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Exploration of endogenous miRNA-200b/c activity and regulation through a functional dual fluorescence reporter

By Paradesi Gollavilli, Beatrice Parma, Aarif Siddiqui, Hai Yang, Vignesh Ramesh, Francesca Napoli, Annemarie Schwab, Ramakrishnan Natesan, Irfan Ahmed Asangani, Thomas Brabletz, Christian Pilarsky, Paolo Ceppi

Posted 21 Jul 2020
bioRxiv DOI: 10.1101/2020.07.19.210997

Since their discovery, microRNAs (miRNA)s have been widely studied in almost every aspect of biology and medicine, leading to the identification of important gene regulation circuits and cellular mechanisms. However, investigations are generally focused on the analysis of their downstream targets and biological functions in overexpression and knockdown approaches, while miRNAs endogenous levels and activity remain poorly understood. Here, we used the cellular plasticity-regulating process of epithelial-to-mesenchymal transition (EMT) as a model to show the efficacy of a fluorescent sensor to separate cells with distinct EMT signatures, based on miR-200b/c activity. The system was further combined with a CRISPR-Cas9 screening platform to unbiasedly identify miR-200b/c upstream regulating genes. The sensor allows to infer miRNAs fundamental biological properties, as profiling of sorted cells indicated miR-200b/c as a molecular switch between EMT differentiation and proliferation, and suggested a role for metabolic enzymes in miR-200/EMT regulation. Analysis of miRNAs endogenous levels and activity could lead to a better understanding of their biological role in physiology and disease. ### Competing Interest Statement The authors have declared no competing interest.

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