肝细胞癌相关DNA甲基化诊断生物标志物的筛选及验证

Screening and validation of diagnostic biomarkers for hepatocellular carcinoma-related DNA methylation

目的筛选肝细胞癌(hepatocellular carcinoma,HCC)相关的甲基化CpG诊断生物标志物。方法下载癌症基因组图谱(the cancer genome atlas,TCGA)中HCC的DNA甲基化和表达数据进行生物信息学分析,获得候选CpG位点。采用焦磷酸测序及qRT-PCR检测50例早期HCC组织样本中候选CpG位点的甲基化率及其所在基因的表达量,并验证其诊断效能。在GSE54503、GSE89852和GSE56588数据集中评估候选CpG位点的诊断效能。结果生物信息学分析筛选出5个候选CpG位点(cg12614630、cg19786751、cg06131338、cg23371746和cg25340966),多变量联合诊断的受试者工作特征(receiver operating characteristic curve,ROC)曲线下面积(area under curve,AUC)为0.993。50例早期HCC组织中cg12614630(GPR182)、cg19786751(ACACB)和cg06131338(ACACB)的甲基化率高于癌旁组织以及对应基因的表达水平均低于癌旁组织,且位点甲基化水平与基因表达水平呈负相关(P<0.05)。ROC曲线分析显示,在早期HCC组织以及GSE54503、GSE89852和GSE56588数据集中,3个CpG位点联合诊断的AUC分别为0.903、0.812、0.844和0.934。结论cg12614630、cg19786751和cg06131338 CpG位点可能是HCC潜在的诊断生物标志物,联合检测这3个CpG位点具有良好的诊断效能。

Objective To screen methylated CpG diagnostic biomarkers for hepatocellular carcinoma(HCC).Methods The DNA methylation and gene expression data of HCC were downloaded from the cancer genome atlas(TCGA)database for bioinformatics analysis to obtain the candidate CpG sites.The methylation rates of candidate CpG sites and the mRNA expression of corresponding genes in 50 early-stage HCC tissue samples were detected by pyrosequencing and qRT-PCR,to verify the diagnostic efficacy.The diagnostic efficacy of candidate CpG sites was evaluated in datasets GSE54503,GSE89852,and GSE56588,respectively.Results Five CpG sites(cg12614630,cg19786751,cg06131338,cg23371746,and cg25340966)were screened out by bioinformatics analysis.The area under curve(AUC)of receiver operating characteristic(ROC)curve of multivariable combined diagnosis was 0.993.The methylation rates of cg12614630(GPR182),cg19786751(ACACB),and cg06131338(ACACB)in 50 early-stage HCC tissues were higher than those in adjacent tissues,while the corresponding gene expression levels were lower than those in adjacent tissues,and site methylation was negatively correlated with gene expression levels(P<0.05).ROC curve analysis found that in the early-stage HCC tissues,GSE54503,GSE89852 and GSE56588,the AUCs of the combined diagnosis of three CpG sites were 0.903,0.812,0.844 and 0.934,respectively.Conclusions The CpG sites cg12614630,cg19786751,and cg06131338 may be potential diagnostic biomarkers for HCC,and the combined diagnosis of the these CpG sites has favorable diagnostic performance.