| ObjectiveEsophageal carcinoma is a kind of malignancies with high morbidity in China.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. 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. Methylation-specific PCR (MSP ) 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 mRNA expression in the cell lines of esophageal carcinoma. This will elucidate the roles of MT-3 hypermethylation in the occurrence and development of carcinoma of the esophagus.Methods1 Five cell lines of esophageal cancer, TE-1, TE-13, TTN, ECA-109 (Which are cell lines of esophageal squamous cell carcinoma) and OE33(Which is cell lines of esophageal adenocarcinoma), were selected. RT-PCR was employed to detect their mRNA expression of MT-3 gene, in order to reveal the relationship between mRNA expression of MT-3 and the occurence of esophageal squamous cell carcinoma.2 Five cell lines of esophageal cancer, TE-1, TE-13, TTN, ECA-109 and OE33, were selected. MSP was employed to detect their methylation in CpG island of MT-3 gene, in order to understand the role of hypermethylation in CpG island of MT-3 in the occurence of esophageal squamous cell carcinoma.3 RT-PCR and MSP was employed to compare the difference of the five cell lines of esophageal cancer before and after treated with 5-Aza-2'-deoxycytidine (5-aza-CdR), a DNA methyltransferase (DNMT) inhibitor. MSP was for comparing the difference of methylation in CpG island of MT-3 gene. RT-PCR was for comparing the difference of mRNA expression of MT-3 gene. This will clarify the relationship between hypermethylation in CpG island of MT-3, mRNA expression of MT-3 and the occurence and development of esophageal squamous cell carcinoma.Results1 Varying extent of MT-3 mRNA expression exists in all the cell lines, among which blood monocyte was the highest, the second one was TE13, and there was no expression of MT-3 detected in OE33.2 MT-3 mRNA expression of all the cell lines of esophageal squamous cell carcinoma in the experiment were obviously weaker than the expression of blood monocyte(P<0.01).3 The result of MSP shows that there were extensive hypermethylation in CpG island of MT-3 in the cell lines of esophageal squamous cell carcinoma, among which TE1 was completely methylated, with no amplification of primer for unmethylation. The other three cell lines of esophageal squamous cell carcinoma were partly methylated.4 After treated with 5-Aza-CdR, the result of MSP shows that the hypermethylation in CpG island of MT-3 in the five cell lines of esophageal carcinoma was partly demethylated, among which TE13 was mostly demethylated and had seldem amplication of primer for unmethylation. OE33 was obviously amplied product of primer for unmethylation.5 After treated by 5-Aza-CdR, the mRNA expression in five cell lines of esophageal carcinoma was obviously enhanced(P<0.01).Conclusion1 Varying extent of MT-3 expression exists in the cell lines of esophageal carcinoma, among which no expression of MT-3 detected in OE33. And the four cell lines of esophageal squamous cell carcinoma have been detected more or less expression of MT-3.2 MT-3 expression of all the cell lines of esophageal squamous cell carcinoma in the experiment were obviously weaker than the expression of blood monocyte, which means there were general decrease of MT-3 expression in the cell lines of esophageal squamous cell carcinoma.3 There were extensive hypermethylation in CpG island of MT-3 in the cell lines of esophageal squamous cell carcinoma in the experiment.4 MSP shows that after treated with 5-Aza-CdR, the hypermethylation of the four cell lines of esophageal squamous cell carcinoma was partly demethylated.5 Extensive hypermethylation in CpG island of MT-3 gene exists in the cell lines of esophageal squamous cell carcinoma. It is the CpG island hypermethylation that causes the reduction of expression of MT-3. After treated with 5-Aza-CdR, the hypermethylation is reversed and the MT-3 mRNA expression will be enhanced accordingly. Therefore, there is a certain correlation between hypermethylation in CpG island of MT-3 gene and the occurrence and development of esophageal squamous cell carcinoma.
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