【作者】 陶然；

【导师】 李兰娟；

【作者基本信息】 浙江大学 ， 内科学， 2012， 博士

【摘要】 肝癌是世界上最常见的恶性肿瘤之一,也是第三位最常见的致死肿瘤。在亚洲,尤其是东亚,是肝癌的高发地区,肝癌的病因复杂,其中大多数是由于肝炎病毒的感染,主要是HBV病毒和HCV病毒。在中国的肝癌患者,大多由于长期的HBV病毒感染,造成慢性肝炎肝硬化后,最终发展为肝细胞肝癌。目前,肝癌的发病机制仍然不十分清楚,对肝癌的诊断,治疗和预后的判断主要还是依赖临床的病理资料和放射学的检查。但是放射学的检查主要受限于小肝癌和早期肝癌的分辨,而拥有相同的病理资料的患者,肝癌的发展也相差较大。另外肝癌目前诊断通常过晚,即使通过肝切,肝移植手术,依然存在着高复发,高转移率的风险,因此通过找到合适的生物分子标志,对患者进行合理的诊断和预后的判断有重要的意义。DNA异常的甲基化作为一个重要表观遗传学的改变,被认为是一项重要的肿瘤发病机制。在肿瘤中表现出整体水平的低甲基化和某些局部区域的高甲基化。在之前的肝癌研究中,GSTP1,RASSF1A,RIZ1,APC, SOCS1和E-cadherin等启动子区域等都发现呈现出异常的高甲基化。随着全基因扫描技术的发展,肿瘤的整体甲基化谱逐渐变得清晰。之前的研究报道发现一系列潜在的肝癌生物标记,但是报道之间能重叠的基因却很少,这除了芯片平台本身存在的差异,也发现不同的病原学背景,病理生理过程和组织学特征的肝癌有着不同的整体甲基化谱。在本次研究中,我们只筛选研究乙肝肝硬化肝癌。在第一部分中将通过全基因的甲基化芯片对肝癌、癌旁和正常肝组织进行扫描,探索可以作为潜在的肝癌诊断的生物标记。第二部分,对比不同预后的肝癌标本的整体甲基化谱,探索可以作为潜在的判断肝癌预后的基因。第一部分纯化的单个乙肝肝癌细胞的全基因甲基化谱目的：之前的研究主要都集中在肝组织的甲基化谱中,肝组织的复杂组成可能会对肝癌真实的甲基化状态产生影响。本研究的目的通过分离纯化乙肝肝癌细胞,来建立乙肝肝癌的整体甲基化谱,探索乙肝肝癌的发生机制,寻找潜在可作为乙肝肝癌诊断的生物标记。方法和结果：纯化分离的单个肝细胞来自乙肝肝癌,通过Illumina Infinium Human Methylation27BeadChips建立整体的甲基化谱。并通过结合亚硫酸氢钠处理和酶解分析法及亚硫酸氢盐测序,对20个在肝癌中显著高甲基化的基因进行验证。在本次研究中,我们发现乙肝肝癌的肝细胞有独特的全基因甲基化谱。无监督的聚类分析显示甲基化谱可以被分为三组：肝癌的肝细胞组,癌旁的肝细胞组和正常的肝细胞组。异常甲基化基因主要富集在间隙连接、钙离子信号通路、细胞粘附分子、自然杀伤细胞介导的细胞毒作用、细胞通讯等信号通路中。在肝癌肝细胞组的20个最显著高甲基化的基因中,7个新发现的基因(WNK2,EMILIN2, TLX3, TM6SF1, TRIM58, HIST1H4F和GRASP)均被证实在肝癌中出现启动子区的DNA高甲基化,而在配对的癌旁中则DNA低甲基化,这些发现在之前肝组织的标本中还没有被报道。结论：乙肝肝癌的肝细胞存在着特异的甲基化谱可与癌旁和正常肝细胞相区别,异常甲基化基因主要富集的信号通路和细胞永生抑制凋亡相关。同时新发现七个基因(WNK2, EMILIN2, TLX3, TM6SF1, TRIM58, HIST1H4F和GRASP)启动子的高甲基化可作为潜在的乙肝肝癌诊断的生物标记。第二部分乙肝肝癌肝移植术后与预后相关的甲基化谱研究目的：由于肝癌的高复发性和转移率,肝移植术后患者的预后依然欠佳。目前尚缺少早期复发肝癌整体甲基化谱的研究,也缺少能准确判断肝癌预后的生物学标记以筛选出高危病例,进一步指导临床治疗。本实验的目的就是要建立乙肝肝癌早期复发病例的整体甲基化谱,筛选出与乙肝肝癌复发相关的异常甲基化的基因,并评估其在肝移植术后乙肝肝癌复发预测中的价值。方法和结果：应用illumina Infinium27k Human DNA methylation BeadChip对两组各三例肝移植术后不同预后的乙肝肝癌组织进行全基因组的甲基化基因扫描,同时以配对的癌旁和正常肝组织作为对照。用随机方差模型找到四组1164个差异甲基化位点,用差异甲基化基因表达趋势分析,筛选出300个与乙肝肝癌复发相关的基因(P<0.05)。通过对特异基因进行疾病差异显著性功能分析、信号通路分析和信号网络分析,我们挑选了16个基因进行进一步筛选。通过临床样本中这些基因的表达量和启动子甲基化验证,发现HIST1H2BH、SOCS2的启动子高甲基化和乙肝肝癌患者肝移植术后的预后密切相关,其中SOCS2可以作为一个独立的预测乙肝肝癌肝移植术后复发的因素。结论：肝移植术后早期复发的乙肝肝癌有着独特的异常甲基化谱,其特有的基因富集通路和生物调控网络,和肿瘤侵袭转移能力十分相关。HIST1H2BH，SOCS2启动子区的异常高甲基化和肝移植术后乙肝肝癌的无瘤生存期密切相关,其中SOCS2可作为预测肝移植术后乙肝肝癌复发的独立危险因素。更多还原

【Abstract】 Hepatocellular carcinoma (HCC) is one of the common malignancies and the third most ordinary cause of cancer-related mortality worldwide. The cause of primary hepatocelluar carcinoma is complicated, in Asia, which is one of the highest HCC incidence areas, most closely related to virus infection, such as hepatitis B virus (HBV) and hepatitis C virus (HCV). It is deduced that most of HCC patients in China were injuries from hepatitis infection and the long term chronic inflammation, accumulated necrosis and regeneration of hepatocytes, finally leaded to cell malignant transformation.The mechanism of hepatocarcinogenesis was still unclear recently, and the judgment on the diagnosis, treatment and prognosis of HCC were still highly depends on pathology and radiology. However, radiological examination is limited to its resolution when dealing with the small HCC and early HCC, and clinicopathologic features could be ambiguous due to the heterogeneous nature of cancer. The high rates of recurrence and metastasis of HCC was still a major obstacle for patient’s long-term survival, even after liver resection and liver transplantation. It’s an urgent need to identify the optimal targets and develop a reliable system for HCC diagnosis and evaluation of early recurrenceAberrant DNA methylation is believed to play an important role in hepatocarcinogenesis as a major epigenetic change, which is associated with global hypomethylation and specific promoter hypermethylation in cancer. Promoter hypermethylation of GSTP1, RASSF1A, RIZ1, APC, SOCS1and E-cadherin were considered as potential biomarkers in HCC. With the development of genome-wide screening, the global methylation profiling of cancer become clearer than ever before. However, in previous studies, although a number of genes were identified as biomarkers of tumor, only a few were confirmed by independent researches. After ruling out the difference between gene array platforms, different gene methylation profiles were found in HCC cases with different etiological backgrounds, pathophysiological processes, and histological characteristics were found with different gene methylation profiles.In this study, we focused on HBV-related HCC(HBHC) secondary to liver cirrhosis. In the first part, we analyzed the aberrant promoter methylation of cancer samples and adjacent liver samples using Methylation BeadChips, to seek for the potential novel biomarkers in HBHC. In the second part, we compared the global methylation profiles of HBHC subclasses with high-risk and low-risk, explored the putative biomarker for the prediction of early recurrence. Part I Methylation Profile of Single Hepatocytes Derived from Hepatitis B Virus-Related Hepatocellular CarcinomaObjective:Previous studies on HCC methylation profiles have focused on liver tissue, and the heterogeneity of liver composition may impact the genuine methylation status of HCC. The aim of the present study was to establish the methylation profile of hepatocytes isolated from primary tissues, to elucidate the mechanism of hepatocarcinogenesis and to identify the potential biomarker for the diagnosis of HBHC.Methods and Findings:The global methylation profile of single hepatocytes isolated from liver tissue of HBHC was analyzed using Illumina Infinium Human Methylation27BeadChips. Combined bisulfite restriction analysis (COBRA) and bisulfite sequencing were used to validate the20significant hypermethylated genes identified.In this study, we found many noteworthy differences in the genome-wide methylation profiles of single hepatocytes of HBHC. Unsupervised hierarchical clustering analysis showed that hepatocyte methylation profiles could be classified according to three cell types:hepatocytes of HCC, adjacent hepatocytes and normal hepatocytes. Aberrant methylated genes were enriched in pathways including gap junction, calcium signaling pathway, nature killer cell mediated cytocoxicity, cell adhesion and cell communication. Among the20most hypermethylated genes in the hepatocytes of HBHC,7novel genes (WNK2, EMILIN2, TLX3, TM6SF1, TRIM58, HIST1H4F and GRASP) were found to be hypermethylated in HBHC and hypomethylated in paired adjacent liver tissues; and these findings have not been reported in previous studies on tissue samples.Conclusion:The methylation profile of purified hepatocytes of HBHC distinguishes significantly from that of adjacent non-cancerous hepatocytes and normal hepatocytes. Aberrant methylated genes in hepatocytes of HBHC were enriched in signal pathways associated with cell survival and resistant to apoptosis. The promoter hypermethylation of seven novel genes found in this study have the potential to be biomarkers for the diagnosis of HBHC. Part Ⅱ The DNA methylation profile for prognosis of Hepatitis B Virus-Related Hepatocellular CarcinomaObjective:The prognosis of HCC patients is unsatisfactory even after liver transplantation (LT) due to the high rate of recurrence and metastasis. Even though the methylation profile of HCC with early recurrence was still of scarcity, it was also the promising biomarkers for HCC prediction of the risk of early recurrence. The aim of the present study was to establish the global methylation profile of HBHC with early recurrence; to find out aberrant methylated genes associated with poor prognosis; and finally to evaluate the effect of those new biomarkers in the prediction.Methods and Findings:In the present study, genome-wide DNA methylation profiles of HBHC subclasses with high-risk and low-risk were established, with adjacent liver tissues and normal liver tissues as control. Random variance model (RVM) f-test was used to filter1164differentially methylated genes from the four groups. Based on change tendency of methylation status under the four groups, we identify300genes associated with poor prognosis of HBHC. After combined the analysis of gene ontology analysis, KEGG pathway and the map of signal net,16genes were selected for future validation. With the verification of gene expression and promoter methylation in clinical samples, promoter hypermethylation of HIST1H2BH and SOCS2were found closely related with the prognosis of HBHC after LT, and SOCS2methylation status was an independent prognostic factor for disease-free survival of HBHC after LT.Conclusion:HBHC subclasses with early recurrence displayed distinct genome-wide DNA methylation profiles, with unique genes enriched in signal pathway and sig-net map associated with tumor invasion and metastasis. Promoter hypermethylation of HIST1H2BH and SOCS2were found closely related with the prognosis of HBHC after LT. And DNA methylation in the promoter region of SOCS2is an independent risk factor influencing HBHC recurrence after LT and thus may be used as a potential biomarker for tumor recurrence of HBHC after LT.更多还原