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Original Research ARTICLE Provisionally accepted The full-text will be published soon. Notify me

Front. Oncol. | doi: 10.3389/fonc.2019.01100

A high-content screening approach to identify microRNAs against Head and Neck Cancer cell survival and EMT in an inflammatory microenvironment.

 Bruno Sangiorgi1, 2,  Ildercílio Mota de Souza Lima1, 2, Josiane Lilian dos Santos Schiavinato1, 2,  Wilson Araújo da Silva Junior1, 2, Dimas Tadeu Covas1, Marco Antônio Zago2 and Rodrigo Alexandre Panepucci1, 2*
  • 1Cell Therapy Center, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil
  • 2Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil

Head and neck squamous cell carcinoma (HNSCC) is among the most common cancer types. Metastasis, the main cause of death by cancer, can be promoted by an inflammatory microenvironment, which induces epithelial-mesenchymal transition (EMT) through a NF-kB-mediated stabilization of Snail. Here, we aimed to explore how microRNAs (miRs) can affect cell survival and EMT in HNSCC cells under an inflammatory microenvironment.

By using an high-content screening (HCS) approach, we evaluated alterations in morphometric parameters, as well as expression/localization of Snail/Slug, in HNSCC cells primed with TNF-α. Based on those quantitation, we established the optimal experimental conditions of EMT induction driven by TNF-α. Those conditions were applied to cells transfected with distinct miRs (N = 31), followed by clusterization of miRs based on alterations related to cell survival and EMT. The signaling pathways enriched with molecular targets from each group of miRs were identified by in silico analyses. Finally, cells were transfected with siRNAs against signaling pathways targeted by miRs with anti-survival/EMT effect and evaluated for alterations in cell survival and EMT.

Overall, we observed that TNF-α, at 20 ng/ml, induced EMT-related changes in cell morphology, Snail/Slug expression and cell migration. Predicted targets of miRs with anti-survival/EMT effect were enriched with targets of NF-kB, PI3K/ATK and Wnt/beta catenin pathways. Strikingly, individual gene silencing of elements from those pathways, namely RELA (NF-kB), AKT1 (PI3K/AKT) and CTNNB1 (Wnt/beta catenin) reduced cell survival and/or expression of Snail/Slug in cells stimulated with TNF-α.

As a whole, our HCS approach allowed for the identification of miRs capable of inhibiting cell survival and EMT considering the presence of an inflammatory microenvironment, also indicating the common signaling pathways and molecular targets most likely to underlie those alterations. These findings may contribute to the development of targeted therapies against HNSCC.

Keywords: Head and neck squamous cell carcinoma, high-content screening, MicroRNAs, epithelial-mesenchymal-transition, Inflammation, NF-kB

Received: 08 Dec 2018; Accepted: 07 Oct 2019.

Copyright: © 2019 Sangiorgi, Mota de Souza Lima, dos Santos Schiavinato, Araújo da Silva Junior, Covas, Zago and Panepucci. 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(s) 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: PhD. Rodrigo Alexandre Panepucci, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirao Preto, Brazil, rapane@gmail.com