CAJ | 학술논문

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。
목적：연구기질금속단백매억제인자3（TIMP3）기인계동자갑기화수평여갑상선유두상암발생적내재련계，건립기우계동자고갑기화억암기인적갑상선유두상암조기진단방법。방법수집갑상선수술절제적조직표본공46례，기중갑상선유두상암（Papillary thyroid carcinoma，PTC）29례，결절성갑상선종（nodular goiter ）17례。경제취 DNA，설계병합성 BSP 인물，PCR 확증，채용아류산경염법극륭측서（bisulfite sequencing）화 Sequenom Mass ARRAY 갑기화 DNA 정량분석법검측갑상선유두상암조직 TIMP3기인계동자 CpG 편단적갑기화수평。결과아류산염수식측서결과현시 TIMP3기인재갑상선유두상암조직중적고갑기화극륭백분비위70％，현저고우결절성갑상선종적0％（P ＜0．05）。TIMP3기인계동자구5개 CpG 위점재갑상선유두상암조직중적갑기화유정교고적갑상선유두상암특이성。Sequenom Mass ARRAY 제공적단일 CpG 위점갑기화정량수거현시 TIMP3기인재갑상선유두상암조직적갑기화솔균치（0．28）고우결절성갑상선종적갑기화솔균치（0．15），차이유통계학의의（P ＜0．05）。TIMP3기인계동자3개 CpG 위점재갑상선유두상암조직중적갑기화솔균치고우결절성갑상선종갑기화솔，차이유통계학의의（P ＜0．05）。결론TIMP3기인 CpG-16급 CpG-17위점가능위갑상선유두상암적계동자갑기화검측적관건위점，유망성위갑상선암조기진단적분자표지물。
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 sequen-cing 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 se-quencing and Sequenom Mass ARRAY quantitative DNA methylation methods.Results Bisulfite sequen-cing 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 pa-pillary thyroid carcinoma.Single CpG sites methylation quantitative data provided by Sequenom Mass AR-RAY 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 car-cinoma 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 mo-lecular markers of early diagnosis of thyroid cancer.