应用二代测序技术检测肝细胞癌组织样本中抑癌基因PTEN基因启动子甲基化率  

Methylation detection of phosphatase and tensin homolog deleted on chromosome ten gene promoter in hepatocellular carcinoma samples by next-generation sequencing

作　　者：荆潇宽 姜棋予 李丛舒 张年荣 柴燕涛 冯帆 李伯安 李炎坤 Jing Xiaokuan;Jiang Qiyu;Li Congshu;Zhang Nianrong;Chai Yantao;Feng Fan;Li Boan;Li Yankun(School of Pharmacy,Hubei University of Science and Technology,Xianning 437100,China;Institute of Infectious Disease,Department of Infectious Disease,the Fifth Medical Center,General Hospital of Chinese People′s Liberation Army,Beijing 100039,China;The Clinical Laboratory Medicine Center,the Fifth Medical Center,General Hospital of Chinese People′s Liberation Army,Beijing 100039,China)

机构地区：[1]湖北科技学院药学院,咸宁437100 [2]中国人民解放军总医院第五医学中心,感染病医学部研究所,北京100039 [3]中国人民解放军总医院第五医学中心,临床检验医学中心,北京100039

出　　处：《中华预防医学杂志》2021年第10期1220-1227,共8页Chinese Journal of Preventive Medicine

摘　　要：目的旨在采用二代测序(NGS)的技术平台检测肝细胞癌(HCC)组织样本中肿瘤抑制基因第10号染色体缺失的磷酸酶和张力蛋白同源等位基因(PTEN)基因启动子区的甲基化率,并对其与接受索拉非尼治疗患者预后相关性进行分析。方法2019年6月至2020年12月期间,对在解放军总医院第五医学中心收集和保存的52例单纯接受索拉非尼治疗患者的HCC成对肿瘤组织与癌旁组织样本,提取样本中的总DNA样品;使用亚硫酸氢盐处理DNA样品并设计特异性引物对PTEN启动子区进行扩增,进一步对扩增产物进行二代测序分析。在对PTEN甲基化的临床意义分析中通过log-rank统计学分析方法计算患者组间生存期是否具有统计学差异。结果肿瘤组织中PTEN启动子区甲基化率(29.17%±9.58%)显著高于癌旁组织(4.17%±2.86%)(t=19.970,P<0.05)。同时,在HCC组织中,PTEN启动子区甲基化率与其表达负相关(F=47.270,P<0.0001;Y=-1800×X+38.03),PTEN甲基化率与患者接受分子靶向药物索拉非尼治疗的预后负相关(χ^(2)=4.313,P<0.05)。结论本研究成功建立了新型PTEN启动子区甲基化的检测方法,PTEN甲基化率可能是HCC诊断及靶向治疗的靶标之一。Objective The purpose of this study is to use the next-generation sequencing(NGS)technology platform to detect the methylation rate of phosphatase and tensin homolog deleted on chromosome ten(PTEN)promoter region in hepatocellular carcinoma(HCC)tissue samples,and to analyze the clinical significance of its correlation with the prognosis of patients receiving sorafenib treatment.Methods The 52 pairs of tumor tissue and para-cancerous tissue samples from HCC patients treated with sorafenib alone,which were collected and preserved in the Liver Tumor Diagnosis and Research Center of the former 302 Hospital of the People′s Liberation Army by the National Natural Science Foundation of China Youth Project with the project batch number 81702986 in 2018,were extracted total DNA from the samples.Then the DNA samples were treated with bisulfite and specific primers were designed to amplify the PTEN promoter region.Finally,the amplified products were analyzed by second-generation sequencing.In the analysis of clinical significance of PTEN methylation,log-rank statistical analysis was used to calculate whether there was a statistical difference in survival between the patient groups.Results The methylation rate of PTEN promoter region in tumor tissues(29.17%±9.58%)was significantly higher than that in paracancer tissues(4.17%±2.86%)(t=19.970,P<0.05).At the same time,in HCC tissues,the methylation rate of the PTEN promoter region is negatively correlated with its expression(F=47.270,P<0.0001;Y=-1800×X+38.03),and the PTEN methylation rate is negatively correlated with the prognosis of patients receiving the molecularly targeted drug Sorafenib(χ^(2)=4.313,P<0.05).Conclusion This study successfully established a new method for detecting methylation in the promoter region of PTEN,and the methylation rate of PTEN can be used as one of the targets of HCC diagnosis and targeted therapy.

关 键 词：甲基化 肝细胞癌 下一代测序技术 

分 类 号：R735.7[医药卫生—肿瘤]