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

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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.