The role of miR-200b/c in balancing EMT and proliferation revealed by an activity reporter

Abstract

Since their discovery, microRNAs (miRNAs) 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 for in vitro and in vivo applications could lead to a better understanding of their biological role in physiology and disease.

Metabolic impairment of non-small cell lung cancers by mitochondrial HSPD1 targeting

“…After 2 weeks of survival (after all control pLKO-infected Cas9-A549 cells treated with the same KHS101 dose underwent complete cell death), the drug treatment was stopped to allow the selected cells to grow and be processed for the DNA preparation and the next-generation sequencing steps. The sequencing results on untreated cells indicated that it was possible to detect a total of 116,700 sgRNAs (all gRNAs excluding those with no counts) present in the library, with a 97% representation, a level similar to previous screenings [ 25 ], while in the treated group this percentage decreased to 56%, as a result of the drug selection. ‘Hits’ were ranked and gene ontology analysis on the 20 highest ranking ‘hits’ (Additional file 1 : Table S8) indicated that the genes whose knockout increased cellular resistance to KHS101 were mainly involved in regulating mitochondrial structure and activity (Fig.…”

“…Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) measurements were determined using the XFe96 Extracellular Flux Analyzer (Seahorse Bioscience/Agilent Technologies). Cells were seeded in specialized culture microplates at a density of 10,000–20,000 cells/well and prepared as describe in [ 25 ]. For Mito Stress Test (Agilent kit 103015–100) 1 μM oligomycin, 1 μM FCCP, 1 μM rotenone and 1 μM antimycin were sequentially injected at regular intervals.…”

“…The CRISPR/Cas9 lentival library was generated as described in [ 25 ]. A549 Cas9 cells were transduced with serial dilutions of a virus to find the MOI of ~ 0.3.…”

“…The first round PCR of Next Generation Sequence (NGS) was performed with 26 separate 100 μL redundant reactions, each containing 5 μg of DNA, 50 μL Q5® Hot Start High-Fidelity 2X Master Mix (NEB # M0494L), and 3 μL of a 10 μM solution of each primer (P5 and P7). The PCR was performed as described in [ 25 ].…”

The miR-200 Family of microRNAs: Fine Tuners of Epithelial-Mesenchymal Transition and Circulating Cancer Biomarkers

“…In turn, miR-200b directly represses expression of the HIF1A mRNA to form a HIF1A-ASCL2-miR-200 feedback loop that contributes to the ability of CRC cells to switch between EMT and MET [ 62 ]. Recent experiments that employed a miR-200b/c-sensitive fluorescent protein sensor reinforced the role for miR-200 in impeding EMT while promoting cell proliferation and implicated metabolic signaling in the control of miR-200b/c expression [ 64 ].…”