胰腺癌转录组m6A修饰异常及其调控机制研究

N6-methyladenosine (m6A) modification in RNAs has recently been found to be heavily involved in tumorigenesis and progression of human cancers. However, the feature and role of m6A modification in RNAs in pancreatic ductal adenocarcinoma (PDAC) is totally unclear. Based on our preliminary profiling and quantification of m6A modification, we found that the overall abundance and profile of m6A modification in PDAC is substantially different from paired non-tumor tissues, and the modification levels for a part of m6A sites were correlated with overall survival time in PDAC patients. Furthermore, we found that methyltransferase like-3 (METTL3), the major enzyme catalyzing m6A formation in RNAs, is overexpressed in PDAC and positively correlated with the abundance of m6A modification. In an experimental model, cells exposed to cigarette smoke condensate (CSC) showed enhanced METTL3 expression and maturation of oncogenic primary miR-125 to mature miR-125. Based on these pilot studies, we propose the current study project, in which we want to systematically profile the landscape of RNA m6A modification and dissect their feature in PDAC. We will explore the functions and involved signaling pathways of m6A modification in the development and progression of PDAC. We will also focus on the regulatory effects of m6A modification on RNA metabolism and protein translation and how m6A related enzymes (e.g., writers and readers) are aberrantly regulated in PDAC. Finally and most importantly, we will explore the potential role of m6A modification as biomarker for early diagnosis, treatment efficiency, and prognosis of disease outcome.

研究表明RNA甲基化修饰即N6-methyladenosine (m6A)与肿瘤发生发展关系密切，但m6A修饰在胰腺癌的表现和功能不清楚。我们前期用胰腺癌及癌旁组织样本检测了转录组m6A修饰，发现癌和非癌组织比较，m6A修饰位点和含量有显著差异，其中部分RNA m6A修饰与胰腺癌患者生存时间相关。我们还发现催化产生m6A的类甲基转移酶-3 (METTL3)在肿瘤组织中高表达，并与m6A含量呈显著正相关。香烟凝集物刺激的细胞METTL3表达显著上调，使癌性miRNA-125通过m6A过量修饰而异常成熟。基于这些前期发现，本项目将进一步系统研究胰腺癌转录组中m6A修饰的整体轮廓及特征；揭示m6A异常修饰在胰腺癌发生发展中的作用及相关信号通路；阐明m6A修饰对RNA代谢及蛋白质翻译的影响；胰腺癌中m6A异常修饰调控的分子机制；筛选与m6A相关的早期诊断、疗效判断以及疾病预后的分子标志物。

本项目利用基于去核糖体RNA的m6A测序 (m6A-seq)方法，从全转录组水平对胰腺癌及其配对的非癌胰腺组织中的m6A修饰进行系统分析，揭示胰腺癌中m6A修饰的总体概况及其特征。机制上我们阐明了参与调控m6A修饰的蛋白METTL3胰腺癌中的作用，揭示了抽烟通过表观遗传学调控促进METTL3表达的调控机制，也进一步对其所影响的下游关键通路和基因的作用进行研究；探讨了CSTF2蛋白在胰腺癌异常m6A调控中的作用以及可能的分子机制。此外，我们也解释了IGF2BP2/3所介导的m6A修饰影响转录本稳定性以及NKAP蛋白所介导的m6A修饰影响microRNA成熟剪切的分子机制。最后，我们系统分析了m6A修饰异常与胰腺癌异质性的关系，探讨利用m6A修饰异常作为胰腺癌分子分型的可能性及其对胰腺癌诊疗和预后的指导作用。