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甲基化特异性PCR联合显微检测技术研究DNA甲基化及微核形成对肝细胞癌发生风险的影响

Effects of DNA methylation and micronucleus formation on pathogenesis of hepatocellular carcinoma by methylation-specific polymerase chain reaction and microscopy

摘要:

目的 探讨肝细胞癌(HCC)患者CDH1 、GSTP1和RASSF1A基因启动子区域异常甲基化以及淋巴细胞微核对肝细胞癌发生风险的影响.方法 应用甲基化特异性PCR(MSP)技术对32例HCC患者肿瘤组织和癌旁组织的CDH1、GSTP1和RASSF1A基因启动子区域进行甲基化检测.未经治疗前抽取HCC患者外周血进行体内淋巴细胞微核试验,比较HCC自发微核率和健康人群对照组(n=50)的差异.结果 32例HCC组织中,CDH1、GSTP1和RASSF1A基因甲基化检出率分别是43.8%(14/32)、68.8% (22/32)和43.8% (14/32),均高于相应的癌旁组织[25%(8/32)、46.9%(15/32)、15.6%(5/32)].只有RASSF1A基因在HCC组织和癌旁组织中的甲基化检出率差异有统计学意义(43.8%比15.6%,P<0.05).81.3%(26/32)的HCC和65.6%(21/32)的癌旁组织中有1个或1个以上基因发生了甲基化.3个基因的甲基化程度与患者年龄、性别、肿瘤型、分化程度等临床病理参数均无统计学关联.HCC组微核率与对照组差异有统计学意义(1.38‰比0.47‰,P<0.05);同时HCC患者自发微核率随着病理分化变差而呈现逐渐增高的趋势,高中、中、中低分化HCC患者微核率分别为1.30‰、1.33‰和1.64%‰.结论 CDH1、GSTP1和RASSF1A基因启动子区域异常甲基化和染色体不稳定存在于HCC的发生发展中.基因启动子区域异常甲基化可能与HCC发生早期密切相关.甲基化检测对于HCC高危人群筛选以及HCC早期辅助诊断具备一定的应用价值.

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Objective To explore the effects of aberrant methylation in the CDH1,GSTP1 and RASSF1A gene promoter CpG region and lymphocyte micronucleus on the pathogenesis of hepatocellular carcinoma(HCC).Methods The methylation-specific polymerase chain reaction(MSP)was employed to investigate the promoter methylation of CDH1,GSTP1 and RASSF1A genes in the tumor and adjacent tissues in 32 patients with HCC.The peripheral blood was sampled from patients with HCC for subsequent micronucleus test for lymphocytes,prior to the treatment.The differences of the spontaneous micronucleus rate in HCC patients and healthy population control group(n=50) were compared.Results Tumor tissues of patients with HCC yielded higher assay sensitivity for detection of methylation of CDH1,GSTP1 and RASSF1A genes[43.8%(14/32),68.8%(22/32) and 43.8% (14/32)] than adjacent tissues [25% (8/32),46.9% (15/32) and 15.6% (5/32)].Nevertheless,the between-group difference in methylation for various genes did not reach statistical significance with exception of RASSF1A gene(43.8% vs 15.6%,P<0.05).Methylation in one or more of the three genes was noted in tumor tissues[81.3%(26/32)] and adjacent tissues[65.6%(21/32)].Additionally,the status of methylation was not statistically associated with the age,sex,type of tumor and tumor differentiation.Patients with HCC,who showed a raised risk of automatic micronucleation with the advanced pathologic differentiation,were featured by a higher rate of micronucleation when compared with normal controls(1.38‰ vs 0.47‰,P<0.05).The frequency of micronucleation was 1.30‰,1.33‰ and 1.64‰o in patients with moderate-to-high,moderate and low differentiation of HCC.Conclusions Genetic instability and promoter methylation of CDH1,GSTP1 and RASSF1A genes may play a vital role in the pathogenesis of HCC.Early stage HCC may be closely linked to aberrant promoter region methylation.Methylation detection is valuable in screening high-risk population and establishing adjunct early diagnosis of HCC.

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