Study population and tissue collection

This retrospective study was conducted with local ethics committee approval (Sapienza Ethics Committee; project code 1184). Twenty-six tissue samples of metastatic DTC were collected from patients who underwent surgical resection between 2002 and 2014 at Agostino Gemelli Hospital in Rome or at Sapienza University of Rome. Formalin-fixed and paraffin embedded (FFPE) tissue samples were reviewed separately by two pathologists who confirmed the diagnosis. FFPE samples were then subjected to molecular analysis after macrodissection of tumor tissues.

Patients were classified according to their radioiodine uptake ability and/or their response to RAI therapy and divided into two cohorts: cohort I included 14 non-responder (NR) patients, while cohort II was composed of 12 responder (R) patients (Table 1). Patients were included in cohort I if they met at least one of the following criteria: i) the metastatic tissue did not concentrate radioiodine at the time of the first I131 treatment; ii) the tumor tissue lost the ability to uptake radioiodine after previous evidence of uptake; iii) radioiodine was concentrated in some lesions but not in others; and/or iv) metastatic disease progressed despite significant RAI uptake [20]. Patients were included in cohort II if they were successfully treated with RAI therapy (i.e., lesions concentrated radioiodine and disappeared or did not progress over time).

Table 1 Baseline Characteristics of Cohorts I and IINucleic acid isolation from tissues

DNA and total RNA (containing miRNAs) were simultaneously extracted from primary DTC tissues using the RecoverAll™ Total Nucleic Acid Isolation Kit (Thermo Fisher Scientific) and quantified with Qubit fluorescence-based assays for dsDNA and RNA (Qubit®, Thermo Fisher Scientific), as previously described [21].

Next-generation sequencing (NGS)-based mutation analysis

NGS analysis was performed on the Ion Gene Studio S5 system (Thermo Fisher Scientific) using two thyroid-specific custom panels, a DNA panel and an RNA panel, as previously described [21, 22].

Briefly, two libraries were prepared from 15 ng of DNA and 10 ng of RNA with the Ion AmpliSeq™ Library Kit Plus using the IonXpress™ Barcode Adapter 1–96 Kit (Thermo Fisher Scientific). The purified libraries were quantified with Ion Library TaqMan® Quantitation Kit on the 7900HT Fast Real Time PCR system (Thermo Fisher Scientific). DNA and RNA pooled libraries were clonally amplified on the Ion One Touch2 System and sequenced on Ion Gene Studio S5 (Thermo Fisher Scientific) according to the manufacturer’s instructions and as previously described [23].

Data analysis was performed using Torrent Suite v.5.10 with Coverage Analysis and Variant Caller plugins and annotated with Ion Reporter 5.12 and the wANNOVAR web server. Gene fusion analysis was performed with Ion Reporter 5.12 software (Thermo Fisher Scientific) using the workflow for gene fusion detection [22].

Analysis of tissue miRNAs

MicroRNA profiling of 754 miRNAs was performed on RNA isolated from FFPE primary thyroid cancer tissues (cohort I and II patients) using TaqMan Array Human MicroRNA A + B Cards v3.0 (Thermo Fisher Scientific). TaqMan arrays were processed as previously reported [24, 25].

Expression Suite software v1.0.3 (Thermo Fisher Scientific) was employed to evaluate cycle threshold (Ct) values for each miRNA and relative miRNA expression using the comparative 2-ΔΔCt method. Data were normalized using U6 as an endogenous control. MicroRNAs with Ct values > 35 were excluded [26]. Row data are available upon request.

Cell cultures

Primary normal and tumor cell cultures were established from the normal and tumoral areas of patients with papillary thyroid cancer who underwent surgical resection, as previously described [27]. They were maintained in culture for 2–5 passages at 37 °C with 5% CO2. The presence of the p.V600E point mutation of the BRAF gene was verified by Sanger sequencing analysis of DNA isolated using NucleoSpin Tissue Mini Kit for DNA from cells and tissue (Macherey Nagel), as previously described [28]. PCR conditions and sequencing primers are available upon request. Mutational status is reported in Supplementary Table 1.

Commercial immortalized thyroid cancer cell lines (TPC1, BCPAP, K1, 8505c, SW1736) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) media (Gibco-BRL Division, Thermo Fisher Scientific, Waltham, MA, USA), according to ATCC® instructions, containing 10% fetal bovine serum (FBS) (Gibco-BRL Division, Thermo Fisher Scientific) and antibiotic–antimycotic solution (Gibco-BRL Division, Thermo Fisher Scientific). The mutational status and histological origin of the cell lines used in this study are reported in Supplementary Table 1. Mutational status of each continuous cell line was verified by Sanger sequencing analysis [8, 28, 29].

Fisher rat thyroid low-serum 5 (FRTL5) cells were cultured in Coon’s modified F12 medium containing 5% FBS (Gibco-BRL Division, Thermo Fisher Scientific, Waltham, MA, USA), 2 mM L-glutamine, antibiotic–antimycotic solution (Gibco-BRL Division, Thermo Fisher Scientific), and the 5H mix (20 µg/ml Gly-His-Lys acetate, 3.62 µg/ml hydrocortisone, 1 µg/ml insulin, and 5 µg/ml transferrin). Every 2 days, the 6H mix containing 1 mUI/ml thyroid-stimulating hormone (TSH) was added to the medium. All cells were incubated at 37 °C in an atmosphere of 5% CO2.

Overexpression of miR-139-5p

Synthetic miR-139-5p (MISSION® microRNA Mimic hsa-miR-139-5p HMI0212) or a negative control (MISSION® miRNA, Negative Control 1, HMC0002) was transfected into primary and immortalized thyroid cancer cell lines at 30 nM of final concentration using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific). Treatments, performed according to the manufacturer’s instructions, were started after 2 h of cell starvation, and maintained for 48 h.

RNA isolation from cells and real-time PCR

RNA was isolated from cells using RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantified with Nanodrop 2000 (Thermo Fisher Scientific). The High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) was used to synthesize cDNA from RNA according to the manufacturer’s instructions (Thermo Fisher Scientific). The expression levels of miR-139-5p and thyroid-specific genes (SLC5A5, TPO, TG, PAX8, TSHR) were analyzed on cDNAs from treated and control cells using TaqMan Gene Expression assays [SLC5A5 (Hs00166567_m1), TG (Hs00174974_m1), PAX8 (Hs00247586_m1), TPO (Hs00892519_m1), TSHR (Hs01053846_m1), and miR-139-5p (005364_mat)] and TaqMan Universal Master Mix according to the manufacturer’s instructions (Thermo Fisher Scientific).

Results were calculated using the 2−ΔΔCt method and normalized to the corresponding endogenous controls, U6 snRNA (TaqMan® Gene Expression Assays Code: 001,093) or GAPDH (TaqMan® Gene Expression Assays Hs02786624_g1). Data were expressed as the mean ± standard deviation (SD) of three replicates.

A commercial pool of total RNA isolated from normal thyroids (Human Thyroid Total RNA, THYN, Takara) has been used as calibrator sample. The pool includes RNA from 3 male Asians, African American, ages 21, 29, 76, respectively, with unknown cause of death [30].

Immunofluorescence

Immunofluorescence experiments were performed using Lab-Tek chamber slides as support (Nunc cell culture division, Thermo Fisher Scientific). Cells were fixed with 4% paraformaldehyde for 20 min at room temperature, permeabilized with Triton X-100 (0.1%), diluted in phosphate-buffered saline (PBS), and incubated in blocking solution (BSA 3% in PBS). Cells were incubated overnight with primary antibodies diluted in PBS and for 1 h with secondary antibodies. Primary antibodies were rabbit anti-NIS polyclonal (Genetek). Secondary antibodies were 488-conjugated anti-rabbit (Thermo Fisher Scientific). Nuclei were Hoechst-counterstained. Images were acquired with an optical microscope, Leica DM IL LED Fluo, using a GFP filter cube.

Iodide uptake assay

Iodide uptake assay was performed as previously described [31]. Briefly, cells were seeded in a 96-well plate at 70% confluence and were treated the next day as described in the cell treatments section. After 48 h of treatment, iodide uptake assay was performed.

We used FRTL5 and BCPAP cell lines as positive and negative controls, respectively. As described in the cell cultures section, the FRTL5 cell line was treated with the medium added with 5H and 6H mixes. BCPAP cell line was treated with suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), as previously described [32].

The culture medium was replaced twice by the uptake buffer (containing 10 mM of HEPES diluted in Hank’s balanced salt solution [HBSS]). At the end of the washing cycle, 80 µl of fresh uptake buffer remained in each well of the 96-well plate. Immediately, 10 µl of 100 µM NaI solution was added to each well. For each experimental condition, a competitive inhibitor of NIS (KClO4 10 µM) was also added in half of the wells as a control to determine non-specific iodide uptake. The assay plate was left at 20 °C for 60 min in the dark.

At the end of incubation, the buffer was discarded, and cells were washed once with ice-cold HBSS (Thermo Fisher Scientific). Then, cells were lysed with 100 µl of 0.1 M NaOH solution. All lysate was used for the Nonradioactive Iodide Assay Kit (Bertin pharma) according to the manufacturer’s instructions. Absorbances obtained from the experiment (at 420 nm) were normalized for the absorbances of the cell in each well (at 630 nm).

Iodide concentration in unknown samples was determined using linear regression of the standard curve provided by the kit. Results were expressed as specific units (µM) of iodide accumulation relative to controls.

Western blot analysis

Cells were lysed using the lysis buffer described by Pecce et al. [29]. Thirty μg of total protein extract were loaded on 12% polyacrylamide gel, transferred to.polyvinylidene difluoride (PVDF) membranes, blocked with 5% non-fat dry milk for 1 h and then incubated overnight with primary antibodies.

For analysis of the nucleus, cytoplasm, and membrane fractions, cells were lysed using Buffer A (containing HEPES pH 7.4 20 mM, glycerol 20%, KCl 50 mM, EDTA 1 mM, and protease inhibitors [Thermo Fisher]). Buffer B (containing 10% NP-40) was added after 15 min. The samples were centrifuged at 5000 rpm for 5 min and the supernatant and pellet were recovered as cytoplasmatic extracts and isolated nuclei, respectively. Nuclear pellets were lysed with Buffer C (containing HEPES pH 7.4 20 mM, glycerol 25%, NaCl 400 mM, EDTA 2 mM, and protease inhibitors [Thermo Fisher Scientific]) and centrifuged at 13,000 rpm for 10 min. The membrane fraction was isolated after incubating the samples for 10 min on ice for cell lysis. After centrifugation (13,000 rpm for 15 min), the pellets were processed with a buffer containing Tris HCl pH 7.4 50 mM, NaCl 150 mM, SDS 1%, TRITON-X 1%, and protease inhibitors (Thermo Fisher Scientific).

The primary antibodies were anti-NIS (Genetek) diluted 1:1000, anti-β-actin (Sigma Aldrich) diluted 1:3000, H3 (Abcam) diluted 1:3000, LDLR (Abcam) diluted 1:1000, which were used as a loading controls. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies: anti-rabbit (diluted 1:5000) or anti-mouse (diluted 1:5000) (Transduction Laboratories, Lexington, KY, USA). The blots were developed with Western Blot ECL Plus detection system (Perkin Elmer, Milan, Italy) and the results were acquired with the ChemiDoc MP system (Bio-Rad). Densitometric analyses were performed using Image Lab software (Bio-Rad).

Statistical analysis

Statistical analyses of the clinicopathological and mutational characteristics of DTC patients and of thyroid cancer cell lines were performed with GraphPad Prism 6.01. Continuous variables were expressed as medians and ranges, and nominal variables as numbers and percentages. Differences between categorical variables were evaluated with the chi-square test. Differences between continuous variables were assessed with the Mann–Whitney test for patient samples and the unpaired t test for cell lines.

Differential miRNA expression levels between NR and R patients were assessed with the Mann–Whitney U test followed by Benjamini–Hochberg correction using the R stats package (R software version 3.1.1). Dendrograms and heat maps were generated with GENE-E software version 3.0.215 (http://www.broadinstitute.org/cancer/software/GENE-E) with Spearman correlation and complete linkage. A p value < 0.05 was considered statistically significant.