Recent studies have shown that miR-146b is the most upregulated microRNA in thyroid cancer and has a central role in cancer progression through mechanisms that remain largely unidentified. As phosphoinositide 3-kinase/protein kinase-B (PI3K/AKT) signaling is a fundamental oncogenic driver in many thyroid cancers, we explored a potential role for miR-146b and its target genes in PI3K/AKT activation. Among the predicted target genes of miR-146b, we found the tumor-suppressor phosphatase and tensin homolog (PTEN). Constitutive overexpression of miR-146b in thyroid epithelial cell lines significantly decreased PTEN mRNA and protein levels by direct binding to its 3′-UTR. This was accompanied by PI3K/AKT hyperactivation, leading to the exclusion of FOXO1 and p27 from the nucleus and a corresponding increase in cellular proliferation. Moreover, miR-146b overexpression led to protection from apoptosis and an increased migration and invasion potential, regulating genes involved in epithelial–mesenchymal transition. Notably, with the single exception of E-cadherin expression, all of these outcomes could be reversed by PTEN coexpression. Further analysis showed that miR-146b directly inhibits E-cadherin expression through binding to its 3′-UTR. Interestingly, miR-146b inhibition in human thyroid tumor xenografts, using a synthetic and clinically amenable molecule, blocked tumor growth when delivered intratumorally. Importantly, this inhibition increased PTEN protein levels. In conclusion, our data define a novel mechanism of PI3K/AKT hyperactivation and outline a regulatory role for miR-146b in suppressing PTEN expression, a frequent observation in thyroid cancer. Both events are related to a more aggressive tumoral phenotype. Targeting miR-146b therefore represents a promising therapeutic strategy for the treatment of this disease.
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We are grateful to Andrea Martinez-Cano for her technical assistance, Javier Perez for the artwork and Dr Kenneth McCreath for helpful comments on the manuscript. We thank Dr Jerónimo Blanco (Catalonian Institute for Advance Chemistry-CSIC) and Dr Eugenia Mato (Institut de Reserca de l’Hospital de la Santa Creu i Sant Pau) Barcelona (Spain) for gifting the CMV-Firefly luc-IRES-EGFP and Cal62-Luc cells, respectively. We also thank the Histology Facility at CNB-CSIC for the histological preparation of biological samples. This work was supported by grants SAF2013-44709-R and SAF2016-75531-R from the Ministerio de Economía y Competitividad (MINECO) of Spain, RD12/0036/0030 from Instituto de Salud Carlos III (ISCIII), Fondo Europeo de Desarrollo Regional (FEDER), and GCB14142311CRES from Fundación Española contra el Cancer. JR-M holds a FPU fellowship from Ministerio de Educación Cultura y Deporte. LW-L was funded by an FPI fellowship from MEC and is currently an investigator of the project PI14/01980 from ISCIII (Spain).