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目的探讨原发性肝癌ras相关结构域家族1A(ras association domain family 1A, RASSF1A)异常甲基化与肿瘤组织中DNA甲基转移酶(DNA methyltransferases,DNMTs)转录水平变化及HBV感染的关系。方法采用甲基化特异性PCR(methylation specific PCR,MSP)技术检测 61例原发性肝癌组织RASSF1A基因异常甲基化情况,RT-PCR法检测其中三种主要DNA甲基转移酶(DNMT1、DNMT3A、DNMT3B)水平的变化。结果61例肝癌标本中RASSF1A基因启动子区异常甲基化45例(73．8％);HBV感染者47例(77．1％),其中MSP阳性32例;无HBV感染者14例,其中MSP阳性13例,RASSF1A基因异常甲基化与HBV感染之间的关系无统计学意义(X2=2．260, P=0．133)。肝癌组、肝癌细胞系组及HBV感染组DNMTs表达水平与正常对照组相比均显著升高; 组内比较显示肝癌组中DNMT3A、DNMT3B在MSP阳性患者中较阴性患者升高(分别为t=3．494, P<0．01:t=4．258,P<0．01),而DNMT1下降(t=3．221,P<0．05);HBV感染组中,MSP阳性组织其 DNMT3B较阴性者升高(t=12．171,P<0．01)。结论肝癌组织DNMTs水平变化与RASSF1A异常甲基化有密切关系;HBV感染与DNMT3B转录水平变化相关。
Objective To investigate the relationship between abnormal methylation of ras association domain family 1A (1A) and DNA methyltransferases (DNMTs) transcription and HBV infection in primary hepatocellular carcinoma. Methods Methylation specific PCR (MSP) was used to detect abnormal methylation of RASSF1A gene in 61 cases of primary hepatocellular carcinoma (HCC). Three major DNA methyltransferases (DNMT1, DNMT3A , DNMT3B) level changes. Results There were 45 cases (73.8%) of abnormal methylation of RASSF1A gene promoter in 61 cases of hepatocellular carcinoma; 47 cases (77.1%) were HBV infected, of which 32 cases were MSP positive, 14 cases were without HBV infection MSP positive in 13 cases, RASSF1A gene abnormal methylation and HBV infection was not statistically significant (X2 = 2.260, P = 0.133). The expression of DNMTs in hepatocellular carcinoma, hepatoma cell line and HBV infection group were significantly higher than those in normal control group. The intra-tumor comparison showed that DNMT3A and DNMT3B were higher in MSP-positive patients than in negative patients (t = 3.494, P <0.01: t = 4.258, P <0.01), but DNMT1 decreased (t = 3.221, P <0.05). In HBV infected group, the expression of DNMT3B More negative than those (t = 12.171, P <0.01). Conclusion The changes of DNMTs level in HCC tissues are closely related to the abnormal methylation of RASSF1A. HBV infection is related to the changes of DNMT3B transcription level.