原发性肝癌组织RASSF1A异常甲基化与DNMTs及HBV的关系

Correlation between aberrant methylation status of ras association domain family 1A and alteration of DNA methyltransferases, hepatitis B virus infection in hepatocellular carcinoma

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 correlation between aberrant methylation status of ras association domain family 1A （RASSF1A）, transcriptional levels of DNA methyltransferases （DNMTs） and hepatitis B virus （HBV） infection in patients with hepatocellular carcinoma （HCC）. Methods HCC samples in 61 cases were collected. Aberrant methylation status of RASSF1A was detected using methylation specific PCR （MSP）. Transcriptional levels of DNMT1 ,DNMT3A and DNMT3B was measured using semiquantitative reverse transcriptase-PCR（RT-PCR）. Results RASSF1A gene with abnormal methylation in initiation zone was found in 45 cases with HCC among 61 patients （73. 8% ）. Further analysis revealed RASSF1A methylated in 32 of 47 HBV infected cases and in 13 of 14 uninfected cases. However, there was no significant association between methylation status of RASSF1A and HBV infection （ X2 = 2. 260, P = 0. 133）. Compared with normal control, DNMTs was up-regulated in all HCC samples, HCC cell lines and HBV infected group. Analysis within each group indicated that DNMT3A and DNMT3B levels of HCC increased in MSP positive cases （ t = 3. 494, P 〈 0. 01 ; t = 4. 258, P 〈 0. 01 respectively）, while DNMT1 decreased when compared with negative ones（t = 3. 221 ,P 〈 0. 05）. DNMT3B in MSP positive cases of HBV infected samples significantly increased when compared with negative ones （ t = 12. 171, P 〈 0. 01 ）. Conclusion Transcriptional levels of DNMTs are highly associated with aberrant methylation of RASSF1A. A correlation between HBV infection and DNMT3B alteration was found in this study.