【摘要】 目的研究肝癌组织中的抑癌基因RASSF1A的表达情况和由于启动区异常甲基化导致其基因外失活的状况,并分析DNA异常甲基化与肝癌临床相关因素之间的关系。方法利用RT-PCR和MS-PCR的方法,结合DNA测序和TaqⅠ酶切消化法,分析24例肝癌标本、4株肝癌细胞株中RASSF1A基因的表达情况,以及其基因启动区异常甲基化的情况。采用甲基化抑制剂5′-Aza-CdR处理肝癌细胞株,观察RASSF1A重新表达的情况。结果66·7%的肝癌组织未表达RASSF1A;4株肝癌细胞株中仅1株检测到RASSF1A的表达。83·3%的肝癌组织及4株肝癌细胞株都发生了异常甲基化,而正常肝组织和正常肝细胞株(L02)中却未发现甲基化。甲基化与肝硬化、乙肝表面抗原、肿瘤分化程度及血管浸润和远处转移有相关性。原来RASSF1A表达失活的3株肝癌细胞株经甲基化抑制剂5′-Aza-CdR处理后,又重新恢复了表达。结论基因转录启动区的异常甲基化是导致肝癌中RASSF1A表达失活的主要原因。检测RASSF1A启动区异常甲基化可作为一种有潜在应用价值的生物分子指标来用于肝癌的早期发现、早期诊断以及预后的判断。更多还原

【Abstract】 Objective To study the expression of tumor suppressor gene RASSF1A and the status of aberrant promoter hypermethylation in hepatocellular carcinoma(HCC),which induces the epigenetic inactivation of tumor suppressor gene,RASSF1A,and analyzing the correlation between aberrant DNA methylation and clinical relative factors in HCC.Methods RT-PCR and MS-PCR strategies,combined with bisulfite DNA sequencing and TaqⅠ digestion,were used for analyzing the status of aberrant promoter methylation of RASSF1A in twenty-four primary HCC samples and adjacent noncanerous tissues,and four HCC cell lines.To assess reactivation of RASSF1A expression,three HCC cell lines were treated by demethylating agent 5′-aza-2′-deoxycytideing(5′-Aza-CdR). Results RASSF1A mRNA was not detected in 66.7% of HCC.Three of four HCC cell lines missed the expression of RASSF1A.By contrast,RASSF1A was expressed in all normal liver and normal liver cell line(L02).MS-PCR analysis demonstrated that RASSF1A promoter region hypermethylation was found in 83.3% of HCC samples,four cell lines were all detected promoter methylation.No methylation was detected in normal liver and normal cell lines(L02).Aberrant promoter hypermethylation of RASSF1A was correlated with hepatocirrhosis,HBsAg,tumor differentiation grade and metastasis, but not with tumor stage and histological type.Three RASSF1A-nonexpressing cell lines were all re-expressed after treated with 5′-Aza-CdR.Conclusion RASSF1A inactivation might be caused by epigenetic and genetic mechanisms in HCC.Testing for RASSF1A methylation should be useful in early detection and diagosis of HCC and could be utilized as a molecular marker to estimate the prognosis of HCC.更多还原