MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells

Lima et al. BMC Cancer (2016) 16:108 DOI 10.1186/s12885-016-2146-z RESEARCH ARTICLE Open Access MiRNA-146b-5p upregulates migration and invasion of different Papillary Thyroid Carcinoma cells Cilene Rebouças Lima, Murilo Vieira Geraldo, Cesar Seigi Fuziwara, Edna Teruko Kimura and Marinilce Fagundes Santos* Abstract Background: Tumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, usually degrading extracellular matrix. Despite significant progress in the knowledge about migration and invasion, there is much more to elucidate about their regulatory mechanisms, especially in cancer cells. MicroRNAs (miRs) were recently described as important regulators of migration. Differential expression of miRs in cancer is frequently associated with progression, invasion and metastasis. In papillary thyroid carcinoma (PTC), miR-146b-5p is highly expressed and positively correlated to the degree of malignancy. Methods: This study aimed to investigate the role of miR-146b-5p on the migratory and invasive behaviors of thyroid cells, using a non tumor rat thyroid follicular cell line (PCCl3) transfected with the miR-146b-5p genomic region, and two PTC cell lines (TPC-1 and BCPAP, bearing distinct oncogenic backgrounds), which express high levels of miR-146b-5p, after miR-146b inhibition by antagomiR and miR-146b overexpression by mimics-miR. Migration and invasion were studied by time-lapse and transwell assays (with and without Matrigel®). Gelatin degradation assays were also employed, as well as F-actin staining. Results: Migration and invasion of PCCl3 were increased 2-3x after miR-146b-5p overexpression (10X) and large lamellipodia were evident in those cells. After miR-146b-5p inhibition, TPC-1 and BCPAP migration and invasion were significantly reduced, with cells showing several simultaneous processes and low polarity. Gelatin degradation was inhibited in TPC-1 cells after inhibition of miR-146b-5p, but was unaffected in BCPAP cells, which did not degrade gelatin. The inhibition of miR-146b-5p in PCCl3 also inhibited migration and invasion, and additional (exogenous) overexpression of this miR in TPC-1 and BCPAP cells increased migration and invasion, without effects on cell morphology or gelatin degradation. The overexpression of SMAD4 in BCPAP cells, a validated target of miR-146b-5p and key protein in the TGF-β signaling pathway, inhibited migration similarly to the effects observed with the antagomiR 146b-5p. Conclusions: miR-146b-5p positively regulates migration and invasion of thyroid normal and tumor follicular cells (independently from their original mutation, either BRAF or RET/PTC), through a mechanism that involves the actin cytoskeleton but not an increased capacity of matrix degradation. Keywords: MicroRNAs, Thyroid, Cancer, Invasion, PTC, Cell migration, miR-146b * Correspondence: Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of São Paulo, Avenida Professor Lineu Prestes 1524, Prédio I, CEP 05508-000 São Paulo, SP, Brazil © 2016 Lima et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lima et al. BMC Cancer (2016) 16:108 Background Tumor invasiveness is directly related to the ability of tumor cells to migrate and invade surrounding tissues, spreading via blood and lymphatic circulation. In tumors, the more pronounced is the migratory phenotype, the higher is its metastatic potential [1]. A complex signal transduction network involving different pathways directly and indirectly controls tumorigenesis and invasion [2]. Highly invasive adherent tumor cells present a mesenchymal phenotype and are able to migrate faster, degrading extracellular matrix on their way. In general, in order to migrate, these cells polarize and form lamellipodia at the cell front, which are large membrane projections rich in branching actin filaments and lacking organelles. New adhesions to the extracellular matrix (ECM) are established, and some of them mature to become anchorage junctions to the actin cytoskeleton. Adhesion maturation is followed by the pulling of the cell body forward and retraction of the rear, partially due to the contraction of actin-myosin II bundles present inside the cell and in the cell cortex [3]. Sometimes filopodia, which are thin spike-like exploratory processes, precede or accompany lamellipodia formation. This migration cycle is regulated by Rho GTPases, central modulators of the cytoskeleton involved in many signaling pathways [4]. The classic regulatory cycle of Rho GTPases involve molecules that regulate GTP binding and hydrolysis, as well as the availability of GTPases to be activated, usually in cell membranes. In the last few years, other important regulatory mechanisms were described, including microRNAs (miRs) [5]. MicroRNAs are small, non-coding RNAs that regulate protein expression and have been implicated in both the promotion and suppression of metastasis [6]. The term ‘metastamir’ describes miRs that are involved in tumor metastasis in different ways, acting either as prometastatic or antimetastatic [7]. The role of miRs is posttranscriptional gene regulation via perfect or imperfect pairing with the 3’ untranslated region (UTR) of target messenger RNAs (mRNAs), leading to mRNA degradation or translation blockage. In tumors, the differential expression of miRs (up or down) is frequently associated with progression, invasion and metastasis. For this reason, miRs have been considered as potentially important tumor hallmarks, and their deregulation is the focus of studies that intend to find tools for early diagnosis, prognosis, monitoring and treatment [6, 7]. An example of tumor which invasive behavior is much less understood than its development is the Papillary Thyroid Carcinoma (PTC). Both in tumor progression and invasiveness, however, miRs are involved [8]. PTC is the most common thyroid type of cancer, representing about 80 % of all malignant tumors in this organ [9, 10]. It is usually a multifocal intra-thyroid tumor (65 % of Page 2 of 13 cases), which can be encapsulated or infiltrative. Small localized PTCs show a 99 % survival rate at 20 years, being considered low risk cancers. Considering the scores usually applied to classify PTCs as low risk, such as age, grade, extent (invasiveness and distant metastasis), size, sex and nodal spread, about 80–85 % of PTCs have excellent prognosis. These s (...truncated)