乳腺癌MCF-7细胞Alu甲基化水平的MSRE-qPCR法检测

Detection of Alu methylation in MCF-7 breast cancer cells by MSRE-qPCR

目的:寻找简单、可靠、经济并适合大批量检测肿瘤组织标本和肿瘤细胞系中Alu甲基化水平的实验方法。方法:用对甲基化敏感的限制性内切酶联合定量PCR(methylation-sensitive restriction endonuclease digestion and quantitative PCR,MSRE-qPCR)法检测乳腺癌细胞系MCF-7中Alu序列的甲基化水平,并与常用的亚硫酸氢盐修饰结合测序法(bisulfite-sequencing PCR,BSP)进行比较。结果:MSRE-qPCR显示Alu甲基化水平在BstUⅠ酶切位点约为33.7%,在HpaⅡ酶切位点约为56.1%,与BSP法比较,两种方法都显示BstUⅠ位点甲基化水平较低,而HpaⅡ位点甲基化水平较高,趋势一致,并提示MCF-7细胞的Alu甲基化水平明显低于正常细胞(84.6%或者更高)。结论:MSRE-qPCR法简单、可靠、经济,适合用于大批量检测Alu甲基化水平。

Objective：to find suitable method for detecting Alu methylation in large samples of tumor tissues and tumor cell lines.Methods：Methylation-sensitive restriction endonuclease digestion and quantitative PCR（MSRE-qPCR）method were used to analyze Alu methylation in MCF-7 breast cancer cells and compared with the traditional bisulfite-sequencing PCR（BSP）method.Results：MSRE-qPCR showed that methylation level of Alu elements in BstUⅠ cutting site was about 33.7%,and that in HpaⅡ cutting site was about 56.1%.The result of MSRE-qPCR was consistent with that of BSP,which also showed relative lower level of Alu methylation in BstUⅠ cutting site and relative higher level of Alu methylation in HpaⅡ cutting site.Methylation levels of Alu elements in MCF-7 cells detected by the two methods were all lower than those in normal cells（84.6% or higher）.Conclusion：MSRE-qPCR method is simple,reliable and inexpensive enough for Alu methylation detection in large samples.