Effect of CpG site selection on SMP30 promoter methylation detection in hepatocellular carcinoma by pyrosequencing

MO Zhijing; ZHENG Shunxin; ZHOU Sufang

doi:10.3969/j.issn.1002-266X.2017.09.001

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.