DNA羟甲基化与肝癌基因表达及临床特征的关联性研究

Liver cancer, hepatic cellular carcinoma (HCC), is one of the common cancers world-wide, and the mechanisms of hepatocellular oncogenesis are unclear. Except for genetic abnormalities, epigenetic abnormalities are also crucial events involved in tumorigenesis, where the epigenetic aberrations could be a key cause of genomic imbalance and deregulatory transcription as a consequence. Of which methylation（5mC）level has been conformed to has close relationship with HCC development and processes. However, the relationship between thydromethylation（5hmC）level and hepatocellular oncogenesis remains unclear...The objective of this project is first to detect the Catalytic hydromethylation/methylation and gene expression at whole genome levels. Afterward, we will map a coordinated transcriptomic and epigenomic landscape of liver cancer through integrating these data using bioinformatics tools. Thesecond part will involve testing the hypothesis that the methylomic/hydro-methylomic and transcriptomic pathways delineated in part 1 indeed play a functional and causal role in the oncogenic and metastatic transformation of liver cells. This will be first done by a systematic siRNA knockdown of critical genes as potential oncogenes and tumor suppressor genes, and then identify the functional pathways causally involved in hepatic cancer. The third part will involve testing of the epigenetic mechanisms of some critical genes and functional cellular pathways leading to hepatic cancer. The fourth part will analyze whether TET gene play role in process of methylomic/hydro-methylomic abnormalities and hepatocellular oncogenesis...In summary, the project's aim is first to chart the transcriptomic and epigenomic (5mC and 5hmC) landscape of liver cancer, identify the critical players andform the basis for human clinical trials in patients with liver cancer. The second is to establish a basis for profound understanding of hepatocellular oncogenesis, and subsequent provide the potential biomarker for diagnosis and therapeutics of liver cancer through integrating epigenomic and transcriptomic data as a systems biology approach.

肝癌是严重影响我国国民健康的恶性肿瘤之一，预后很差，机制并不十分清楚。肿瘤发病除了与遗传学机制相关外，表观遗传学异常也起到重要作用。其中，甲基化（5mC）修饰状态与肝癌的发生发展有密切关系，但与之对应的去甲基化过程中羟甲基化（5hmC）水平与肝癌的关系并不明确。本课题拟研究：（1）肝癌及癌旁组织中羟甲基化和甲基化水平的差异及其两者之间关联性；（2）差异羟甲基化或甲基化与基因表达的关系及其与临床特征的关联性；（3）利用RNA干扰/基因转染技术筛选和鉴定潜在癌基因和抑癌基因，并研究这些潜在肝癌相关基因对肿瘤形成、转移等特性的作用；（4）探讨细胞内去甲基化的分子机制，尤其是羟甲基化相关酶蛋白（TET）与DNA羟甲基化水平异常的关系，及其在肝癌发生发展过程中的作用。通过本项目的实施，为发现肝癌早诊分子标志物和药物靶点奠定理论基础。

肝癌是严重影响我国国民健康的恶性肿瘤之一，预后差，其发病机制并不十分清楚。过去普遍认为，积累染色体改变、基因扩增和突变等与HCC有关。近年来人们发现表观遗传学改变，特别是胞嘧啶5位上异常的DNA甲基化（5mC）与肿瘤发生密切相关。研究报道，癌细胞中的DNA低甲基化，可导致染色体不稳定和致癌基因激活；抑癌基因（TSGs）的高甲基化有助于肝癌的形成。因此，DNA甲基化的准确检测，可以帮助人们深入理解肝癌发生机制，可能对HCC的临床诊断具有潜在的应用价值。.然而，羟甲基胞嘧啶（5hmC）分布的差异，可能会使异常5mC的观察结果复杂化。以前的技术，例如重亚硫酸盐处理和限制性内切酶为基础的技术，无法区分5mC和5hmC，5hmC在样品中的存在，可降低DNA甲基化检测的准确度。.为了全面检查5mC和5hmC 在HCC中的状态，我们选择16例肝癌手术患者的癌和癌旁组织，提取高质量的DNA。采用超高效液相色谱/串联质谱（UPLC-MS/MS）和新开发的单碱基高通量测序方法（羟甲基化和甲基化敏感标签测序，HMST-seq）同步检测这两种表观修饰水平。.结果发现，肝癌组织中甲基化水平明显高于癌旁组织，羟甲基化水平明显低于癌旁组织。此外，结果还显示5mC更易富集启动子区域，而5hmC易于富集在EXONs区域。为了解释这个现象的原因，我们利用RT-PCR技术检测TET1,TET2,TET3（负责将5mC氧化为5hmC）和UHRF1（负责招募DNMT1活化半甲基化从而起到维持DNA甲基化的水平）基因在肝癌和癌旁组织中的表达差异。结果显示TET1,TET2在肝癌明显低表达，而UHRF1在肝癌明显高表达，提示一些关键酶基因和共刺激因子表达水平的变化可能是肝癌组织中高甲基化及低羟甲基化水平的潜在原因。.整合肿瘤相关信号通路分析，我们确定了6个具有明显表观遗传畸变的甲基化或羟甲基化作用关键基因。利用体外细胞学实验结合siRNA技术，发现其中三个新的基因ECM1、ATF5和EOMES具有明显的抑制肝癌细胞增殖生长的作用，提示这三个基因具有潜在的抗癌功能。.本研究结果可促进人们对于HCC发生和发展的分子机制的理解，增强5hmC检测在未来的临床应用前景。研究成果发表在国际著名学术期刊Genome Biology等。