10个肺癌相关基因多重甲基化特异性PCR检测方法的建立

Establishment of multiplex methylation specific PCR assay for detecting methylation in 10 lung cancer-related genes

目的建立10个肺癌相关基因的多重甲基化特异性聚合酶链反应(PCR)检测方法并运用于临床标本的检测。方法选取19例肺癌患者病灶组织标本,患者中男性16例,女性3例;平均年龄57.3岁。1例肺部良性病变患者组织标本为男性,年龄52岁。通过质粒构建及甲基化转移酶和亚硫酸盐修饰制备10个基因的甲基化和非甲基化标准品。通过选择甲基化特异性引物来识别甲基化模板,挑选10对甲基化特异性引物并平均分成2组,经最佳聚合酶及最佳退火温度的选择,建立多重甲基化特异性PCR体系,并应用于19例肺癌组织的检测,计算10个基因的甲基化率。结果在成功构建10个基因的甲基化和非甲基化标准品的基础上,建立了它们的多重甲基化特异性PCR检测方法。临床标本检测结果显示10个肺癌相关基因的甲基化率分别为p16INK4A 92%,H-cadherin 89%,E-cadherin和DAPK 76%,TIMP-3 63%,RARβ50%,MGMT 39%,RASSF1A 21%,GSTP1 11%,hMLH1 0%。2次实验结果重复性较好。结论这种分2组多重甲基化特异性PCR的方法能一次性检测10个基因甲基化状态,可以应用于临床标本的检测。

Objective To explore a new multiplex methylation specific polymerase chain reaction（PCR） method for analyzing the methylation status of 10 lung cancer genes.Methods A total of 19 hospitalized patients with lung cancer were selected,including 16 males and 3 females with mean age of 57.3 years old.A 52-year-old male patient with pulmonary benign lesion was also enrolled in the present study.The methylation and unmethylaiton standards of 10 lung cancer-related genes were set up by treating the plasmids encoding the genes with DNA methyltransferase and sulfite.Ten pairs of methylation specific primers were selected for the multiplex-methylation specific PCR and the condition for the PCR were optimized by choosing the best optimal polymerase and annealing temperature.The methylation rate of 10 lung cancer-related genes from the patients was detected using the multiplex methylation specific PCR.Results The methylation and unmethylaiton standards of 10 lung cancer genes were set up successfully and the muliplex-methylation specific PCR were able to test the methylation status of the 10 genes simultaneously with perfect consistency.The methylation rate is 92 %,89 %,76 %,63 %,50 %,39 %,21 %,11 %,and 0 % for p16INK4A,H-cadherin,E-cadherin and DAPK,TIMP-3,RARβ,MGMT,RASSF1A,GSTP1 and hMLH1,respectively.Conclusion It is demonstrated that the new multiplex methylation specific PCR could be utilized to evaluate the methylation status of 10 lung cancer-related genes simultaneously and have the potential in clinical diagnosis.