山东医药2017，Vol.57Issue(9)：1-4,4.DOI:10.3969/j.issn.1002-266X.2017.09.001
CpG位点选择对焦磷酸测序法检测肝癌SMP30启动子甲基化的影响
Effect of CpG site selection on SMP30 promoter methylation detection in hepatocellular carcinoma by pyrosequencing
摘要
Abstract
Objective To determine appropriate CpG sites for senescence marker protein 30 (SMP30) promoter methylation detection in hepatocellular carcinoma using pyrosequencing.Methods Genomic DNA was isolated from human hepatocellular carcinoma cell lines SMCC-7721 and BEL-7404.The amplification and sequencing primers were designed for SMP30 gene sequence 1 and sequence 2,and the methylation status of CpG sitesin SMP30 gene sequence 1 and sequence 2 was determined by pyrosequencing technology.Sanger sequencing was used to validate the SNP.Results In SMCC-7721 and BEL7404 cell lines,the 6 CpG sites in SMP30 gene sequence 1 were low methylatied,which was not consistent with the low expression of SMP30 gene,and the quality of the sequencing results all failed.The 6 CpG sites in the SMP30 gene sequence 2 were highly methylated,which was consistent with the low expression of SMP30 gene,the first 2 CpG sites passed,and the last 4 CpG sites failed.The 5th and 6th CpG sites were ignored and only the first 4 CpG sites beforethe poly (T) structure in SMP30 gene sequence 2 were detected,the percemage of methylation in BCC-7721 cell line was 100％,90％,100％ and 80％.The percentage of methylation in BEL-7404 cell line was 100％,92％,100％ and 77％.Except for the 3th CpG site,the rest of the CpG sites passed.Sanger sequencing showed that SMCC-7721 and BEL-7404 cell lines had SNP sites before the 3th CpG site,and all of them were A.Condmion Pyrosequencing methylation analysis of CpG sites near the upstream of transcription start site and before the poly (T) structure in SMP30 promoter is more accurate.关键词
肝癌/衰老标记蛋白30/甲基化/焦磷酸测序Key words
liver carcinoma/senescence marker protein 30/methylation/pyrosequencing分类
医药卫生引用本文复制引用
莫之婧,郑顺心,周素芳..CpG位点选择对焦磷酸测序法检测肝癌SMP30启动子甲基化的影响[J].山东医药,2017,57(9):1-4,4.基金项目
国家自然科学基金委员会资助项目(81460432). （81460432）
