【摘要】 目的观察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,分析了24例肝癌标本、4株肝癌细胞株(SMMC7721、HepG2、BEL7402和BEL7703)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SMMC7721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和早期诊断,以及预后的判断。更多还原

【Abstract】 ObjectiveTo 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 analyze the correlation between aberrant DNA methylation and elinical relative factors in HCC.MethodsRT-PCR and MS-PCR strategies,combined with bisulfite DNA sequencing and TaqⅠdigestion,were used for analyzing the status of aberrant promot- er methylation of RASSF1A,in 24 primary HCC and adjacent noncanerous tissues,and 4 HCC cell lines. The reactivation of RASSF1A expression was assessed after the 3 HCC cell lines were treated with demethylating agent 5’-aze-2’-deoxyeytideing (5’-Aza-CdR).ResultsRASSFIA mRNA was not de- tected in 66.7% of the HCC.Three of 4 HCC cell lines missed the expression of RASSF1A.By contrast, RASSFIA was expressed in all normal liver tissues and normal liver cell lines (L02).MS-PCR analysis demonstrated that RASSFIA promoter region hypermethylation was found in 83.3% of HCC and 4 cell lines.No methylation was detected in normal liver tissues 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 types.Three RASSF1A-nonexpressing cell lines were all re-expressed after treatment with 5’-Aza-CdR.ConclusionRASSF1A inactivation might be caused by epigenetic and genetic mechanisms in HCC.Testing for RASSFIA methylation should become useful in HCC early detection and diagnosis and could be utilized as a molecular marker to estimate the prognosis of HCC.更多还原