启动子甲基化状况对唾液腺恶性多形性腺瘤体外细胞系细胞E-cadherin表达的影响  

Promoter methylation level affects the expression of E-cadherin in salivary malignant pleomorphic adenoma cell lines

作　　者：夏亮[1] 张春叶[1] 胡宇华[1] 钱佳骏 李江[1] 田臻[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院.口腔医学院口腔病理科上海市口腔医学重点实验室,上海200011

出　　处：《中国口腔颌面外科杂志》2016年第4期308-314,共7页China Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金(81272976;81372910)

摘　　要：目的:通过体外改变人唾液腺恶性多形性腺瘤(malignant pleomorphic adenoma,MPA)细胞系细胞E-cadherin基因启动子甲基化情况,探讨启动子甲基化对E-cadherin表达的影响。方法:采用合适浓度的5-Aza-Cd R和TGF-β1处理MPA细胞系SM-AP4和SM-AP1细胞,分别使用蛋白免疫印迹法、亚硫酸盐测序法、实时荧光定量PCR检测用药前、后E-cadherin蛋白表达、E-cadherin基因启动子甲基化状况、m RNA表达;同时采用划痕实验、Transwell小室检测用药前、后细胞迁移能力的变化。应用SPSS13.0软件包对数据进行独立样本t检验。结果:经5-Aza-Cd R处理后,SM-AP4细胞E-cadherin启动子区甲基化率显著降低(P<0.01),E-cadherin蛋白和m RNA表达显著升高(P<0.05),相应的细胞迁移能力显著降低(P<0.05)。而经TGF-β1处理后的SM-AP1细胞表达Vimentin蛋白,E-cadherin启动子区甲基化率显著升高(P<0.01),E-cadherin蛋白和m RNA表达显著降低(P<0.05),相应的细胞迁移能力显著提高(P<0.05)。结论 :DNA甲基化是调节MPA体外细胞系细胞中E-cadherin表达的重要机制之一,E-cadherin表达升高对MPA细胞转移具有潜在的抑制作用。PURPOSE： To investigate the effect of promoter methylation on the expression of E-cadherin by changing the E-cadherin gene promoter methylation level in human salivary malignant pleomorphic adenoma cell lines in vitro.METHODS： After appropriate concentrations of 5-Aza-Cd R and TGF-β1 treatment, E-cadherin promoter methylation,protein and m RNA expression were detected by bisulfite sequencing, Western blotting, fluorescence quantitative real-time PCR respectively. Wound-healing test and Transwell test were used to identify the corresponding changes of SM-AP4 and SM-AP1 cells when E-cadherin expression was altered. SPSS 13.0 software package was used for statistical analysis.RESULTS： After treatment with 5-Aza-Cd R, E-cadherin promoter methylation rate was decreased significantly in SMAP4 cell（P0.001）. E-cadherin protein and m RNA expression was significantly elevated（P〈0.05） and the corresponding ability of cell migration significantly decreased（P〈0.05）. After TGF-β1 treatment in SM-AP1, vimentin expression was induced. E-cadherin promoter methylation rate significantly increased（P 0.001） and E-cadherin protein and m RNA expression significantly decreased（P〈0.05）. The corresponding migration ability of SM-AP1 cell significantly increased（P〈0.05）. CONCLUSIONS： DNA methylation is an important mechanism to regulate the expression of E-cadherin in MPA, which can improve the level of E-cadherin protein expression and decrease the ability of tumor metastasis by reducing the methylation level of E-cadherin gene promoter.

关 键 词：E-cadherin 5-AZA-CDR TGF-β1 DNA甲基化 恶性多形性腺瘤 唾液腺 

分 类 号：R739.87[医药卫生—肿瘤]