RASSF1A、MINT31启动子区高甲基化与食管上皮内瘤变和食管鳞癌的关系

Correlation of RASSF1A and MINT31 Promoter Hypermethylation with Esophageal Intraepithelial Neoplasia and Esophageal Squamous Cell Carcinoma

背景:食管上皮内瘤变(EIN)是食管正常或慢性炎症组织向鳞癌转变过程中必经的病理阶段,目前对EIN的发生、发展尚缺乏临床实用的早期评估指标。目的:探讨肿瘤相关基因RASSF1A、MINT31启动子区高甲基化和DNA甲基转移酶1(DNMT1)表达与EIN和食管鳞癌发生、发展的关系。方法:以甲基化特异性PCR(MSP)检测30例食管鳞癌、80例EIN和20例正常食管组织中的RASSF1A、MINT31甲基化状态,以亚硫酸氢盐修饰后测序(BSP)验证MSP结果。以免疫组化方法检测EIN和食管鳞癌组织中的DNMT1蛋白表达。结果:正常食管组织、EIN、食管鳞癌组织中的RASSF1A和MINT31甲基化率分别为5.0%和5.0%、32.5%和26.2%、60.0%和46.7%,组间两两比较差异均有统计学意义(P<0.05)。BSP方法证实,MSP示RASSF1A甲基化阳性的EIN和食管鳞癌组织,RASSF1A启动子区存在甲基化位点。低级别、高级别EIN和食管鳞癌组织中的DNMT1蛋白表达阳性率分别为32.5%、55.0%和76.7%,组间两两比较差异均有统计学意义(P<0.05)。DNMT1高表达与RASSF1A甲基化相关,与MINT31甲基化无关。结论:RASSF1A、MINT31启动子区高甲基化和DNMT1表达上调在EIN和食管鳞癌的发生、发展中起重要作用,其检测或许有助于EIN的评估。

Background： Esophageal intraepithelial neoplasia （EIN） is the crucial step when normal or chronic inflammatory esophageal tissue evolving to esophageal squamous cell carcinoma （ESCC）, however, no practical molecular biomarkers are available for the early appraisal of EIN in clinical practice. Aims： To investigate the correlation of RASSF1A and MINT31 （tumor-related genes） promoter hypermethylation, as well as expression of DNA methyltransferase 1 （DNMT1）, with the development and progression of EIN and ESCC. Methods： Methylation-specific PCR （MSP） was used to determine the methylation patterns of RASSF1A and MINT31 in 30 ESCC, 80 EIN and 20 normal esophageal tissues, and the results of MSP were verified by bisulfite sequencing PCR （BSP）. Expression of DNMT1 protein in EIN and ESCC was measured by immunohistochemistry. Results： RASSF1A and MINT31 promoter methylation rates were increased gradually from normal esophageal tissue, EIN to ESCC （5.0% and 5.0% vs. 32.5% and 26.2% vs. 60.0% and 46.7%）, significant difference was observed between any two stepwise groups （P〈0.05）. In EIN and ESCC with MSP-proved RASSF1A promoter hypermethylation, methylation loci could be detected in RASSF1A promoter by BSP. The positivity rates of DNMT1 protein in low-grade, high-grade EIN and ESCC were 32.5%, 55.0% and 76.7%, respectively, with significant difference between any two stepwise groups （P〈0.05）. Overexpression of DNMT1 was positively correlated with RASSF1A promoter hypermethylation, but not MINT31 promoter hypermethylation. Conclusions： RASSF1A and MINT31 promoter hypermethylation, as well as overexpression of DNMT1, play important roles in the development and progression of EIN and ESCC. Their measurements might be useful for the appraisal of EIN.