摘要
肝细胞癌(以下简称肝癌)是临床上最常见的恶性肿瘤之一,其发病率和在肿瘤相关死亡中占据的比例均很高,严重威胁着人类的健康,因此,肝癌的防治成为肿瘤研究的重要课题。肝癌的发生发展是一个涉及多基因多步骤的复杂过程,其分子机制仍未完全阐明,抑癌基因启动子区异常高甲基化可导致抑癌基因失活,是肝癌发生的重要原因之一。本文通过检索相关文献,分析肝癌发生的分子途径,选取Wnt途径中WIF-1基因作为研究对象,检测其在肝癌中的甲基化和表达水平,结合临床资料探讨其临床意义。
目的：探讨WIF-1基因启动子区甲基化的状态与WIF-1 mRNA表达和肝癌发生发展的关系,评价检测肝癌WIF-1基因启动子区甲基化和基因表达的临床意义。
方法：收集53例成对的肝癌及相应癌旁组织,采用实时定量甲基化特异性PCR (Q-MSP)技术对组织中WIF-1基因的甲基化水平进行相对定量检测；应用实时定量RT-PCR (Q-RT-PCR)检测53例成对肝癌及相应癌旁组织、22例肝炎后肝硬化、4例慢性肝炎和11例正常肝组织的WIF-1 mRNA的表达水平,分析WIF-1甲基化状态与mRNA表达水平的关系以及两者与临床资料间的相关性。
结果：Q-MSP检测结果表明WIF-1基因在肝癌组织和癌旁组织中甲基化阳性例数分别为20例(37.7%)和3例(5.7%),癌组织中WIF-1基因甲基化频率显著高于癌旁组织(P<0.001)；ROC分析表明WIF-1基因甲基化水平的差异可以高效地鉴别癌组织与非癌组织(AUC=0.714,P<0.001)；Kaplan-Meier生存曲线分析显示甲基化阳性病例组的无瘤生存期显著低于甲基化阴性病例组(P<0.05)；WIF-1基因的甲基化状态与性别、年龄、肿块数目、是否肝硬化、Child-pugh分级及TNM分期等因素均未见明显相关(P>0.05)。
对WIF-1 mRNA在各组组织中表达水平的分析显示：正常肝组织与肝硬化、肝癌、癌旁组织组间的表达均存在统计学差异(P<0.05),正常肝组织组基因表达水平高于除慢性肝炎的其它各组；肝癌组基因表达水平显著低于癌旁组织组(P<0.05)。WIF-1基因表达水平与临床资料相关性分析未见明显相关性,高表达组和低表达组的无瘤生存期未见统计‘学差异(P>0.05)。
53例成对肝癌及癌旁组织标本中,肝癌组织相对于癌旁组织WIF-1表达显著下调的有31例,其中,在甲基化阳性、阴性标本里分别为11例(55%)和20例(60.6%)。肝癌与癌旁组织WIF-1 mRNA表达的下调与甲基化状态之间未见相关性(P>0.05)。
结论：检测WIF-1基因的甲基化状况有助于肝癌的诊断与预后评估,可能作为一种具有临床诊断价值的分子标志物。WIF-1基因在肝癌组织中的表达下调除受基因启动子甲基化影响外可能还存在其他机制。
Hepatocellular carcinoma (HCC) is one of the most common life-threatening malignancies in the world with a high mortality and morbility. Carcinogenesis and development of HCC is a complicated process with multiple gene participated and multiple phase developed, and the molecular mechanism of hepatocarcinogenesis remains unclear. Aberrant methylation of tumor suppressor genes is an important reason of tumorigenesis.So, we selected WIF-1 gene as a target for testing the methylation and expression level in tissue samples and estimating their clinical significance.
AIM:To investigate the level of promoter methylation and geneexpression of WIF-1 in hepatocellular carcinoma. To estimate the value of aberrant methylation and expression of WIF-1 as a biomarkerfor diagnosis and prognosis of HCC.
METHODS:We collected 53 tumor and adjacent non-tumor tissuesamples, and tested methylation status of WIF-1 in these samples quantitatively by quantitative methylation specific polymerase chain reaction (Q-MSP).The expression of WIF-1 was determined by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). Then we investigated the correlation among methylation status, mRNA expression, and clinicopathological data.
RESULTS:The results of Q-MSP analysis indicated that the methylation of WIF-1 in HCC increased significantly compared with adjacent non-tumor tissues(P< 0.001).ROC analysis demonstrated methylation of WIF-1 could distinguish the tumor lesions from non-malignant tissuesefficiently (AUC=0.714, P<0.001).The methylation of WIF-1 had no significant correlation with patients gender, age, tumor number, liver cirrhosis status, Child-pugh grade, and TNM stage(P>0.05). Kaplan-Meier survival analysis showed the patients with lower methylation had a better disease-free survival time(P<0.05).
The analysis of Q-RT-PCR showed that normal liver tissues had a significantly higher expression of WIF-1 than other kinds of tissues except for chronic hepatitis tissues.Tumor tissues compared with adjacent non-tumor tissueshad a lower expression level of WIF-1(P<0.05).
Among 53 HCCs and their corresponding nontumorous liver tissues, the prominent down-regulation of WIF-1 (by>2-fold) was observed in 31 of 53 HCCs compared with nontumorous liver tissues.The number of samples with prominent down-regulation were 11(55.0%) in aberrant hypermethylated HCCs and 20(60.6%) in the tumor samples without hypermethylation, respectively.
CONCLUSION:The promoter hypermethylation of WIF-1 is a common event in HCC and may plays an important role at the stage of hepatocarcinogenesis, and the methylation level of WIF-1 may server as a potential biomarker for diagnosis and prognosis of HCC. WIF-1 methylation and other factors mediated the expression of WIF-1.
引文
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DNA甲基化是肿瘤发生中的早期事件,对一些肿瘤特异基因的甲基化状态进行筛查有望用于肿瘤的早期诊断。DNA甲基化是一种可逆过程,应用DNA甲基化抑制剂可以使一些重要基因发生去甲基化,恢复正常功能。因此,敏感性、特异性更高的肝细胞癌甲基化基因、更方便的检测方法、特异性更强的去甲基化药物等需要进一步去研究。而且肿瘤表观遗传治疗的有效性已在体外及动物实验中得到证实,部分临床试验也取得了非常满意的结果,但是肝细胞癌的发生发展是一个多因素参与的复杂过程,不能简单地以DNA的甲基化来解释,所以DNA甲基化如何与其他致癌因素协同作用促进肝细胞癌的发生发展仍需进一步研究。目前,肿瘤表观遗传治疗有可能成为肿瘤综合治疗的一部分,对提高化疗、放疗的敏感性,减少肿瘤的复发和转移起一定作用。而且更深入地研究DNA甲基化和组蛋白密码的关系有望在基因调控和肝细胞癌发生上获得新的突破,并可能提出新的治疗靶点,将成为肝细胞癌基因治疗今后研究的一个方向。
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