Pri-miRNA加工机器的筛选及功能机制研究

MiRNAs (microRNAs) are a class of non-coding RNA molecules in ～22 nucleotide length. Primary microRNAs are first processed into ～70nt pre-miRNAs by Drosha/DGCR8 complex. The resulting precursor hairpins are further cleavaged by Dicer/TRBP/AGO complex in cytoplasm. Currently, the detailed composition and dynamic regulation of miRNA processing machinery in cytoplasm have been well documented. However, the architecture of Dorsha/DGCR8 processing machinery and the regulatory cascades is still unclear. In this application, we proposed to conduct an unbiased screening with CRISPR-Cas9 genome-wide knockout libraries in three pri-miRNA processing reporter cell lines, in which pri-miRNAs were inserted into the 3’UTR of green fluorescence proteins. We hope to identify new genes involed in pri-miRNA processing and further to uncover the whole architecture of miRNA processing machinery in nucleus. This proposal will decipher the regulatory cascade of miRNA processing and the pathological mechanism of abonormal miRNAs processing in cancer cells. This study may also provide a novel strategy for the diagnosis and treatment of cancer.

MiRNAs（microRNAs）是一类长度～22nt的非编码RNA，在细胞分化、个体发育和癌症发生中发挥重要作用。Pri-miRNA（primary microRNA）在细胞核内经Dorsha/DGCR8复合体加工而形成～70nt的pre-miRNA（precursor microRNA），进而在细胞质中被Dicer/TRBP/AGO复合物切割成熟。细胞质内加工机器的组成及动态调控研究比较清楚，但是细胞核内Dorsha/DGCR8加工机器的具体组份及协同调控机制尚不明了。本项目计划利用三个pri-miRNA加工的GFP报告细胞系，借助CRISPR-Cas9敲除技术在全基因组水平上筛选调控pri-miRNA加工的新基因，阐明其调控pri-miRNA加工的新机理。本项目有望解析pri-miRNA加工的精细级联调节网络，揭示癌细胞中miRNA加工异常的病理机制，为癌症的诊断和治疗提供新策略。

微小RNA（miRNA）是一类小的非编码RNA，它们共同调节哺乳动物细胞中一半以上的蛋白质编码基因。Drosha及其辅因子DGCR8形成微处理器复合体，该复合体通过切割活性切割初级miRNA转录本（pri-miRNA）启动miRNA生物发生。这种过程的失调在癌症发展中起重要作用。尽管其重要性，但对微处理器活性的体内调节了解甚少。本项目利用pri-miR-1-2 和 pri-miR-125b-1两种GFP报告系统，通过CRISPR-Cas9全基因组筛选手段，筛选出一些miRNA加工重要的调控因子。其中，我们利用功能基因组学方法解析AQR在miRNA加工过程中的作用和功能。并且我们还发现AQR通过该机制能够抑制肾癌细胞的增殖。揭示了AQR在肾癌发生发展过程中扮演着重要的作用，从而为肾癌患者的治疗提供了新的思路和靶点。