| Objective: China is a high-incidence country of esophageal cancer, especially in southern Hebei and northern Henan province. Its morbidity and mortality are considered to be the highest in the world. For this reason, it is especially important to study the mechanism of esophageal cancer occurrence, so as to prevent or treat it effectively in the region. Just as most of other malignant tumors, the carcinogenesis of esophageal epithelium is a result of long-term cooperation of activations of oncogenes with inhibitions of anti-oncogenes. Hypermethylation of anti-oncogenes is another aberrant gene change in addition to mutation, deletion and displacement. Compared with other gene abnormal change, studies on gene hypermethylation not only is of great value in the etiology, early diagnosis and prognosis evaluation of malignant disease, but also plays important role in gene therapy of malignancies. It has been known that the carcinogenesis of the esophagus is associated with aberrant methylation of several genes, such as p16 (INK4a), etc. MT-3 gene encodes metallothionein-3 (MT-3) in cells, which not only have many important physiological functions, but also inhibit cell proliferation. It has been shown that MT-3 gene is aberrantly methylated in gastric cancer; however, the methylation status of MT-3 in carcinoma of the esophagus is still unknown so far. Combined bisulfite restrict analysis (COBRA) and reverse transcription-polymerase chain reaction (RT-PCR) were employed in this study to detect the hypermethylation of MT-3 and its effect on MT-3 expression in cell lines, fresh tissues and formalin-fixed paraffin-embedded tissues of carcinoma of the esophagus, in order to elucidate the roles of MT-3 hypermethylation in the occurrence and development of carcinoma of the esophagus. Meanwhile, studies on DNA degradation of ordinary formalin- fixed paraffin-embedded tissues in our hospital and the methods to extract DNA from these samples were also conducted.Methods1. Four cell lines of esophageal cancer, OE21,OE19,OE33 and TE7, were selected. DNAs and RNAs were extracted from cell lines before and after treated with 5-Aza-2'-deoxycytidine (5-aza-CdR), a DNA methyltransferase (DNMT) inhibitor. COBRA analysis by restriction enzyme of BstU I digestion was employed to compare the difference of methylation in CpG island of MT-3 gene. RT-PCR was employed to compare the difference of mRNA expression of MT-3 gene. 2. Sixty-eight cases of esophageal cancer were selected; samples of tumor tissues and normal tissues from upper margin were collected. COBRA analysis by restriction enzyme of BstU I digestion was employed to study the status of MT-3 methylation. Real-time RT-PCR was employed to study the status of mRNA expression of MT-3 gene. The results of COBRA and RT-PCR were compared according to clinopathological parameters. 3. Twenty ordinary formalin treated blocks of carcinoma of the esophagus were selected from our hospital with ten processed in 2000 and ten in 1984. Ten buffer formalin treated blocks of carcinoma of the esophagus processed in 2000 were received from Royal Adelaide Hospital, Australia. Three fresh samples of esophageal cancer were also collected as control group. After extracted from these samples with the method of proteinase K digestion and NaCl salting-out, the DNA was analyzed by electrophoresis and PCR amplification, in order to clarify the degradation of DNAs in these samples. 4. Ten blocks of carcinoma of the esophagus resected in our hospital in 2000 were selected. The DNAs of these samples were obtained by three methods: DNA extraction with phenol:chloroform after proteinase K digestion, salting-out by NaCl after proteinase K digestion, extraction by phenol: chloroform after heating under the influence of pH. Then, the DNAs were analyzed by electrophoresis and PCR amplification, the aim of which was to find the ideal method to extract DNA from unbuffered formalin-fixed, paraffin-embedded tissues. 5. Fifteen blocks of carcinoma of the esophagus resected in our hospital betw...
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