应用亚硫酸氢盐修饰后PCR联合TA克隆测序对E-cadherin启动子区甲基化水平的检测

Detection Methylation of E-cadherin Gene Promoter by Combination of PCR with TA Clone Sequencing after Bisulfite Modification

E-cadherin是一种细胞粘附因子,通过增强细胞之间的粘附而起到抑制肿瘤转移的作用.Ecadherin基因启动子区的高甲基化是导致其在众多肿瘤细胞中表达下调甚至缺失的主要原因之一.本实验首先抽提SGC-7901细胞(胃腺癌细胞)、A549细胞(肺腺癌细胞)、MCF-7细胞(乳腺癌细胞)等3个肿瘤细胞株的全基因组DNA,然后对抽提的DNA进行亚硫酸氢盐修饰和纯化回收,根据修饰后的DNA序列设计引物并对其进行PCR扩增.然后将PCR扩增产物与pUC-T TA载体连接并转化入感受态大肠杆菌DH5α中进行培养,对筛选出的含有阳性重组子的菌落进行测序.测序结果显示,3个肿瘤细胞株的E-cadherin基因启动子区的CpG岛都呈现了高度的甲基化,亚硫酸氢盐的修饰效率达到了99.2%.综上研究表明,亚硫酸氢盐修饰后PCR(BSP)联合TA克隆测序可以对肿瘤细胞某基因启动子区CpG岛的甲基化水平进行精确量化,研究所使用的3个肿瘤细胞株均可作为研究肿瘤细胞E-cadherin基因甲基化的细胞模型.

E-cadherin is a adhesion molecule which can suppress tumor invasion and metastasis by enhancing the adhesion between cells and cells. Hypermethylation of E-cadherin gene promoter is one of the most important causes which lead to E-cadherin downexpression or deficiency, a common phenomenon in a variety of tumor cells. The first step was to isolate genomic DNA from three tumor cells（SGC-7901,A549 and MCF7）and then converted the isolated DNA using bisulfite. Then we amplified the bisulfite converted DNA with specific primers, ligated the PCR products into pUC-T TA plasmid vectors and then transfected into E.coli DH5α. At last the positive recombinants were chosen and sequenced. The results demonstrated that high methylation was in all three tumor cells and the transformation rate from cytosine to thymine except the CpG sites reached 992%. Accurate quantification of methylation status of some genes can be obtained by the method of BSP combined with TA clone sequencing and all the three tumor cells can be used as cell models for the study of methylation of E-cadherin gene.