DNA中甲基、羟甲基、醛基胞嘧啶多位点检测及胞嘧啶羟甲基修饰的方法研究

With the development of research of DNA methylation, in particular the discovery hydroxymethylcytosine and formylcytosine, the epigenetic modification of DNA have arisen increasing concern of researchers. However, the specific sites and function of epigenetic modifications are not clear as the result of the limited research tools and methods. In this project, we intend to establish (1) the simultaneous detection methods of methylcytosine, hydroxymethylcytosine and formylcytosine with high specifity and sensitivity, (2) and the hydroxymethylation of cytosine, (3) as well as the application of these methods in the research of function and disease diagnosis of epigenetic modifications, especially the hydroxymethylcytosine. We will apply the methods for the clinical samples, based on the previous experimental results on the detection of methylcytosine. The establishment of these methods will facilitate the study of methylcytosine, hydroxymethylcytosine and formylcytosine distribution, so that we can understand the effect of epigenetic regulation in human disease. This project is of great significance in the screening the DNA methylation sites as the marker for the diagnosis and research of disease.

随着DNA甲基化研究的不断深入，特别是羟甲基、醛基胞嘧啶的发现，DNA的表观遗传修饰的相关研究越来越受到研究者重视。但受限于研究手段，关于表观遗传修饰的具体位点及其相应功能还不是很清楚。本课题拟针对DNA中甲基、羟甲基、醛基胞嘧啶，在前期已有实验结果的基础上，主要研究内容为：（1）针对甲基、羟甲基、醛基胞嘧啶的高特异性和敏感性的多位点同步检测方法，（2）及DNA特定胞嘧啶位点的羟甲基修饰化学新方法，（3）并进行表观遗传修饰位点特别是羟甲基胞嘧啶位点在基因调控中的功能以及疾病诊断中的研究。本课题的实施将为表观遗传、基因调控及肿瘤诊断及研究提供强有力的技术支持。

我们围绕DNA甲基化及DNA修饰碱基检测的相关重要方向，开发了新型DNA甲基化多位点同步检测方法，circRNA检测，多种miRNA同时检测等多种核酸检测方法。这些检测方法有着良好的灵敏性和特异性。而且我们都将这些方法应用到细胞、组织和血液的实际样本的检测中，也有着很好的检测效果。我们通过对比肿瘤样本和正常样本，发现人类癌细胞系HepG-2和MCF中E-钙粘着蛋白启动子区域（肿瘤抑制基因）中三个CpG位点的甲基化状态。在HepG-2细胞中，E-cadherin基因通过其启动子的CpG岛的超甲基化而沉默。此外，E-cadherin基因启动子中的CpG位点在MCF-7细胞中未甲基化。肝癌细胞Hepg2和正常肝细胞LO2中的hsa_circ_0001445的含量，发现肝癌细胞Hepg2中hsa_circ_0001445的含量明显低于正常肝细胞L02。肝癌组织和相对应的癌旁正常组织中hsa_circ_0001445的含量，肝癌组织hsa_circ_0001445的含量明显低于相对应的癌旁正常组织。宫颈癌细胞Hela含有较多的miR-21，表现为高表达，而正常的肝细胞L02含有较低的miR-21或检测不出含有miR-21。 这些核酸修饰位点状态或者含量的区别，有希望作为疾病的诊断标志物。