应用BSP克隆测序检测肝细胞癌组织中Dynamin 3基因启动子区域甲基化水平

目的检测原发性肝细胞癌组织Dynamin 3(DNM3)基因启动子区域36个CpG的甲基化水平,分析其与肝细胞癌患者临床病理特征的关系,探讨DNM3基因甲基化在肝癌形成过程中的作用。方法提取30对患者肝细胞癌组织和癌旁组织DNA,经亚硫酸氢盐处理后,进行巢式PCR并联合克隆测序检测DNM3基因的甲基化水平。结果肝细胞癌患者的甲基化事件发生率为83.3%(25/30)。癌组织的DNM3基因启动子区域平均甲基化率为37.8%,癌旁组织为12.0%,两者比较差异有统计学意义(P0.05)。DNM3基因甲基化诊断肝癌的受试者工作特征曲线(ROC)曲线下面积(AUC)为0.831,对肝癌有较好的诊断价值。结论 DNM3作为肝细胞癌的一种抑癌基因,其启动子区域高度甲基化可能会促进肝细胞癌的形成。

Analysis of the methylation status of Dynamin3 Gene Promoter by bisulfite genomic sequencing PCR combined with TA clone sequencing technology in hepatocellular carcinoma

Objective To compare the methylation status of 36 CpG sites in dynamin 3(DNM3) promoter between hepatocellular carcinoma and paracancerous tissues ,analyze the associations between clinicopathological characteristics and DNM3 methylation ,ex‐plore its role in HCC formation processes .Methods Genomic DNA of 30 pairs of liver cancer tissues and paracancerous tissues was extracted ,then DNM3 gene methylation levels was detected by nested PCR combined cloning sequencing after bisulfite treatment of genomic DNA .Results The methylation level of DNM3 gene in hepatocellular carcinoma is high .The incidence of DNM3 methyla‐tion in hepatocellular carcinoma was 83 .3% (25/30) .The average methylation rate of cancer tissues was 37 .8% ,and the average methylation rate of adjacent non‐tumor tissues was 12 .0% .There were significant statistical difference in DNA methylation be‐tween the tumor and non‐tumor tissues(P<0 .05) .The AUC of ROC for DNM3 gene was 0 .831 ,which showed great value for the diagnosis of hepatocellular carcinoma .Conclusion DNM3 as a tumor suppressor gene in hepatocellular carcinoma ,the hypermethyl‐ation of its promoter might promote the formation of hepatocellular carcinoma .