The RUNX3 gene is a member of the Runt domain family of transcription factors that are master regulator of gene expression in major development pathways. Recently, lack of RUNX3 function was found to be associated with carcinogenesis and progression of gastric cancer, while gene mutation was rare found, hint there is other inactive way. DAPK is a novel calcium/calmodulin-dependent enzyme with serine/threonine kinase activity. It is an essential mediator involved in the IFN induced programmed cell death and in the TNF-Fas induced apoptosis. It was found that DAPK mRNA and protein expression is frequently lost in various human cancer cell lines. In this study, we investigated the methylation status of RUNX3 and DAPK gene in primary gastric cancer, adjacent nonneoplastic mucosa and lymphonode. In order to find the relationship between the methylation status of these genes and clinicopatholgic characters.Materials and methodsSamples : 38 samples of primary gastric cancer, corresponding adjacent nonneoplastic tissues and lymphonode were collected from patients diagnosed with primary gastric cancer and exairesis at the Department of Surgical Oncology, The First Hospital Affiliated to China Medical University during Feb, 2005 to Aug, 2005 Each fresh specimens were dissected into two part, one was immediately stored at 70℃until to be used in the experiments, the other was subjected to histologist diagnosis. Among these patients, 26 were male and 12 female. The age range was from 36 to 80 (mean age 57) years. No cancer cell was found in all of tissue samples adjacent to the tumor. No chemotherapy or radiotherapy was given to any patients before operation. Normal controls were obtained from human peripheral blood which had no any diseases of digestive system or tumor.Methods: Frozen tissues were digested with protease K, and genomic DNA was obtained by conventional phenol/chloroform and ethanol extraction.4μg Genomic DNA was treated with sodium bisulfite as described previously. And then genomic DNA was desalted using the Wizard DNA purification resin, according to the manufacturer's instructions. Modified DNA amplification with specific-primers was carried out, A methylation positive control (DNA had been methylated with Sss I), an unmethylation positive control (DNA hadn't been methylated with Sss I )and a negative control (distilled water without DNA) were included in each amplification. Products were analyzed on 2.5% agarose gels, stained with GeneFiner and photographed.Assessment: It means methylation when got methylation products, while there was only unmethylation product without methylation product, the specimen was judged as unmethylation, neither methylation nor unmethylation could get means there was something wrong in our work.Statistical analysis: The Fisher's exact test was used to compare the methylation rate in different tissues, All statistical analyses were performed using software SPSS 13.0 and the differences were considered to be statistically significant when P<0.05 .Results1. DNA methylation of RUNX3,DAPK gene in corresponding nonneoplastic: RUNX3 methylation frequency was 21.1%,DAPK methylation frequency was 42.1%, and it was significant correlation with old age(>60 years, P <0.05),the methylatin rates of these two genes in nonneoplastic were significant lower than either that in tumor tissue or that in lymphnode (each P <0.01) . 2. DNA methylation of RUNX3,DAPK gene in tumor tissue: 36 (94.7%) cancer samples showed CpG island hypermethylation of at least one gene, and both genes were hypermethylated in 60.5% of all cancer samples. Methylated RUNX3 was detected in cancer samples in 73.7% individuals, and when the size of tumors were above 5 centimeter the frequency of methylation significantly increased (P <0.05) . DAPK gene methylation frequency was 81.6%.3. DNA methylation of RUNX3,DAPK gene in lymphnode: methylation of RUNX3 was present in 65.8%,and 35 in all(92.1%) got RUNX3 unmethylation production, DAPK methylation frequency was 70.2%.4. The relationship between DNA methylation and clinicopathologic parameters in patients with gastric cancer: No significant correlation between methylation status of both genes and clinicopathologic characteristics (such as Borrmann type, differentiation, growth pattern) was observed (P>0.05).Conclusion1. RUNX3 and DAPK gene methylation are common events in gastric cancer development. DAPK methylation frequency in tumor was significant correlation with old age, it may increase the risk of getting gastric cancer, and RUNX3 methylation would hasten tumor grow.2. Methylation seems to provide an ideal set of cancer specific markers for early detection of or for monitoring micrometastasis. Reverse the methylation status may be used as a novel approach for prevention gastric carcinogenesis and progression.