甲状腺癌中CYP1A1和CYPIB1基因启动子区甲基化分析

Analysis of promoter methylation of CYP1A1 and CYP1B1 gene in thyroid cancer

目的研究甲状腺癌中药物代谢基因CYP1A1、CYP1B1启动子区异常甲基化及其与临床病理特征之间的关系。方法提取201例甲状腺癌及23例正常甲状腺石蜡包埋组织基因组DNA，采用甲基化特异性PCR（MSP）检测上述基因启动子区甲基化状态，对比甲状腺乳头状癌（PTC）及正常对照中CYP1A1、CYP1B1基因甲基化的发生率。在体外实验中利用甲基化转移酶抑制剂5-氮-2′-脱氧胞苷（5-Aza-dC）处理5种甲状腺癌细胞系，用实时定量PCR（RT-PCR）方法检测5-Aza-dC处理前后CYP1A1、CYP1B1基因的表达变化。用logistic回归方法分析CYP1A1、CYP1B1基因的异常甲基化与临床特点之间的相关性。结果在各种病理类型的甲状腺癌中均存在CYP1A1、CYP1B1基因甲基化的情况；在PTC中CYP1A1基因启动子区甲基化水平高于对照组，CYP1B1基因启动子区甲基化水平低于对照组；CYP1A1基因在PTC中的表达低于对照组（P〈0.01），CYP1B1基因在PTC中的表达高于对照组（P〈0.01）；利用5-Aza-dC处理甲状腺癌细胞系后可使CYP1A1基因的表达水平在K1及C643细胞系中较对照分别升高6.92及8.30倍；CYP1B1基因在K1细胞中表达较对照升高7.62倍；CYP1B1基因启动子区甲基化与淋巴结转移呈负相关。结论在PTC中基因启动子区甲基化调控CYP1A1及CYP1B1基因的表达，CYP1B1启动子区异常甲基化与PTC的淋巴结转移有关。

ObjectiveTo investigate the promoter methylation of drug metabolism genes CYP1A1 and CYP1B1 in thyroid cancer and its relationship with clinical pathological characteristics.Method201 cases of thyroid cancer and 23 cases of normal thyroid tissues were involved. Methylation-specific PCR（MSP） was performed to analyze promoter methylation of CYP1A1 and CYP1B1 genes in the above tissues to detect the frequency of methylation positive, compare the promoter methylation level of CYP1A1 and CYP1B1 genes in papillary thyroid carcinomas（PTC） and the controls. Five thyroid cancer cell lines were treated with methyltransferase inhibitor 5-Aza-dC for 5 days, and real time PCR （RT-PCR） was performed to evaluate the mRNA expression of CYP1A1 and CYP1B1 genes. Logistic regression was used to analyze the correlation between the aberrant methylation and the clinical features.ResultsAberrant methylation status in promoter region of CYP1A1 and CYP1B1 genes were detected in all kinds of thyroid cancers. Compared with control tissues, the methylation in promoter regions of CYP1A1 in PTCs was significantly higher, while that in promoter regions of CYP1B1 was lower （P〈0.05）. In vitro, 5-Aza-dC treatment significantly increased the CYP1A1 gene mRNA expression for 6.92 and 8.30 times in K1 and C643 cell lines respectively and restored CYP1B1 gene mRNA expression for 7.62 times in K1 cell line. Compared with the controls, PTCs with methylation in promoter regions of CYP1B1 had decreased lymphatic metastasis rate.ConclusionThe methylation in promoter regions of CYP1A1 and CYP1B1 gene may regulate their mRNA expressions. Aberrant methylation of the promoter region of CYP1B1 is associated with lymph node metastasis in PTC.