【作者】 王宁；

【导师】 杜智；

【作者基本信息】 天津医科大学 ， 外科学， 2012， 硕士

【摘要】 肝细胞癌(以下简称肝癌)是在世界范围内的常见恶性肿瘤之一,严重威胁人类健康。因此,肝癌的防治成为肿瘤研究的重要课题。我们前期研究发现,Dickkopf-3(DKK3)基因的甲基化可作为一种诊断或预后的候选标志物；但同时也注意到,与DKK3甲基化在癌组织中明显的组织特异性相比,其mRNA表达在肝细胞癌与癌旁组织中的差异并不明显,而且与其基因的甲基化状态也未发现相关性。为进一步阐明DKK3基因表达调控方式及其在肿瘤发生的作用,本研究通过对Wnt途径拮抗剂家族中DKK3基因在肝细胞癌中的蛋白表达及亚细胞定位分析,探讨了DKK3甲基化状态与蛋白表达的相关性及其蛋白表达的临床意义。目的：检测Wnt途径拮抗剂家族中DKK3基因在肝细胞癌中的蛋白表达及亚细胞定位,分析基因甲基化状态与DKK3蛋白表达的相关性,探讨该蛋白表达在肝细胞癌中的临床意义。方法：采用细胞免疫荧光和免疫组织化学方法,分别检测肝癌细胞系SMMC-7721、HepG2、正常肝细胞系HL-7702和外周血单核细胞(PBMC)以及43例成对肝细胞癌与癌旁组织中DKK3的蛋白表达；甲基化特异性PCR技术检测其中31对癌及癌旁组织中DKK3基因的甲基化状态,统计学分析DKK3基因甲基化与蛋白表达二者之间的关系；结合病例资料分析DKK3蛋白表达与临床病理特征的关系。结果：DKK3蛋白在肝细胞癌组织中的高表达率为67.4%(29／43),显著高于相应癌旁组织中的41.8%(18／43)(P=0.017)。对于单个病例的成对组织来说,癌与癌旁组织中DKK3表达程度相似(同为低表达或高表达)的有24例(55.81%),癌组织中DKK3表达高于癌旁组织的有15例(34.88%),而低于癌旁组织的仅有4例(9.30%),这一比较也可表明,相对于癌旁组织,DKK3在癌中的表达呈增高趋势；而且在SMMC-7721、HepG2等肝癌细胞系中也都呈明显细胞核内表达的趋势。肝细胞癌组织中DKK3基因甲基化发生率为90.3%(28／31),显著高于相应的癌旁组织中的64.5%(20／31)(P=0.015),统计学分析表明,DKK3基因的甲基化状态与蛋白表达无明显相关性(P>0.05)。肝细胞癌组织中DKK3表达与患者复发(P=0.022)、A／G(白蛋白／球蛋白)及ALP(碱性磷酸酶)状态(P=0.025；0.038)有关,而与患者的其它临床病理特征(性别、年龄、肿块数目、是否肝硬化、Child-pugh分级、肿瘤大体类型、肿瘤生长方式、肿瘤直径及TNM分期等)未见明显相关(P>0.05)。Kaplan-Meier生存曲线显示,DKK3高表达的肝细胞癌患者总体生存率为48.3%,无瘤生存率为20.7%；而DKK3低表达的肝细胞癌患者总生存率为71.4%,无瘤生存率为64.3%。相比之下,DKK3低表达的肝细胞癌患者总生存率和无瘤生存率明显高于高表达者(总体生存率P=0.049；无瘤生存率P=0.001)。结论：肝细胞癌组织中DKK3蛋白的表达高于癌旁组织,且成明显细胞核内表达趋势,其该基因的蛋白表达与其甲基化状态无关。这些结果表明DKK3在肝细胞癌中的作用并非典型Wnt途径拮抗剂所起的肿瘤抑制基因的功能,而且定性方法检测到的甲基化状态也并非一定会导致蛋白表达的完全失活。此外,DKK3在癌组织中表达可以作为一个判断肝细胞癌预后的指标。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the most common malignancies threatening human health worldwidely. Our previous study of Dickkopf-3(DKK3) gene methylation and clinical significance in hepatocellular carcinoma suggest that methylation of the DKK3gene can be used as a protential marker for diagnosis or prognosis. Meanwhile we also noted that, contrary to the significant specificity of DKK3methylation in cancer tissues, the differences of DKK3’s mRNA expression between cancer and corresponding adjacent tissues seemed not obvious, moreover no correlation between gene methylation and mRNA’s expression was found. To clarify the regulation of DKK3gene expression and its role in tumorigenesis, our study analyze the correlation between DKK3gene methylation and its protein expression, and further investigated clinical implication of DKK3expression in HCC by examining the expression and subcellular localization of DKK3in HCC.AIM:To examine the expression and subcellular localization of Wnt antagonist Dickkopf-3in hepatocellular carcinoma, analyze the correlation between DKK3gene methylation and its protein expression, and investigate clinical implication of DKK3expression in HCC.METHODS:First, DKK3protein expression in liver cancer cell line SMMC-7721, HepG2, normal liver cell line HL-7702, peripheral blood mononuclear cell (PBMC) and43pairs of HCC and corresponding adjacent tissues were examined by immunofluorescence or immunohistochemistry. Then, DKK3methylation status were assayed by methylation-specific PCR in31cases of HCC and paired adjacent tissues, and the correlation between DKK3methylation and its protein expression were statistically analyzed; Finally, The correlation between DKK3protein expression and clinical pathological data was analyzed.RESULTS:The high expression level of DKK3protein occurred in67.4%(29/43) of HCC tissues; such frequency was significantly higher than that of41.8%(18/43) in the paired adjacent tissues (P=0.017). For the single case of paired tissue, DKK3expression has similar levels in HCC tissues and adjacent tissues is24cases(55.81%), there is15cases(34.88%) that DKK3expression is higher in HCC tissues than adjacent tissues, but there is only4cases(9.30%) that DKK3expression is lower in HCC tissues than adjacent tissues. This comparison may indicate that, DKK3protein tended to express in cell nucleus in HCC tissues and SMMC-7721, HepG2cell lines. DKK3gene methylation were detected in90.3%(28/31) of HCC tissues, which was significantly higher than that in64.5%(20/31) of the corresponding adjacent tissues (P=0.015). There was no correlation between DKK3methylation status and its protein expression(P>0.05). There was correlation between DKK3expression in HCC tissues and relapse(P=0.002), A/G and ALP(P=0.025;0.038), but no correlation between DKK3expression in HCC tissues and other clinical and pathological features of the patients(gender, age, tumor number, liver cirrhosis, the Child-pugh classification, tumor gross type, tumor growth pattern, tumor size and TNM stage, et al)(P>0.05). Kaplan-Meier survival curve showed that, the overall survival of patients with high expression of DKK3is48.3%, the free-disease survival of patients with high expression of DKK3is20.7%; the overall survival of patients with low expression of DKK3is71.4%, the free-disease survival of patients with low expression of DKK3is64.3%. In contrast, patients with low expression of DKK3had higher overall or free-disease survival than those with high expression (overall survival P=0.049; free-disease survival P=0.001).CONCLUSIONS:The expression of DKK3protein in liver tissues was higher than the paired adjacent tissues, and its subcellular location tended to express in cell nucleus. In addition, there was no correlation between DKK3methylation status and its protein expression. These results suggest that DKK3may not play a tumor-suppressor role as one of typical Wnt antagonist in hepatocellular carcinoma, and the methylation status is not sufficient to lead to complete gene inactivation. Finally, DKK3protein expression in HCC tissues may be used as a prognostic maker for hepatocellular carcinoma.更多还原