Cesar Seigi Fuziwara
Tumorigenic role of microRNA miR-17-92 cluster via deregulation of TGFbeta signaling pathway in thyroid follicular cells
Fuziwara CS, Kimura ET
Department of Cell and Developmental Biology, University of Sao Paulo, Brazil
Introduction: MicroRNAs (miRNAs) are important regulators of physiological processes including cell proliferation and apoptosis, acting as posttranscriptional regulators of gene expression. TGFbeta (TGFβ) antimitogenic signaling pathway is involved in high iodine anti-proliferative effect in thyroid follicular cells, and we have previously shown that the induction of miR-17-92 cluster of miRNAs by BRAF oncogene leads to resistance to TGFβ-mediated cell cycle arrest. In this study, we aim to clarify the role of miR-17-92 in the biology of normal thyroid follicular cell under the influence of TGFβ signaling transduction.
Methods: We used PCCl3-miR-17-92 cells that over-express miR-17-92 (pCDNA3.1-Neo-miR-17- 92) and PCCl3-0 (empty plasmid) as control. TGFβ signaling reporter assay was performed with 3TP-lux plasmid that contains responsive binding sites to TGFβ signaling. MiR-17-92 expression was detected by real-time and protein expression of TGFβ signaling components by Western-blotting (WB). Cell viability was measured by MTT assay, proliferation by cell counting, cell migration by transwell assay and resistance to doxorubicin by measuring cell apoptosis.
Results: We observed an impaired PCCl3-miR-17-92 response to TGFβ at 1 and 2ng/mL, 0.53-fold and 0.45-fold, respectively, when compared to the PCCl3-0 in 3TP-lux assay. WB revealed reduced Tgfbr2, Smad4 and Smurf2 protein levels in PCCl3-miR-17-92 cells. In the cell cycle assay TGFβ-treated PCCl3-miR-17-92 does not exhibit G1-arrest in the cells. Furthermore, PCCl3-miR-17-92 cells escape from the antiproliferative effect of high iodine concentration treatment (10-3M) and show resistance to doxorubicin-induced apoptosis. The enforced expression of miR-17-92 also increased the cell viability of PCCl3-miR-17-92 cells in 18% and cell migration in more than 3-fold.
Conclusions: The miR-17-92 over-expression enhances the PCCl3 proliferation, abrogating the anti-proliferative effect of high dose iodine and impairing the TGFβ inhibitory signaling, by targeting components of TGFβ pathway. Moreover, the miR-17-92 influence in the migration and resistance to chemotherapy suggest an important role of these miRNA in the tumorigenic behavior in thyroid follicular cells.
Grants from FAPESP:2014/50521-0 and NapmiR