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MicroRNA miR-146b-5p regulates signal transduction of TGF-β by repressing SMAD4 in thyroid cancer

Abstract

MicroRNAs (miRNA) are small non-coding RNAs involved in post-transcriptional gene regulation that have crucial roles in several types of tumors, including papillary thyroid carcinoma (PTC). miR-146b-5p is overexpressed in PTCs and is regarded as a relevant diagnostic marker for this type of cancer. A computational search revealed that miR-146b-5p putatively binds to the 3′ untranslated region (UTR) of SMAD4, an important member of the transforming growth factor β (TGF-β) signaling pathway. The TGF-β pathway is a negative regulator of thyroid follicular cell growth, and the mechanism by which thyroid cancer cells evade its inhibitory signal remains unclear. We questioned whether the modulation of the TGF-β pathway by miR-146b-5p can contribute to thyroid tumorigenesis. Luciferase reporter assay confirmed the direct binding of miR-146b-5p on the SMAD4 3′UTR. Specific inhibition of miR-146b-5p with a locked nucleic acid-modified anti-miR-146b oligonucleotide significantly increased SMAD4 levels in the human papillary carcinoma cell lines, TPC-1 and BCPAP. Moreover, suppression of miR-146b-5p increased the cellular response to the TGF-β anti-proliferative signal, significantly decreasing the proliferation rate. The overexpression of miR-146b-5p in normal rat follicular PCCL3 cells decreased SMAD4 levels and disrupted TGF-β signal transduction. MiR-146b-5p overexpression in PCCL3 cells also significantly increased cell proliferation in the absence of thyroid-stimulating hormone and conferred resistance to TGF-β-mediated cell-cycle arrest. Additionally, the activation of thyroid most common oncogenes RET/PTC3 and BRAF in PCCL3 cells upregulated miR-146b-5p expression. Our results confirm the oncogenic role of miR-146b-5p in thyroid follicular cells and contribute to knowledge regarding the modulation of TGF-β signal transduction by miRNAs in PTCs.

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References

  • Adeniran AJ, Zhu Z, Gandhi M, Steward DL, Fidler JP, Giordano TJ et al. (2006). Correlation between genetic alterations and microscopic features, clinical manifestations, and prognostic characteristics of thyroid papillary carcinomas. Am J Surg Pathol 30: 216–222.

    Article  PubMed  Google Scholar 

  • Bartel DP . (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281–297.

    Article  CAS  PubMed  Google Scholar 

  • Bhaumik D, Scott GK, Schokrpur S, Patil CK, Campisi J, Benz CC . (2008). Expression of microRNA-146 suppresses NF-kappaB activity with reduction of metastatic potential in breast cancer cells. Oncogene 27: 5643–5647.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cahill S, Smyth P, Finn SP, Denning K, Flavin R, O'Regan EM et al. (2006). Effect of ret/PTC 1 rearrangement on transcription and post-transcriptional regulation in a papillary thyroid carcinoma model. Mol Cancer 5: 70.

    Article  PubMed  PubMed Central  Google Scholar 

  • Calin GA, Croce CM . (2006). MicroRNA signatures in human cancers. Nat Rev Cancer 6: 857–866.

    Article  CAS  PubMed  Google Scholar 

  • Cerutti JM, Ebina KN, Matsuo SE, Martins L, Maciel RM, Kimura ET . (2003). Expression of Smad4 and Smad7 in human thyroid follicular carcinoma cell lines. J Endocrinol Invest 26: 516–521.

    Article  CAS  PubMed  Google Scholar 

  • Chou CK, Chen RF, Chou FF, Chang HW, Chen YJ, Lee YF et al. (2010). miR-146b is highly expressed in adult papillary thyroid carcinomas with high risk features including extrathyroidal invasion and the BRAF(V600E) mutation. Thyroid 20: 489–494.

    Article  CAS  PubMed  Google Scholar 

  • Costamagna E, Garcia B, Santisteban P . (2004). The functional interaction between the paired domain transcription factor Pax8 and Smad3 is involved in transforming growth factor-beta repression of the sodium/iodide symporter gene. J Biol Chem 279: 3439–3446.

    Article  CAS  PubMed  Google Scholar 

  • D'Inzeo S, Nicolussi A, Ricci A, Mancini P, Porcellini A, Nardi F et al. (2010). Role of reduced expression of SMAD4 in papillary thyroid carcinoma. J Mol Endocrinol 45: 229–244.

    Article  CAS  PubMed  Google Scholar 

  • Dykxhoorn DM . (2010). MicroRNAs and metastasis: little RNAs go a long way. Cancer Res 70: 6401–6406.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ellenrieder V . (2008). TGFbeta regulated gene expression by Smads and Sp1/KLF-like transcription factors in cancer. Anticancer Res 28: 1531–1539.

    CAS  PubMed  Google Scholar 

  • Ermak G, Cancasci VJ, Davies KJ . (2003). Cytotoxic effect of doxycycline and its implications for tet-on gene expression systems. Anal Biochem 318: 152–154.

    Article  CAS  PubMed  Google Scholar 

  • Fabien N, Fusco A, Santoro M, Barbier Y, Dubois PM, Paulin C (1994). Description of a human papillary thyroid carcinoma cell line. Morphologic study and expression of tumoral markers. Cancer 73: 2206–2212.

    Article  CAS  PubMed  Google Scholar 

  • Fagin JA . (2002). Minireview: branded from the start-distinct oncogenic initiating events may determine tumor fate in the thyroid. Mol Endocrinol 16: 903–911.

    CAS  PubMed  Google Scholar 

  • Franzen A, Piek E, Westermark B, ten Dijke P, Heldin NE . (1999). Expression of transforming growth factor-beta1, activin A, and their receptors in thyroid follicle cells: negative regulation of thyrocyte growth and function. Endocrinology 140: 4300–4310.

    Article  CAS  PubMed  Google Scholar 

  • Fusco A, Berlingieri MT, Di Fiore PP, Portella G, Grieco M, Vecchio G . (1987). One- and two-step transformations of rat thyroid epithelial cells by retroviral oncogenes. Mol Cell Biol 7: 3365–3370.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Garcia-Jimenez C, Santisteban P . (2007). TSH signalling and cancer. Arq Bras Endocrinol Metabol 51: 654–671.

    Article  PubMed  Google Scholar 

  • He H, Jazdzewski K, Li W, Liyanarachchi S, Nagy R, Volinia S et al. (2005). The role of microRNA genes in papillary thyroid carcinoma. Proc Natl Acad Sci USA 102: 19075–19080.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Heinemeyer T, Wingender E, Reuter I, Hermjakob H, Kel AE, Kel OV et al. (1998). Databases on transcriptional regulation: TRANSFAC, TRRD and COMPEL. Nucleic Acids Res 26: 362–367.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Heldin CH, Landstrom M, Moustakas A . (2009). Mechanism of TGF-beta signaling to growth arrest, apoptosis, and epithelial-mesenchymal transition. Curr Opin Cell Biol 21: 166–176.

    Article  CAS  PubMed  Google Scholar 

  • Hurst DR, Edmonds MD, Scott GK, Benz CC, Vaidya KS, Welch DR . (2009). Breast cancer metastasis suppressor 1 up-regulates miR-146, which suppresses breast cancer metastasis. Cancer Res 69: 1279–1283.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jemal A, Siegel R, Xu J, Ward E . (2010). Cancer statistics, 2010. CA Cancer J Clin 60: 277–300.

    Article  PubMed  Google Scholar 

  • Jhiang SM, Caruso DR, Gilmore E, Ishizaka Y, Tahira T, Nagao M et al. (1992). Detection of the PTC/retTPC oncogene in human thyroid cancers. Oncogene 7: 1331–1337.

    CAS  PubMed  Google Scholar 

  • Kawaguchi A, Ikeda M, Endo T, Kogai T, Miyazaki A, Onaya T . (1997). Transforming growth factor-beta1 suppresses thyrotropin-induced Na+/I- symporter messenger RNA and protein levels in FRTL-5 rat thyroid cells. Thyroid 7: 789–794.

    Article  CAS  PubMed  Google Scholar 

  • Kimura ET, Kopp P, Zbaeren J, Asmis LM, Ruchti C, Maciel RM et al. (1999). Expression of transforming growth factor beta1, beta2, and beta3 in multinodular goiters and differentiated thyroid carcinomas: a comparative study. Thyroid 9: 119–125.

    Article  CAS  PubMed  Google Scholar 

  • Kimura ET, Nikiforova MN, Zhu Z, Knauf JA, Nikiforov YE, Fagin JA . (2003). High prevalence of BRAF mutations in thyroid cancer: genetic evidence for constitutive activation of the RET/PTC-RAS-BRAF signaling pathway in papillary thyroid carcinoma. Cancer Res 63: 1454–1457.

    CAS  PubMed  Google Scholar 

  • Lazzereschi D, Nardi F, Turco A, Ottini L, D'Amico C, Mariani-Costantini R et al. (2005). A complex pattern of mutations and abnormal splicing of Smad4 is present in thyroid tumours. Oncogene 24: 5344–5354.

    Article  CAS  PubMed  Google Scholar 

  • Massague J . (1998). TGF-beta signal transduction. Annu Rev Biochem 67: 753–791.

    Article  CAS  PubMed  Google Scholar 

  • Massague J . (2008). TGFbeta in cancer. Cell 134: 215–230.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Matsuo SE, Fiore AP, Siguematu SM, Ebina KN, Friguglietti CU, Ferro MC et al. (2010). Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues. Arq Bras Endocrinol Metabol 54: 406–412.

    Article  PubMed  Google Scholar 

  • Matsuo SE, Leoni SG, Colquhoun A, Kimura ET . (2006). Transforming growth factor-beta1 and activin A generate antiproliferative signaling in thyroid cancer cells. J Endocrinol 190: 141–150.

    Article  CAS  PubMed  Google Scholar 

  • Morris III JC, Ranganathan G, Hay ID, Nelson RE, Jiang NS . (1988). The effects of transforming growth factor-beta on growth and differentiation of the continuous rat thyroid follicular cell line, FRTL-5. Endocrinology 123: 1385–1394.

    Article  CAS  PubMed  Google Scholar 

  • Moustakas A, Heldin CH . (2005). Non-Smad TGF-beta signals. J Cell Sci 118: 3573–3584.

    Article  CAS  PubMed  Google Scholar 

  • Nicolussi A, D'Inzeo S, Santulli M, Colletta G, Coppa A . (2003). TGF-beta control of rat thyroid follicular cells differentiation. Mol Cell Endocrinol 207: 1–11.

    Article  CAS  PubMed  Google Scholar 

  • Nikiforova MN, Chiosea SI, Nikiforov YE . (2009). MicroRNA expression profiles in thyroid tumors. Endocr Pathol 20: 85–91.

    Article  CAS  PubMed  Google Scholar 

  • Nikiforova MN, Tseng GC, Steward D, Diorio D, Nikiforov YE . (2008). MicroRNA expression profiling of thyroid tumors: biological significance and diagnostic utility. J Clin Endocrinol Metab 93: 1600–1608.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pacifico F, Crescenzi E, Mellone S, Iannetti A, Porrino N, Liguoro D et al. (2010). Nuclear factor-\{kappa\}B contributes to anaplastic thyroid carcinomas through up-regulation of. J Clin Endocrinol Metab 95: 1421–1430.

    Article  CAS  PubMed  Google Scholar 

  • Paire A, Bernier-Valentin F, Selmi-Ruby S, Rousset B . (1997). Characterization of the rat thyroid iodide transporter using anti-peptide antibodies. Relationship between its expression and activity. J Biol Chem 272: 18245–18249.

    Article  CAS  PubMed  Google Scholar 

  • Pallante P, Visone R, Croce CM, Fusco A . (2010). Deregulation of microRNA expression in follicular-cell-derived human thyroid carcinomas. Endocr Relat Cancer 17: F91–104.

    Article  CAS  PubMed  Google Scholar 

  • Palona I, Namba H, Mitsutake N, Starenki D, Podtcheko A, Sedliarou I et al. (2006). BRAFV600E promotes invasiveness of thyroid cancer cells through nuclear factor kappaB activation. Endocrinology 147: 5699–5707.

    Article  CAS  PubMed  Google Scholar 

  • Pei XH, Xiong Y . (2005). Biochemical and cellular mechanisms of mammalian CDK inhibitors: a few unresolved issues. Oncogene 24: 2787–2795.

    Article  CAS  PubMed  Google Scholar 

  • Pfaffl MW . (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ricarte-Filho JC, Fuziwara CS, Yamashita AS, Rezende E, da-Silva MJ, Kimura ET . (2009). Effects of let-7 microRNA on cell growth and differentiation of papillary thyroid cancer. Transl Oncol 2: 236–241.

    Article  PubMed  PubMed Central  Google Scholar 

  • Riesco-Eizaguirre G, Rodriguez I, De la Vieja A, Costamagna E, Carrasco N, Nistal M et al. (2009). The BRAFV600E oncogene induces transforming growth factor beta secretion leading to sodium iodide symporter repression and increased malignancy in thyroid cancer. Cancer Res 69: 8317–8325.

    Article  CAS  PubMed  Google Scholar 

  • Saavedra HI, Knauf JA, Shirokawa JM, Wang J, Ouyang B, Elisei R et al. (2000). The RAS oncogene induces genomic instability in thyroid PCCL3 cells via the MAPK pathway. Oncogene 19: 3948–3954.

    Article  CAS  PubMed  Google Scholar 

  • Santoro M, Melillo RM, Carlomagno F, Fusco A, Vecchio G . (2002). Molecular mechanisms of RET activation in human cancer. Ann NY Acad Sci 963: 116–121.

    Article  CAS  PubMed  Google Scholar 

  • Siegel PM, Massague J . (2003). Cytostatic and apoptotic actions of TGF-beta in homeostasis and cancer. Nat Rev Cancer 3: 807–821.

    Article  CAS  PubMed  Google Scholar 

  • Taganov KD, Boldin MP, Chang KJ, Baltimore D . (2006). NF-kappaB-dependent induction of microRNA miR-146, an inhibitor targeted to signaling proteins of innate immune responses. Proc Natl Acad Sci USA 103: 12481–12486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Taton M, Lamy F, Roger PP, Dumont JE . (1993). General inhibition by transforming growth factor beta 1 of thyrotropin and cAMP responses in human thyroid cells in primary culture. Mol Cell Endocrinol 95: 13–21.

    Article  CAS  PubMed  Google Scholar 

  • Wang J, Knauf JA, Basu S, Puxeddu E, Kuroda H, Santoro M et al. (2003). Conditional expression of RET/PTC induces a weak oncogenic drive in thyroid PCCL3 cells and inhibits thyrotropin action at multiple levels. Mol Endocrinol 17: 1425–1436.

    Article  CAS  PubMed  Google Scholar 

  • Xia H, Qi Y, Ng SS, Chen X, Li D, Chen S et al. (2009). microRNA-146b inhibits glioma cell migration and invasion by targeting MMPs. Brain Res 1269: 158–165.

    Article  CAS  PubMed  Google Scholar 

  • Yang G, Yang X . (2010). Smad4-mediated TGF-beta signaling in tumorigenesis. Int J Biol Sci 6: 1–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhong H, Wang HR, Yang S, Zhong JH, Wang T, Wang C et al. (2010). Targeting Smad4 links microRNA-146a to the TGF-beta pathway during retinoid acid induction in acute promyelocytic leukemia cell line. Int J Hematol 92: 129–135.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Dr Konstantin D Taganov for donation of pcDNA3.1-miR-146b plasmid, Dr Joan Massagué for donation of p3TP-Lux plasmid, Dr Nancy Carrasco for donation of anti-NIS antibody, Dr Massimo Santoro for BCPAP, and Dr James A Fagin for PCCL3, PCCL3-rtTA, PTC3-5, TPC-1, and ARO cell lines used in this study. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Correspondence to E T Kimura.

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Geraldo, M., Yamashita, A. & Kimura, E. MicroRNA miR-146b-5p regulates signal transduction of TGF-β by repressing SMAD4 in thyroid cancer. Oncogene 31, 1910–1922 (2012). https://doi.org/10.1038/onc.2011.381

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