High Iodine Blocks a Notch/miR-19 Loop Activated by the BRAF V600E Oncoprotein and Restores the Response to TGFβ in Thyroid Follicular Cells

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Abstract
Background: Excess iodine inhibits thyroid follicular cell proliferation associated with TGFβ pathway activation, although thyroid cancers are frequently refractory to TGFβ signaling. The TGFβ pathway is predicted to be regulated by miR-17-92 cluster microRNAs. MicroRNAs are small noncoding RNAs that inhibit target mRNA translation and have emerged as potent modulators of tumorigenesis. Although the BRAF V600E mutation is the most prevalent alteration in thyroid cancer, the impact of iodine intake on BRAF-mediated oncogenesis remains unclear. Therefore, the aim of this study was to investigate the influence of high iodine on miR-17-92 transcriptional regulation and expression in thyroid cells expressing activated BRAF. Methods: Rat thyroid follicular cells that conditionally express BRAF V600E under doxycycline stimulation (PC-BRAF V600E -6) were derived from the PCCl3 line. These cells were treated with doxycycline for two days, in the absence or presence of 10 μM sodium iodide. The thyroid cancer cell lines BCPAP and KTC2 were also analyzed. Expression of the miR-17-92 cluster and Notch1 was analyzed by quantitative polymerase chain reaction, and expression of these genes was modulated by anti-miR or anti-Notch1 siRNAs transfection. Protein expression was assessed by Western blot. Luciferase assays were used to quantify Smad4 3′-UTR/ miR-19 interaction and Notch signaling activation. TGFβ responsiveness was evaluated by cell cycle analysis of TGFβ-treated cells. Results: High iodine blocked BRAF V600E -induced upregulation of miR-17-92 , including miR-19a/b . miR-17-92 promoter region analysis revealed a putative binding site for Hes1, a transcription factor responsive to Notch signaling. Notch-1 overexpression resulted in miR-19 upregulation in normal thyroid cells, while Notch-1 knockdown blocked BRAF-induced miR-19 expression. Moreover, in anaplastic thyroid cancer cells, Notch-1 knockdown reduced miR-19 . Expression of BRAF V600E decreased Smad4 protein in normal thyroid cells. Smad4 was validated as a miR-19 target by luciferase assays, which revealed reduced luminescence associated with miR-19 interaction in Smad4 3′-UTR. Iodine treatment restored Smad4 levels in BRAF-activated cells, resulting in enhanced G1-cell cycle arrest in response to TGFβ. Moreover, this effect was mimicked in papillary thyroid cancer cells treated with anti-miR-19. Conclusion: High iodine abrogates BRAF V600E -induced activation of miR-19 , a newly identified Smad4 regulator, through Notch pathway inhibition and restores responsiveness to TGFβ signaling. Our results indicate that iodine exerts protective effects in thyroid cells, attenuating acute BRAF oncogene-mediated microRNA deregulation.

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