肝癌抑癌基因GSTP1、p16、RIZ1、RASSF1A异常甲基化检测及其临床意义

Study on aberrant methylation detection and clinical significance of suppressor gene GSTP1, p16,RIZ1 and RASSF1A in hepatocellular carcinoma

目的研究肝细胞癌中的GSTP1、p16、RIZ1、RASSF1A 4种基因启动子区域甲基化状态的表达差异,并探讨其在肝癌发生中的作用。方法运用甲基化特异性聚合酶链反应(methylation-specific PCR,MSP)技术,分别检测35例肝癌组织及其相应的癌旁组织和20例正常对照肝组织中4种候选抑癌基因(GSTP1、p16、RIZ1、RASSF1A)启动子区域的甲基化表达情况,并结合临床病理资料进行分析。结果在肝细胞癌、癌旁及正常对照肝组织中检测到GSTP1基因启动子区的甲基化率分别是57.1(20/35),25.7%(9/35),0%(0/20);p16基因启动子区甲基化率分别是54.3%(19/35),37.1%(13/35),0%(0/20);RIZ1基因启动子区甲基化率分别是68.6%(24/35),14.3%(5/35),0%(0/20);RASSF1A基因启动子区甲基化率分别是88.6%(31/35),74.3%(26/35),10%(2/20),其中癌组织中GSTP1和RIZ1基因甲基化率均显著高于相应的癌旁组(P<0.01)和正常对照肝组织(P<0.01),癌组织中p16、RASSF1A启动子区甲基化率高于癌旁组织,但二者无统计学意义(P>0.05),但明显高于正常对照肝组织(P<0.01)。在肝癌组织中肿瘤有包膜侵犯者GSTP1基因甲基化阳性率明显高于无包膜侵犯者(P<0.05);乙肝表面抗原(HbsAg)阳性的肝癌患者p16基因甲基化阳性的比率要显著高于HbsAg阴性者(P<0.05);而RIZ1、RASSF1A与临床病理资料间均没有统计学意义(P>0.05)。结论 RIZ1、GSTP1基因甲基化状态具有肝癌特异性,或可作为临床肝癌辅助诊断的分子标记物。慢性HBV感染可能是导致p16甲基化而失活的原因,GSTP1基因启动子甲基化可能与肝细胞癌的侵袭性有关。

Objective To investigate the aberrant methylation status of GSTP1,p16,RIZ1 and RASSF1A in hepatocellular carcinoma,and to assess their role in hepatocarcinogenesis.Mehthods Methylation status of GSTP1,p16,RIZ1 and RASSF1A genes promoter were detected by methylation specific PCR （MSP） in 35 cases of HCC and adjacent non-cancerous tissues,20 normal liver tissues as control.The relationship between methylation and clinicopathologic factors were analyzed.Rusults GSTP1 promoter methylation rates were 57.1％ （20/35）,25.7％ （9/35）,0％ （0/20） and p16 promoter methylation rates were 54.3％（19/35）,37.1％ （13/35）,0％ （0/20） in HCC,adjacent non-cancerous tissues and normal control tissues,respectively.While RIZ 1 promoter methylation ratios were 68.6％ （24/35）,14.3％ （5/35）,0％ （0/20） and RASSF1A promoter methylation ratios were 88.6％ （31/35）,74.3％ （26/35）,10％ （2/20） in HCC,adjacent non-cancerous tissues and normal control tissues,respectively.The rates of methylation of GSTP1 and RIZ1 genes in HCC were significantly higher than those in adjacent non-cancerous tissues （P＜0.01） and normal control tissues （P＜0.01）.The rates of methylation of p16 and RASSF1A genes in HCC were higher than those in adjacent non-cancerous tissues,but showed no significance （P＞0.05）.The rates of methylation of p16 and RASSF1A genes in HCC were significantly higher than those in normal control tissues （P＜0.01）.The rate of methylation of GSTP1 gene was significantly higher in tumor with capsular invasion than that in tumor without capsular invasion （P＜0.05）; the rate of methylation of p16 gene was significantly higher in tumor with HbsAg positive than that in tumor with HbsAg negative （P＜0.05）;promoter methylation of RIZ1 and RASSF1A had no statistical correlation with the clinicopathologic factors （P＞ 0.05）.Conclusion The methylation status of GSTP1 and RIZl promoter has specificity to HCC,they may serve as the molecular biology markers for the auxiliary diagnosis of HCC.Chronic HBV infection may be lead to promoter methylation of p16,and the promoter methylation of GSTP1 probably has effect on the invasion of HCC.