| IntroductionThe function loss of tumor suppressor gene by multiple pathway-s correlates with DNA methylation as well as gene mutation and loss of heterozygosity(LOH). The association of methylation with carcino-genesis and progression has been studied heatedly. Methylational in-activation should also be considered as a mechanism of tumor suppressor gene silencing , as in the case of PI6, E - cadherin, RBI, BRCA1 ,HMLH1 ,MGMT et al. E - cadherin is a related invasive suppressor gene in tumor progression . In 1998, Guilford et al found three families of gastric cancer with germline E - cadherin mutation in New Zealand, which brought about the attention of many researchers of gastric cancer. Researchers found that E - cadherin mutation didnt frequently associate with sporadic gastric cancer. Some reporters thought DNA methylation correlated more frequently with sporadic gastric cancer. Because gastric cancer is attributed to the accumulation of multiple genes in multiple stages, it is important to study the role of genes in different stages to understand the molecular mechanism of carcinogenesis. So we performed the mutation detection, the analysis of promoter methylation and protein expression of E - cadherin gene.The role of E - cadherin mutation and methylation in gastric carcino-genesis has been studied systemically.Materials and Methods1. samples; 130 specimens of gastric tissues with early stage cancer and advanced gastric cancer, including 40 cases of early cancer from high incidence area, 30 cases of early cancer, 40 cases dys-plasia and 20 cases advanced gastric cancer from non ?high incidence area were obtained from the department of oncology of China Medical University.2. DNA extracted from paraffin embedded specimens and frozen tissue.3. PCR-DGGE: Primers of 6 J,8,9 exons with "GC" clamp were designed to perform PCR amplification. DGGE and seo^iencing were applied to detect the E - cadherin mutation.4. MSP; E - cadherin promoter methylation was evaluated by means of methylation specific PCR, including 20 cases of early cancer, 23 cases of dysplasia, 20 cases of advanced gastric cancer and 10 cases of normal mucous.5. E - cadherin protein expression: Immunohistochemical staining for E - cadherin was performed by the avidin - biotin - peroxidase method with the same specimens as methylation detection.Result1. A mutation of the 9th exon was found in advanced stage gastric cancer. The third base of the 432nd code altered, aspartic acidchanged into lysine ( A AC AAG) .2. E -;c&dherin prohlotertvas methylated' for dysplasia, early gastric cancer and advanced gastric cancer and positive rate was 78.3% ,80% and 90% respectively.3. All of advanced gastric carcinomas (20/20) examined were completely E - cadherin - negative in immunohistochemistry stain. Fourteen of 20 early gastric carcinomas were E - cadherin - negative. And 23 dysplasia were all E - cadherin positive.DiscussionOne mutation of 9th exon was found in advanced gastric cancer. No mutation was found in early gastric cancer and dysplasia. The new mutation undiscovered before located in the third domain of extracellular and resulted 432nd code alteration, aspartic acid changed into lysine ( AAC AAG) , which should affect the adhesion function of cells. The finding of new mutation is correlated with the use of denaturing gel gradient electrophoresis (DGGE) for mutation detection. The application of DGGE is limited because it requires special primer with "GC" clamp. We firstly applied DGGE to the detection of E - cadherin gene mutation. Practice proved primers with "GC" clamp designed by this way were effective. Any kind of point mutation can be detected by PCR - DGGE. The high frequency of E - cadherin promoter hypermethylation in dysplasia, early gastric cancer and progressive gastric cancer indicated that it may be an important event in the carcinogenesis and development of gastric cancer. Although methyla-tion of some genes is associated with gene silencing and demethylation assoc... |
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