基于微流控的高通量靶向测序mmPCR-seq技术探究A-to-I RNA编辑对肿瘤发生发展的影响及其分子机制

Chemical modifications on RNA, including A-to-I RNA editing, have been recently appreciated as an important regulatory feature. RNA editing is critical to life and alteration in editing level of an individual substrate can also lead to serious pathological consequences. The overall level of A-to-I RNA editing, as well as the expression levels of adenosine deaminase-ADARs were abnormal in many kinds of cancer, suggesting that RNA editing is implicated in cancer. RNA-seq is often used for the quantification of RNA editing, however RNA-seq is intrinsically limited by the large dynamic range of RNA expression, which leads to inaccurate quantification of allelic ratios for genes with low-to-moderate expression levels. Based on microfluidics and multiplex PCR, mmPCR-seq is able to accurately quantify RNA editing levels of any gene, independent of its gene expression level. In this project we will use mmPCR-seq to quantify the mRNA and miRNA editing levels in more than 300 pairs of matched normal and tumor samples from multiple tumor types. Then we will further explore the molecular mechanisms of A-to-I RNA editing in cancer by combining the bioinformatics and molecular cell biology analyses. Utilizing mmPCR-seq and clinical data analysis, this project will help to reveal the role of RNA editing in cancer.

RNA层面的基因调控，包括RNA编辑是近代生物学研究的重大热点之一。A-to-I RNA编辑不仅参与调控生物体正常生长发育过程，而且其整体水平及催化酶ADARs在多种肿瘤中均出现异常，提示RNA编辑影响肿瘤的发生发展。RNA-seq常被用于RNA编辑检测，但因高丰度转录本消耗过多测序序列，无法精确定量中低表达量转录本的编辑水平。mmPCR-seq通过将多重PCR和微流控技术相结合，具有低成本高通量靶向精确定量包括中低表达量RNA编辑水平特性。本项目利用mmPCR-seq精确定量超过300例各种肿瘤样本及对应癌旁组织mRNA及miRNA上已知编辑位点的编辑水平。然后进一步采用生物信息学与分子细胞生物学相结合的方法从多个层次探讨A-to-I RNA编辑影响肿瘤发生发展的分子机制。本项目将mmPCR-seq和临床数据分析相结合，有助于深入揭示RNA编辑尤其中低丰度转录本异常编辑对肿瘤形成的影响。

因项目计划书中A-to-I RNA编辑对肿瘤发生发展的影响及其分子机制研究内容与其他课题组2019年发表文章有较多重叠，导致创新性不足。我们将研究计划进行了重新调整，把研究目标聚焦于另外一种重要的RNA修饰：5甲基胞嘧啶（m5C）。我们开发出一套全转录组水平高置信度鉴定m5C修饰方法：mRNA BS-seq技术，成功构建出哺乳动物m5C修饰精细图谱，并发现mRNA上两类拥有独特序列和结构特征的m5C修饰位点，分别由NSUN2和NSUN6催化。此外，本项目亦是首次通过构建特定底物催化突变体蛋白，揭示NSUN6非催化及其特定催化底物（tRNA或mRNA）m5C修饰的功能差异，为其他多底物RNA修饰writer蛋白，如METTL16、TRMT6/TRMT61A及PUS家族等研究开拓新方向。