Abstract
Abstract Background: Thyroid follicular cells proliferation is regulated by TGFβ anti-mitogenic signal. However, thyroid cancer cells become refractory to this inhibitory signal. TGFβ signaling deregulation is observed in thyroid cancer, which shows high frequence of MAPK genetic alteration. BRAF mutation (BRAFT1799A), the most prevalent alteration in thyroid cancer, induces MAPK activtion and thyroid oncogenesis. In this process, deregulation of microRNAs potentiates oncogene activation. MicroRNAs are small non-coding RNAs that regulate protein levels by pairing to 3-UTR of mRNA and blocking translation. miR-17-92 cluster of microRNAs, miR-17-5p/3p, miR-18a, miR-19a/b, miR20a and miR-92a, regulates several tumor-suppressor genes and is predicted to target TGFβ pathway components. Therefore, we aim at analyzing the influence of miR-17-92 in TGFβ signaling during oncogene activation in thyroid follicular cells and its implication for thyroid cancer biology. Methods: The BRAF9-6 cells, derived from normal rat thyroid follicular cell PCCl3, express BRAFT1799A under doxicyclin (dox) stimulation. The PCCl3-miR-17-92 cells, derived from PCCl3 cells, over-express miR-17-92 cluster (pCDNA3.1-Neo). PCCl3- is the control cell transfected with empty plasmid. MiR-17-92 expression was detected by real-time qPCR. Cell viability was measured using MTT assay and proliferation by cell counting (24-72h). TGFβ signaling components, Smad4 and Tgfbr2, protein levels was analyzed by Western-blotting. TGFβ responsiveness assay was analyzed by luciferase gene reporter assay after transfection of 3TP-lux plasmid and treatment with 1-2ng/mL recombinant TGFβ for 24 hours. Results: BRAF-oncogene activation (dox) in thyroid follicular cells induced a high expression of miR-17-92 cluster components and reduced Smad4 and Tgfbr2 protein levels. In order to analyze the particular influence of miR-17-92 in TGFβ signaling, we constructed a cell line, PCCl3-miR-17-92, that over-expressed miR-17-92 in normal thyroid follicular cells. Over-expression of miR-17-92 inhibited Tgfbr2 and Smad4 protein levels that reflected in impaired responsiveness to recombinant TGFβ inhibitory signal. Using the 3TP-lux reporter gene assay, that contains TGFβ signaling activation responsive sequences, we observed that recombinant TGFβ treatment in PCCl3- cells resulted in a 8.0 and 12.3-fold increase in luciferase activity at concentrations of 1ng/ml and 2ng/mL of recombinant TGFβ, respectively, while a 4.3 and 5.6-fold increase is observed in PCCl3-miR-17-92 cells. Moreover, PCCl3-miR-17-92 showed increases cell proliferation and cell viability in 18% and 9%, respectively, in comparison to PCCL3-. Conclusion: miR-17-92 over-expression impairs normal thyroid follicular cells responsiveness to TGFβ and increases cell proliferation and viability, which could imply in TGFβ inhibitory signal refractoriness observed in thyroid cancer cells. Citation Format: Cesar Seigi Fuziwara, Felipe E. Martins, Edna T. Kimura. MicroRNA miR-17-92 impairs TGFβ signaling responsiveness in BRAF oncogene-activated thyroid cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5210. doi:10.1158/1538-7445.AM2014-5210
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