甲状腺乳头状癌与基质金属蛋白酶抑制因子3(TIMP3)基因启动子甲基化的关系研究  

Tissue inhibitor of matrix metalloproteinase3 (TIMP3) gene promoter methylation in papillary thyroid carcinoma

作　　者：阿布来提.麦麦提艾力 哈德提.别克米托夫 哈力木拉提.木尔提扎 艾力.塞丁 西尔扎提.苏来曼 夏米西努尔.伊力克 

机构地区：[1]新疆医科大学基础医学院生物学教研室,乌鲁木齐830011 [2]新疆医科大学第一附属医院病理科,乌鲁木齐830054 [3]新疆医科大学第一附属医院血管甲状腺外科,乌鲁木齐830054 [4]新疆维吾尔自治区人民医院肿瘤外一科,乌鲁木齐830000 [5]新疆医科大学基础医学院生物化学教研室,乌鲁木齐830011

出　　处：《新疆医科大学学报》2014年第7期841-845,852,共6页Journal of Xinjiang Medical University

基　　金：国家自然科学基金(30860275)

摘　　要：目的研究基质金属蛋白酶抑制因子3(TIMP3)基因启动子甲基化水平与甲状腺乳头状癌发生的内在联系,建立基于启动子高甲基化抑癌基因的甲状腺乳头状癌早期诊断方法。方法收集甲状腺手术切除的组织标本共46例,其中甲状腺乳头状癌(Papillary thyroid carcinoma,PTC)29例,结节性甲状腺肿(nodular goiter)17例。经提取DNA,设计并合成BSP引物,PCR扩增,采用亚硫酸氢盐法克隆测序(bisulfite sequencing)和Sequenom Mass ARRAY甲基化DNA定量分析法检测甲状腺乳头状癌组织TIMP3基因启动子CpG片段的甲基化水平。结果亚硫酸盐修饰测序结果显示TIMP3基因在甲状腺乳头状癌组织中的高甲基化克隆百分比为70%,显著高于结节性甲状腺肿的0%(P<0.05)。TIMP3基因启动子区5个CpG位点在甲状腺乳头状癌组织中的甲基化有呈较高的甲状腺乳头状癌特异性。Sequenom Mass ARRAY提供的单一CpG位点甲基化定量数据显示TIMP3基因在甲状腺乳头状癌组织的甲基化率均值(0.28)高于结节性甲状腺肿的甲基化率均值(0.15),差异有统计学意义(P<0.05)。TIMP3基因启动子3个CpG位点在甲状腺乳头状癌组织中的甲基化率均值高于结节性甲状腺肿甲基化率,差异有统计学意义(P<0.05)。结论 TIMP3基因CpG-16及CpG-17位点可能为甲状腺乳头状癌的启动子甲基化检测的关键位点,有望成为甲状腺癌早期诊断的分子标志物。Objective To study the inner link between the methylation level of tissue inhibitor of matrix metalloproteinase 3 （TIMP3） gene promoter and papillary thyroid carcinoma for an early diagnostic method of papillary thyroid carcinoma based on promotor hypermethylation of tumor suppressor genes. Methods 46 cases of thyroid lesions fresh tissue samples were collected, including 29 cases of papillary thyroid carcinoma （PTC） and 17 cases of nodular goiter. Genomic DNA were extracted; bisulfite sequencing primer （BSP） were designed and synthesized by means of primer design software PCR amplification; promoter CpG sites methylation level of TIMP3 gene in papillary carcinoma were analyzed by bisulfite sequencing and Sequenom Mass ARRAY quantitative DNA methylation methods. Results Bisulfite sequencing data showed the hypermethylation percentage of TIMP3 gene cloning in thyroid carcinoma was 70%, significantly higher than the nodular goiter 0% （P 〈0.05）. Total twelve CpG sites of TIMP3 gene showed methylation in papillary thyroid carcinoma, including five CpG sites with a high degree of specificity of papillary thyroid carcinoma. Single CpG sites methylation quantitative data provided by Sequenom Mass ARRAY showed TIMP3 gene the mean （0.28） of methylation rate in papillary thyroid carcinoma and the mean （0.15） of nodular goiter methylation rates had a significant difference （P 〈0.05）. When compared with nodular goiter, the mean of TIMP3 gene of three CpG sites methylation rate in papillary thyroid carcinoma had significant difference （P 〈0.05）. Conclusion According to BSP sequencing and Sequenom Mass ARRAY quantitative DNA methylation analysis, CpG-16 and CpG-17 of TIMP3 gene may be the key sites of papillary thyroid carcinoma promoter methylation testing, and they were expected to become molecular markers of early diagnosis of thyroid cancer.

关 键 词：甲状腺乳头状癌 TIMP3 启动子甲基化 亚硫酸盐修饰测序 Sequenom Mass ARRAY 

分 类 号：R736.1[医药卫生—肿瘤]