Identification of miR-146b-3p as a key player in thyroid cancer dedifferentiation

Abstract

Next-generation sequencing expression analysis of papillary thyroid cancer performed in our laboratory, revealed the existence of a set of abundant miRNAs differentially expressed in this pathology. MiR-146b-3p was unveiled as one of the most upregulated microRNAs (miRs) in this neoplasm, surpassing the minimum threshold amount to repress target mRNAs. Furthermore, our previous results showed that this miR binds to the 3'UTR of NIS, inhibiting its expression and consequently its activity. As NIS is a gene regulated by thyroid transcription factors, the aim of this work was to study the effect of miR-146b-3p on the thyroid cancer dedifferentiation process, focusing on the most relevant event for the clinical outcome: the loss of NIS function that leads to radioiodide therapy refractority. By computational predictions, we identified PAX8 and FOXE1 as potential targets of miR-146b-3p according to miRanda algorithm. We validated these predictions through functional studies in PCCl3, NThy-ori and, hNISMDCK cells. Mir-146b expression vector was transfected and reporter constructs containing 3'UTRs, miR specific binding sites, and NIS promoter were generated. The levels of mRNA and protein were determined by RT-qPCR and Western Blot; direct mRNA destabilizing effect was measured in luciferase assays. Overexpression of miR-146b-3p resulted in NIS, PAX8, and FOXE1 mRNA and protein silencing. Furthermore, we observed that as NIS, the PAX8 inhibition is specifically exerted through a direct binding to their 3'UTRs. Also, preliminary data suggest that FoxE1 and PAX8 silencing induced by miR-146b-3p overexpression was sufficient to repress NIS promoter activation. The general outcome was a significant decrease in iodide uptake in miR-146b-3p overexpressing cells. These data suggest that tumor overexpression of miR-146b-3p widely contributes to thyroid dedifferentiation and especially to NIS loss of function through at least two complementary mechanisms: (i) direct postranscriptional level, and (ii) through PAX8 and FOXE1 silencing.