Abstract
Abstract Background: BRAF mutation (T1799A) is the most prevalent genetic alteration in thyroid malignancy, associated with aggressive and poor prognosis thyroid cancer. Iodine excess exerts anti-proliferative effects in thyroid cells associated to TGFβ pathway enhancement, although refractoriness to TGFβ inhibitory signal is observed in thyroid cancer. We previously showed that high iodine dose protects thyroid cells from BRAF-induced genomic instability and also, delays oncogenic effects of RET/PTC3 oncogene. MicroRNAs are small non-coding RNAs that regulate protein levels by pairing to 3-UTR of mRNA and blocking translation. MiR-19 belongs to cluster miR-17-92 which attenuates several tumor-suppressor genes and is predicted to target TGFβ pathway components. Therefore, we aim to analyze the influence of iodine on microRNA-mediated TGFβ signaling in BRAFT1799A activated thyroid follicular cells. Methods: BRAF9-6 cells, derived from normal rat thyroid follicular cell, which express BRAFT1799A under doxacyclin stimulation were treated with iodine-containing medium at 10-5M (NaI group) for 5 days. Dox treatment was performed for 2 days (Dox-group), in the absence or presence of iodine (Dox+NaI-group). Control was performed without Dox/NaI. MiR-19 expression was performed by real-time qPCR and Smad4 target validation was performed by Western-blotting and luciferase-assay. TGFβ responsiveness was evaluated by analyzing cell cycle of TGFβ-treated cells using flow-cytometry. Results: BRAFT1799A induction in thyroid cells increased miR-19a and miR-19b levels in 634.3-fold/141.6-fold, respectively (Dox vs Control), while adding iodine prior to BRAFT1799A induction resulted in 2.36-fold/0.66-fold levels (Dox+NaI vs Control). Iodine per se treatment did not change miR-19 levels. BRAFT1799A induction reduced Smad4 protein which was restored by iodine treatment. In parallel to the low Smad4 levels during BRAFT1799A-activation, we observed an impairment of responsiveness to recombinant TGFβ treatment. Moreover, the iodine-containing medium restored the inhibitory signal and rendered cells arrested in G1-phase. Bioinformatic search for miR-19 potential targets revealed Smad4 as a predicted target which was validated by luciferase-assay that showed reduced luminescence of Smad4 3’-UTR plasmid in the presence miR-19. Conclusion: Iodine is a modulator of oncogene activated mir19 expression, which target Smad4 and regulates the efficiency of TGFβ anti-mitogenic action on the BRAFT1799A induced thyroid follicular cells (Supported by FAPESP and CNPq Grant). Citation Format: Cesar S. Fuziwara, Edna T. Kimura. Iodine regulates miR-19-mediated inhibition of Smad4 and restores TGFBeta responsiveness in BRAF-activated thyroid cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2013-4169
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