Whole-genome CRISPR/Cas9-mediated identification of miR-200 repressors

自发现以来，microRNA（miRNA）在生物学和医学的几乎每个领域都得到了广泛的研究，导致了几个重要途径和生物学机制的鉴定。在癌症中，miRNA在功能上与一些最致命的特征相关，如化学抗性、改变的代谢途径、转移形成、上皮向间质转化（EMT）过程和癌症干性。然而，研究通常集中在下游途径上，因为没有工具以无偏见的全基因组方式研究miRNA调控因子。该提案的主要目的是优化和使用一种新的高通量方法，通过我们最近开发的基于质粒的荧光传感器系统来识别miRNAs的上游调控因子，该系统允许测量和监测活细胞中miRNAs的表达。筛选将结合miRNA报告系统与CRISPR/Cas9全基因组编辑技术，FACS分选和下一代测序方法进行。miR-200家族，其成员已被表征为EMT和癌症干细胞的强大抑制剂，将成为调查的对象。该项目分为两个部分：1）我将进行高通量筛选，并在独立实验中验证miR-200阻遏物候选物。2)我将在相关的体外细胞模型和来自癌症患者的临床注释样本中确定所鉴定的miR-200阻遏物对EMT的意义。这些发现可能在分子肿瘤学领域具有潜在的高度相关性，因为所确定的途径可以被特异性抑制剂靶向，并在实体瘤治疗中作为抗EMT（抗转移）药物进行测试。该项目也代表了一个前所未有的概念验证示范的miRNA传感器作为一种新的工具，用于无偏见地识别上游调控的miRNA。

Since their discovery, microRNAs (miRNA)s have been widely studied in almost every field of biology and medicine, leading to the identification of several important pathways and biological mechanisms. In cancer, miRNAs have been functionally associated with some of the most lethal features, like chemoresistance, altered metabolic pathways, metastasis formation, the process of epithelial-to-mesenchymal transition (EMT) and cancer stemness. However, investigations are generally focused on the downstream pathways, as there are no tools to investigate miRNAs regulators in unbiased whole-genome fashion. The main aim of this proposal is to optimize ¬¬-and use a novel high-throughput approach to identify upstream regulators of miRNAs by a plasmid-based fluorescent sensor system we recently developed, which allows to measure and monitor the expression of miRNAs in living cells. Screenings will be performed combining the miRNA reporter system with CRISPR/Cas9 whole-genome editing technology, FACS-sorting and Next-Generation Sequencing approaches. The miR-200 family, whose members have been characterized as powerful suppressors of EMT and cancer stem cells, will be the object of the investigation. The project is divided in 2 parts: 1) I will perform the high-throughput screenings and validate the miR-200 repressor candidates in independent experiments. 2) I will determine the significance of the identified miR-200 repressors on EMT in relevant in vitro cellular models and in clinically-annotated samples from cancer patients. The findings could be potentially highly relevant in the field of molecular oncology, as the identified pathways could be targeted by specific inhibitors and tested in the treatment of solid tumors as anti-EMT (anti-metastatic) agents. This project could also represent an unprecedented proof-of-concept demonstration of the miRNA sensor as a novel tool for the unbiased identification of upstream regulators of miRNAs.