CDH1基因在SD大鼠舌黏膜癌变过程中甲基化和突变的研究

目的:通过甲基化和外显子突变研究探索CDH1基因在舌鳞状细胞癌发生过程中的作用。方法:采用质量分数为0.004%的4-硝基喹啉-1-氧化物(4-nitroquinoline-1-oxide,4NQO)饮水饲养90只无特定病原体(specific pathogen free,SPF)SD大鼠以诱发舌黏膜癌变全过程,分别于第10、14、18、22、24周分批处死大鼠,取舌黏膜组织行病理分级,并提取基因组DNA。利用甲基化特异性PCR(methylation-specific PCR,MS-PCR)检测CDH1启动子甲基化水平;利用聚合酶链式反应(polymerase chain reaction,PCR)扩增CDH1外显子1-16,提纯后测序以检测突变。结果:病变各阶段均未检测出CDH1启动子甲基化产物,CDH1 mRNA第2106位发生碱基G→T错义突变。结论:4NQO饮水诱导SD大鼠舌黏膜癌变的发生、发展可能与CDH1外显子突变有关而与CDH1启动子甲基化无关。

Study on the Methylation and Mutation of CDH1 in the Process of SD Rats Tongue Carcinogenesis.

Objective: To detect the CDH1 gene methylation and mutation in exon at different pathological stages of tongue mucosa carcinogenesis. Methods: Seventy-five SD rats were administered by 0.004% 4-nitroquinoline-1-oxide (4NQO) in drinking water, and 15 SD rats were kept in the same conditions without 4NQO as negative control group. After 10, 14, 18, 22 and 24 weeks, the rats were euthanatized, and the tumorsin the tongue mucosa were collected for histological examination and DNA extraction. Then, methylation specific polymerase chain reaction (MS-PCR) was performed to detect the levels of CDH1 promoter methylation, and PCR amplification of CDH1 exons 1-16 was performed to detect CDH1 exons mutations in the various stages of tongue squamous cell carcinoma (TSCC). Results: There were no CDH1 promoter methylation specific products detected in different stages of SD rats TSCC. In sequenced exons 1-16 of all samples, only exon 13 mutation was detected at the site of mRNA 2106 (G→T). Conclusion: CDH1 mutations in exons may be associated with the development of SD rats TSCC which induced by 4NQO, but CDH1 promoter methylation was not.