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Front. Genet. | doi: 10.3389/fgene.2018.00045

Cell-based reporter system for high-throughput screening of microRNA pathway inhibitors and its limitations.

Katerina Podolska1,  David Sedlak2, Jana Kubikova1,  Ctibor Skuta2,  Katerina Solcova1, Radek Malik1,  Petr Bartunek2 and  Petr Svoboda1*
  • 1Institute of Molecular Genetics (ASCR), Czechia
  • 2CZ-OPENSCREEN, Institute of Molecular Genetics (ASCR), Czechia

MicroRNAs (miRNAs) are small RNAs repressing gene expression. They contribute to many physiological processes and pathologies. Consequently, strategies for manipulation of the miRNA pathway are of interest as they could provide tools for experimental or therapeutic interventions. One of such tools could be small chemical compounds identified through high-throughput screening with reporter assays. While a number of chemical compounds have been identified in such high-throughput screens, their application potential remains elusive. Here, we report our experience with cell-based high-throughput screening of a library of 12,816 chemical compounds to identify miRNA pathway modulators. We used human HeLa and mouse NIH 3T3 cell lines with stably integrated or transiently expressed luciferase reporters repressed by endogenous miR-30 and let-7 miRNAs and identified 163 putative miRNA inhibitors. We report that compounds relieving miRNA-mediated repression via stress induction are infrequent; we have found only two compounds that reproducibly induced stress granules and relieved miRNA-targeted reporter repression. However, we have found that this assay type readily yields non-specific (miRNA-independent) stimulators of luciferase reporter activity. Furthermore, our data provide partial support for previously published miRNA pathway modulators; the most notable intersections were found among anthracyclines, dopamine derivatives, flavones, and stilbenes. Altogether, our results underscore the importance of appropriate negative controls in development of small compound inhibitors of the miRNA pathway. This particularly concerns validation strategies, which would greatly profit from assays that fundamentally differ from the routinely employed miRNA-targeted reporter assays.

Keywords: miRNA, High-Throughput Screening, Let-7, miR-30, Argonaute

Received: 04 Dec 2017; Accepted: 31 Jan 2018.

Edited by:

Hervé Seitz, Centre national de la recherche scientifique (CNRS), France

Reviewed by:

Gyorgy Hutvagner, University of Technology Sydney, Australia
Patricia Richard, Columbia University, United States  

Copyright: © 2018 Podolska, Sedlak, Kubikova, Skuta, Solcova, Malik, Bartunek and Svoboda. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Petr Svoboda, Institute of Molecular Genetics (ASCR), Videnska 1083, Prague, 142 20, Czechia, svobodap@img.cas.cz