Nineteen thyroid cancer patients diagnosed with anaplastic thyroid cancer were recruited from the Second Artillery General Hospital of PLA and Tongji Hospital. The use of human tissue samples followed internationally recognised guidelines as well as local and national regulations. Research carried out on humans follow international and national regulations. Medical ethics committee approved the experiments undertaken. Informed consent was obtained from each individual. Thyroid cancer cell lines ARO and FRO was kindly donated by Dr Su Kai (Shanghai Cancer Center, China). Briefly, cells were maintained in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and penicillin/streptomycin at 37°C in a humidified atmosphere with 5% CO₂.

MRTF-A expressing plasmid/empty vector and shMRTF-A plasmids/scramble were donated by Shao-Xin Huang (Boston University, Boston, MA, USA) and was as described previously (28). Pre-miR-206/control miR and anti-miR-206/scramble were purchased from Ambion, Inc. (Ambion, Austin, TX, USA). For transfection experiments, the cells were cultured in serum-free medium without antibiotics at 60% confluence for 24 h, and then transfected with transfection reagent (Lipofectamine 2000, Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After incubation for 6 h, the medium was removed and replaced with normal culture medium for 48 h, unless otherwise specified.

Western blot analysis was performed as described before (29). In brief, after incubation with primary antibody anti-MRTF-A (1:500; Abcam, Cambridge, MA, USA) and anti-β-actin (1:500; Abcam) overnight at 4°C, IRDye™-800 conjugated anti-rabbit secondary antibodies (Li-COR, Biosciences, Lincoln, NE, USA) were used for 30 min at room temperature. The specific proteins were visualized by Odyssey™ Infrared Imaging System (Gene Co., Lincoln, NE, USA).

For transwell migration assays, 2.5×10⁴–5.3×10⁴ cells were plated in the top chamber with the non-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences, San Jose, CA, USA). For invasion assays, 1.25×10⁵ cells were plated in the top chamber with Matrigel-coated membrane (24-well insert; pore size, 8 mm; BD Biosciences). In both assays, cells were plated in medium without serum or growth factors, and medium supplemented with serum was used as a chemoattractant in the lower chamber. The cells were incubated for 24 h and cells that did not migrate or invade through the pores were removed by a cotton swab. Cells on the lower surface of the membrane were stained with the Diff-Quick Staining Set (Dade) and counted.

Cells (5×10⁵) were seeded onto each 35-mm glass bottom dish (MatTek Co., Ashland, MA, USA) and cultured at 37°C with 5% CO₂ for 24 h. The confluent monolayer of cells was wounded. Monolayers of cells were wounded with pipette tips. After washing with warm PBS, the cells were incubated in fresh culture medium. The wounded areas were photographed at the beginning (0 h, top panels) and the end (10 h, bottom panels) of the assay with Nikon inverted microscope (Eclipse TE-2000U, Nikon, Japan) equipped with a video camera (DS-U1, Nikon, Japan).

Cells seeded on 96-well plates, were stained at indicated time point with 100 ml sterile MTT dye (0.5 mg/ml, Sigma, St. Louis, MO, USA) for 4 h at 37°C, followed by removal of the culture medium and addition of 150 ml of dimethyl sulphoxide (DMSO) (Sigma). The absorbance was measured at 570 nm, with 655 nm as the reference wavelength.

Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miRNA Isolation kit (Ambion). cRNA for each sample was synthesized by using 3′ IVT Express kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s protocols. The purified cRNA was fragmented by incubation in fragmentation buffer (provided in the 3′IVT express kit) at 95°C for 35 min and chilled on ice. The fragmented labeled cRNA was applied to MicroRNA2.0 array (Affymetrix) and hybridized in Genechip hybridization oven 640 (Affymetrix) at 45°C for 18 h. After washing and staining in Genechip fluidics station 450 (Affymetrix), the arrays were scanned by using Genechip scanner 3000 (Affymetrix). The gene expression levels of samples were normalized and compared by using Partek GS 6.5 (Partek, Inc., St. Louis, MO, USA). Average-linkage hierarchical clustering of the data was applied by using the Cluster (Eisen et al, Stanford, Stanford University, CA, USA; http://rana.lbl.gov) and the results were displayed by using TreeView (Eisen et al, Stanford, Stanford University, CA, USA; http://rana.lbl.gov).

The analysis of potential microRNA target sites were performed using common prediction algorithms - TargetScan (http://www.targetscan.org) and PicTar (http://pictar.mdc-berlin.de/).

For immunofluorescence analyses of cells were plated on glass coverslips in 6-well plates and transfected with 30 nM pre-miR-206 or control miR. At 36 h after transfection, coverslips were stained with the mentioned anti-MRTF-A antibodies. Alexa Fluor 488 goat anti-rabbit IgG antibody was used as secondary antibody (Invitrogen). Coverslips were counterstained with DAPI (Invitrogen-Molecular Probes, Eugene, OR, USA) for visualization of nuclei. Microscopic analysis was performed with a confocal laser-scanning microscope (Leica Microsystems, Bensheim, Germany). Fluorescence intensities were measured in a few viewing areas for 200–300 cells per coverslip and analyzed using ImageJ 1.37v software (http://rsb.info.nih.gov/ij/index.html).

Total RNA was isolated from cells using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized from the total RNA using M-MLV reverse transcriptase (Promega, Madison, WI, USA) and random hexamer primers (Sangon, Shanghai, China). The thermal cycle profile was as follows: denaturation for 30 sec at 95°C, annealing for 45 sec at 52–58°C depending on the primers used, and extension for 45 sec at 73°C. PCR products were visualized on 2% agarose gels stained with ethidium bromide under UV transillumination. qRT-PCR was performed with a Power SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s protocol. The primer sequences for MRTF-A: forward primer 5′-ATGGAGCTGGTGGAGAAGAATATC-3′ and reverse 5′-GAAGGAGGAACTGTCTGCTACC-3′.

Total RNA from cultured cells, with efficient recovery of small RNAs, was isolated using the mirVana miRNA Isolation kit (Ambion). Detection of the mature form of miRNAs was performed using the mirVana qRT-PCR miRNA Detection kit, according to the manufacturer’s instructions (Ambion). The U6 small nuclear RNA was used as an internal control.

The 3′-untranslated region (3′-UTR) of human MRTF-A mRNA was cloned in pRL-TK (Promega) using PCR-generated fragment. Site-directed mutagenesis of the predicted target-site of miR-206 in the MRTF-A-3′-UTR was carried out using Quik change-mutagenesis kit (Stratagene, Heidelberg, Germany), with MRTF-A-WT-luc as a template. For reporter assays, cells was transiently transfected with WT or mutant reporter plasmid and microRNA or anti-microRNA using Lipofectamine 2000 (Invitrogen). Reporter assays were performed 36 h post-transfection using the Dual-luciferaseassay-system (Promega), and normalized for transfection efficiency by cotransfected Renilla-luciferase.

Anoikis resistance was evaluated by seeding 7.5×10⁴ cells in ultralow attachment plates (Corning). After 24 h of anchorage-independent culture, cells were transfected as indicated and resuspended in 0.4% trypan blue (Sigma) and cell viability was assessed.

Data are presented as mean ± SEM Student’s t-test (two-tailed) was used to compare two groups (P<0.05 was considered significant), unless otherwise indicated (χ² test).

In an attempt to identify MRTF-A expression between thyroid cancer tissues and adjacent normal tissues, we performed western blotting in cancer tissues versus normal tissues. Protein was isolated from 6 pairs of thyroid cancer tissues and normal tissues (patients no. 1–6). However, we did not detect any difference between cancer tissues and adjacent normal tissues (Fig. 1A). Because MRTF-A was recently implicated in tumor invasion and metastasis, we hypothesized that MRTF-A was also associated with invasion and metastasis in thyroid cancer (8). Thus, using western blot assay, we detected MRTF-A expression between primary thyroid cancer tissues and metastatic thyroid cancer tissues obtained from further 6 patients (patients no. 7–12). Cancer metastasis has occurred in all the 6 patients. Although we did not detect any difference between cancer tissues and normal tissues, we found that MRTF-A protein was significantly increased in metastatic thyroid cancer tissues, compared with primary thyroid cancer (Fig. 1B). All the results implied that MRTF-A was associated with metastasis of anaplastic thyroid cancer.

In an attempt to identify the role of MRTF-A in regulating migration and invasion of ARO cells, the cells were transfected with MRTF-A expressing plasmids. After stable transfection, MRTF-A protein expression was detected by western blotting and the results showed that MRTF-A protein was increased by MRTF-A expressing plasmids in the cells (Fig. 2A). Next, we performed migration and invasion assay to detect migration and invasion of ARO cells transfected with MRTF-A expressing plasmids and empty vectors. Ectopic MRTF-A promoted motility and invasion by ~4-fold in the cells (Fig. 2B). To confirm the results, wound-healing assay was performed. Wound-healing assay showed that MRTF-A significantly promoted motility in the cells (Fig. 2C). To further show the effects of MRTF-A on metastasis, we performed anoikis assays to analyze its effects on anoikis resistance. Also, MRTF-A-transfected cells exhibited ~150% increased resistance to anoikis-mediated cell death (Fig. 2D). Having demonstrated that MRTF-A overexpression promoted migration and invasion in ARO cells, to provide further evidence that the roles of MRTF-A were involved in migration and invasion, we studied the effects of shMRTF-A, an inhibitor of MRTF-A. After stable transfection, MRTF-A expression was detected by western blotting. The results showed that shMRTF-A significantly downregulated MRTF-A expression in ARO cells (Fig. 2E). Next, we performed migration and invasion assay to detect migration and invasion of ARO cells transfected with shMRTF-A plasmids and scramble. Silencing MRTF-A inhibited motility and invasion by ~0.6-fold (Fig. 2F). To further confirm the roles of shMRTF-A on metastasis, we performed anoikis assays to analyze its effects on anoikis resistance. Contrary to MRTF-A overexpression, shMRTF-A-transfected cells exhibited ~30% decreased resistance to anoikis-mediated cell death (Fig. 2G).

We also performed MTT assay to detect whether it affected proliferation in ARO cells. Although MRTF-A was associated with metastasis in thyroid cancer patients and promoted migration and invasion in thyroid cancer ARO cells, neither overexpressing MRTF-A nor silencing it affected the cell viability (Fig. 2H and I).

The consequences of MRTF-A expression were not unique to ARO cells: MRTF-A protein was not only increased by MRTF-A expressing plasmids (Fig. 3A), but also promoted invasion (Fig. 3B), motility (Fig. 3B), and anoikis resistance (Fig. 3C), yet did not affect proliferation (Fig. 3D), in FRO human thyroid cancer cells. Hence, MRTF-A promotes in vitro surrogates of metastatic ability in thyroid cancer.

Having demonstrated that MRTF-A expression is specifically upregulated in metastatic thyroid cancer and it promotes metastasis-relevant traits in vitro, MRTF-A expression in metastatic thyroid cancer was studied for clarifying the mechanisms promoting MRTF-A expression in the disease. MicroRNAs (miRNAs) are a new class of small (~22 nucleotide) noncoding RNAs and negatively regulate protein-coding gene expression by targeting mRNA degradation or translation inhibition (9–11). Downregulation of specific miRNA can contribute to oncogene overexpression (30). Thus we evaluated whether MRTF-A was upregulated by defection of specific miRNA in metastatic thyroid cancer.

In an attempt to identify the level of miRNA expression in primary thyroid cancer and metastatic thyroid tissues, we performed miRNA profiling. RNAs isolated from 3 pairs of primary tumors and metastatic tumors were hybridized to a custom miRNA microarray platform. After hybridization, quantification, and normalization, we found that miR-206, miR-31 and miR-96 were significantly decreased in the metastatic tumors compared with primary tumors >100-fold (Fig. 4A).

As further confirmation, we used two common prediction algorithms - TargetScan (http://www.targetscan.org) and PicTar (http://pictar.mdc-berlin.de/) to analyze 3′-UTR of MRTF-A. All 2 algorithms predicted that miR-206 could target 3′-UTR of MRTF-A (Fig. 4B). In order to further study whether that miR-206 expression was associated with metastasis of thyroid cancer, we performed real-time PCR to detect miR-206 expression in 5 metastatic tumors and 14 primary tumors. Consistent with the results of miRNA microarray, real-time PCR demonstrated that miR-206 expression was significantly downregulated in metastatic tumors (Fig. 4C). Target sites on 3′-UTR of MRTF-A are shown in Fig. 4D. We reasoned that miR-206 could downregulate MRTF-A expression by targeting its 3′-UTR in thyroid cancer and that MRTF-A was overexpressed in thyroid cancer, due to lack of miR-206.

In an attempt to identify the role of miR-206 in regulating MRTF-A expression in ARO cells, cells were transfected with pre-miR-206 and control miR. After transfection, miR-206 expression was detected by real-time PCR and the results showed that miR-206 was increased by the pre-miR-206 in the cells (Fig. 4E). To confirm the reason, we performed immunofluorescence analyses in ARO cells transfected with pre-miR-206 or control miR. The results showed that MRTF-A protein was evidently suppressed in the cells transfected with pre-miR-206 (Fig. 4F). We next performed RT-PCR and western blotting to detect MRTF-A expression in ARO cells transfected with pre-miR-206 or control miR. The results showed that MRTF-A protein (Fig. 4G) and mRNA (Fig. 4H) were significantly downregulated in the cells transfected with pre-miR-206. Consistent with the results of RT-PCR, real-time PCR demonstrated that MRTF-A mRNA was reduced in ARO cells transfected with pre-miR-206, compared with control miR-transfected groups (Fig. 4I).

To further demonstrate the direct regulation of MRTF-A by miR-206, we constructed luciferase reporters with the targeting sequences of wild-type (MRTF-A-WT-luc) and mutated MRTF-A 3′-UTRs (MRTF-A-MUT-luc) (Fig. 4J). Both the wild-type and mutant reporters were introduced into ARO cells.

Luciferase reporter assay showed that the luciferase activities of MRTF-A-WT-luc plasmids were significantly suppressed in the cells transfected with pre-miR-206, implying that miR-206 targeted 3′-UTR of MRTF-A mRNA (Fig. 4K). In order to further identify that miR-206 targeted 3′-UTR of MRTF-A by the predicted sites, we mutated 3 bases in the predicted sites (Fig. 4J). In addition, mutant reporters were introduced into ARO cells, as expected the luciferase activities of MRTF-A-MUT-luc were not suppressed by miR-206 in ARO cells (Fig. 4K).

Having demonstrated that miR-206 overexpression inhibited MRTF-A-WT-luc plasmids by the predicted sites, we next studied whether silencing miR-206 could affect activity of the MRTF-A-WT-luc plasmids. Thus luciferase reporters assay was performed again and the results showed that contrary to pre-miR-206, anti-miR-206 significantly promoted luciferase activity of MRTF-A-WT-luc in ARO cells (Fig. 4L). Moreover, mutant reporters were also introduced into ARO cells, but the luciferase activities of MRTF-A-MUT-luc were not affected by anti-miR-206 in ARO cells (Fig. 4L). All the data illustrated that miR-206 degraded MRTF-A by targeting the specific sites predicted in silico in anaplastic thyroid cancer cells.

Having demonstrated that miR-206 was decreased in the metastatic tumors compared with primary tumors and it could degrade metastasis-relevant MRTF-A expression, we reasoned that it was also associated with metastasis-relevant traits in thyroid cancer cells. It was confirmed that miR-206 could be increased by pre-miR-206 (Fig. 4E). Next, we performed migration and invasion assay to detect migration and invasion of ARO cells transfected with pre-miR-206 and control miR. Ectopic miR-206 inhibited motility and invasion by about 2-fold (Fig. 5A). To further show the effects of miR-206 on metastasis, we performed anoikis assays to analyze its effects on anoikis resistance. Also, miR-206 overexpressing cells exhibited 40% decreased resistance to anoikis-mediated cell death (Fig. 5B).

Having demonstrated that miR-206 overexpression inhibits metastasis-relevant traits in ARO cells, to provide further evidence that miR-206 was involved in ARO cell migration and invasion, we studied the effects of anti-miR-206, an inhibitor of miR-206. After stable transfection, miR-206 expression was detected by real-time PCR. The results showed that anti-miR-206 significantly downregulated miR-206 expression in ARO cells (Fig. 5C). Next, we performed migration and invasion assay to detect migration and invasion of ARO cells transfected with anti-miR-206 and scramble. Silencing miR-206 promotes motility and invasion by more than 2-fold (Fig. 5D). To further confirm that the roles of anti-miR-206 on migration and invasion, we performed anoikis assays to analyze its effects on anoikis resistance. Contrary to miR-206 overexpression, anti-miR-206-transfected cells exhibited more than 2-fold increased resistance to anoikis-mediated cell death (Fig. 5E).

Because miR-206 directly degraded MRTF-A through its 3′-UTR, we reasoned that ectopic expression of MRTF-A by transfection of the cDNA that did not contain the predicted target of 3′-UTR should escape the regulation of miR-206 and thus attenuate or eliminate miR-206 function. To this end, we transfected MRTF-A expressing plasmids or empty vector (mock) into control miR or pre-miR-206-treated ARO cells. Immunoblot analysis revealed that transfection of MRTF-A expressing plasmids eliminated the effect of miR-206 on MRTF-A protein (Fig. 6A).

Because overexpression of miR-206 in ARO cells inhibited motility and invasion, and in order to identify whether MRTF-A could abrogate or attenuate the roles of miR-206 on motility and invasion, control miR or pre-miR-206-treated ARO cells were transfected with either MRTF-A expressing plasmids or empty vectors (mock), we performed migration and invasion assay and then found that pre-miR-206-treated ARO cells displayed >50% decrease in migration and invasion compared to control miR-treated cells (Fig. 6B). Restoration of MRTF-A sufficed to reverse the loss of migration and invasion (Fig. 6B) as well as resistance to anoikis-mediated cell death (Fig. 6C) observed in pre-miR-206-treated cells. Thus, we concluded that miR-206 inhibited migration and invasion via degrading MRTF-A expression.