Human clinical PTC tissues from 30 patients were collected at Jiangsu Cancer Hospital (Nanjing, China) between January 2015 and December 2016. The stage following surgery was evaluated according to the 7th edition of American Joint Committee on Cancer Tumor Node Metastasis staging system (21). The adjacent normal tissues were isolated at the same time as matched controls. Written informed consent was obtained from all the patients and the research protocols were approved by the Ethics Committee of Jiangsu Cancer Hospital.

The human PTC cell lines (CGTH W-3 and PTC-uc3) and human thyroid follicular epithelial cells (Nthyori 3-1) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), and incubated at 37°C in a humidified incubator with 5% CO₂.

RNA was extracted from tissues or cells using TRIzol reagent (Thermo Fisher Scientific, Inc.) following the manufacturer’s protocol. Total RNA (2 µg) was reversed transcribed into cDNA using the TaqMan microRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). qPCR was performed in a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using SYBR Premix Ex Taq kit (Takara Biotechnology Co., Ltd., Dalian, China) or TaqMan probes (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer’s instructions. The thermo-cycling conditions were as follows: 95°C for 5 min followed by 45 cycles at 95°C for 10 sec and 55°C for 30 sec, then a melting curve analysis every 0.2°C from 55°C to 95°C for 2 min was obtained. Expressions were normalized to the levels of the internal reference gene GAPDH. Specific PCR primers were synthesized by Invitrogen and the sequences were as follows: miR-214 forward, 5′-TAT ACA TCA AAC AGC AGG CAC A-3′ and reverse, 5′-CAT TCG ATC TTC TCC ACA GTC TC-3′; PSMD10 forward, 5′-CGA GCT CTG GAA GGT TAA ACA GCT TGG A-3′ and reverse, 5′-GCT CTA GAA GAC TCA CAA CAG CCA CAG AA-3′; GAPDH forward, 5′-AAG AAG GTG GTG AAG CAG GC-3′ and reverse, 5′-TCC ACC ACC CTG TTG CTG TA-3′. The expression levels of miR-214 and PSMD10 were assessed using the 2^−∆∆Cq method (22).

miR-214 mimics (5′-ACA GCA GGC ACA GAC AGG CAG U-3′) miR-214 inhibitors (5′-ACU GCC UGU CUG UGC CUG CUG U-3′) mimics negative control (NC; 5′-UUC UCC GAA CGU GUC ACG UTT-3′) inhibitors NC (5′-CAG UAC UUU UGU GUA GUA CAA-3′) and PDMS10 small interfering (si) RNA (PDMS10-siRNA; 5′-CTG GCC GGG ATG AGA TTG TAA AAG-3′) were obtained from GenePharma Co., Ltd. (Shanghai, China). Cells were transfected at a final concentration of 50 nM using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) for 24 h, according to the manufacturer’s protocols.

Cell Counting Kit-8 (CCK-8) assays were performed to evaluate cell proliferation, following the manufacturer’s protocol. Briefly, transfected cells were added to 96-well plates (1×10⁴ cells/well) and then CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added. The optical density at 450 nm was recorded on a microplate reader (Multiscan FC; Thermo Fisher Scientific, Inc.) at different times.

Flow cytometry analysis (C6 type flow cytometer; BD Biosciences, San Jose, CA, USA) using a FACSCalibur (version 6.0; BD Biosciences) was conducted to evaluate cells apoptosis. For apoptosis analysis, cells were collected 48 h post-transfection and stained with fluorescein isothiocyanate (FITC)-Annexin V (5 µl; Nanjing KeyGen Biotech Co., Ltd., Nanjing, China), and propidium iodide (PI; 5 µl).

Following transfection for 48 h, cells were harvested, washed with PBS and fixed in 70% ethanol over-night at 4°C. Then, 10 mg/l RNase A was added and incubated in 37°C for 30 min. The cells were subsequently incubated in 10 g/ml PI at 4°C for 30 min in the dark. Cell cycle distribution was analyzed by flow cytometry. The results were analyzed using ModFit software v3.3 (BD Biosciences).

Cells were seeded in 6-well plates and grown to ~80% confluency. Cell monolayers were wounded (width, 2 mm) with sterile plastic pipette tips and washed with PBS to remove cell debris. Images with a magnification of ×40 were captured at 0, 24 and 48 h following wounding using a light microscope. A total of 5 fields were captured per sample and the distance of cell migration into the wound area was calculated using Image J software 1.48u (National Institutes of Health, Bethesda, MD, USA).

Diluted matrigel (BD Biosciences) was incubated for 2 h at 37°C in the upper chamber of Transwell chambers (Corning Incorporated, Corning, NY, USA). Then, cells (1×10⁵/well) were added into the upper chamber. The bottom chamber was filled with medium containing 20% FBS and incubated for 24 h. Then, noninvasive cells on the upper surface were gently wiped off with cotton swabs. Invaded cells were fixed with 4% paraformaldehyde (Sigma-Aldrich; Merck KGaA) and stained in 10% crystal violet (Sigma-Aldrich; Merck KGaA). Images of the invasive cells at the bottom of the chamber were captured under an inverted light microscope (TE 2000-S; Nikon Corporation, Tokyo, Japan); the quantification of the invaded cells used the mean number of cells in five fields.

TargetScan (www.targetscan.og) (23) was used to predict the binding site of miR-214 and PSMD10 3′ untranslated region (UTR) using miR-214 as a keyword on the 12th February 2017. The wild-type or mutant 3′UTR of PSMD10 (Shanghai GeneChem Co., Ltd., Shanghai, China) was cloned into pGL3 vector (Promega Corporation, Madison, WI, USA) to confirm direct target association. The wild-type contained binding sites of PSMD10 3′UTR with miR-214. Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) was used for transfection for 24 h at 37°C. Cells (1×10⁴/well) were incubated for 48 h. Then the luciferase activity was measured after 48 h using a dual-luciferase reporter assay system (Promega Corporation). Luciferase ratio was calculated for each construct. The activity was normalized to the corresponding Renilla luciferase activity.

RIPA lysis buffer was used to extract total proteins. The supernatant was collected by centrifuging at 13,282 x g at 4°C for 10 min. Protein concentration was tested using the Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Inc.). The samples (30 µg) were separated on SDS-PAGE (10%) and then blotted onto a polyvinylidene fluoride membrane. The membrane was blocked with 5% non-fat milk at 25°C for 1 h, and then incubated with primary antibodies overnight at 4°C, including matrix metalloproteinase (MMP)-2 (cat. no. 40994; Cell Signaling Technology Inc., Danvers, MA, USA; 1:1,000), MMP-9 (cat. no. 13667; Cell Signaling Technology; 1:1,000), E-cadherin (cat. no. 14472; Cell Signaling Technology; 1:2,000), Vimentin (cat. no. 5741; Cell Signaling Technology; 1:1,000), Fibronectin (cat. no. 4705; Cell Signaling Technology; 1:1,000), PSMD10 (cat. no. 12985; Cell Signaling Technology; 1:1,000), phosphorylated (p)-protein kinase B (AKT; cat. no. 4060; Cell Signaling Technology; 1:2,000), AKT (cat. no. 2920; Cell Signaling Technology; 1:1,000), p-glycogen synthase kinase (GSK)-3 β (cat. no. 9323; Cell Signaling Technology; 1:1,000), GSK-3 β (cat. no. 5676; Cell Signaling Technology; 1:1,000) and GAPDH (cat. no. 5174; Cell Signaling Technology; 1:2,000). The membrane was incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (cat. no. 4410; Cell Signaling Technology; 1:5,000) at 25°C for 2 h. The immunoreactivity was visualized using the ECL Western blotting kit (Beyotime Institute of Biotechnology, Beijing, China). The amount of protein was normalized to GAPDH and analyzed using ImageJ software 1.48u (National Institutes of Health).

PTC cells (1×10⁴ cells/well) were plated in 24-well culture plates. After 12 days, cells were washed and fixed with 4% paraformaldehyde at 25°C for 30 min, and then stained in 10% crystal violet. The number of colonies containing ≥50 cells was counted under a light microscope.

Data were represented as means ± standard deviation (n=3). GraphPad Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform all the statistical analyses. When only two groups were compared, Student’s t-test was conducted. One-way analysis of variance followed Bonferroni’s correction as a post-hoc test was applied to compare differences between multiple groups. P<0.05 was considered to indicate a statistically significant difference.

To explore the expression status of miR-214 in PTC, the expression levels of miR-214 were examined in 30 pairs of human PTC tissues and matched adjacent normal tissues by RT-qPCR. The results demonstrated that miR-214 expression was significantly downregulated in PTC tissues compared with adjacent normal tissues (Fig. 1A). In addition, the correlation of miR-214 expression to various clinical features, including age, gender, tumor size, lymph node metastasis and TNM stage, was investigated in PTC. The results revealed that decreased expression of miR-214 was associated with lymph node metastasis, tumor size and advanced TNM stage (Table I).

Next, the expression levels of miR-214 were determined in one human thyroid follicular epithelial cell line (Nthyori 3-1) and two PTC cell lines (CGTH W-3 and PTC-uc3). The results demonstrated that miR-214 was markedly downregulated in the PTC cell lines (CGTH W-3 and PTC-uc3) compared with the normal Nthyori 3-1 cell line (Fig. 1B). These data indicated that the expression of miR-214 was decreased in both PTC tissues and cells, suggesting that miR-214 may be important in PTC development and metastasis.

CGTH W-3 and PTC-uc3 cells were transfected with miR-214 mimics or miR-214 inhibitors or corresponding negative control (NC) to evaluate the effect of miR-214 in PTC. The results indicated that miR-214 mimics increased expression of miR-214 compared with the mimics NC group in CGTH W-3 and PTC-uc3 cells, while its expression was significantly decreased in the miR-214 inhibitors-transfected group compared with the inhibitors NC group (Fig. 2A).

CCK-8 assay results demonstrated that transfection with miR-214 mimics significantly decreased the proliferation of CGTH W-3 and PTC-uc3 cells compared with the mimics NC group (Fig. 2B). By contrast, transfection of miR-214 inhibitors resulted in the opposite effect (Fig. 2B). Flow cytometry analysis demonstrated that miR-214 mimics clearly promoted cell apoptosis compared with the mimics NC group, while miR-214 inhibitors markedly decreased apoptosis compared with the inhibitors NC group (Fig. 2C). In addition, cell cycle phase distribution analysis revealed that miR-214 mimics significantly increased the % of cells in the G₀/G₁ phases of the cell cycle in CGTH W-3 and PTC-uc3 cells compared with the mimics NC group (Fig. 2D). By contrast, inhibition of miR-214 decreased the % of cells in the G₀/G₁ phases in both CGTH W-3 and PTC-uc3 cells compared with the inhibitors NC group (Fig. 2D), suggesting that miR-214 affects PTC cell cycle regulation.

The effects of miR-214 on PTC cell migration and invasion were evaluated next. The results demonstrated that miR-214 mimics decreased cell migration in CGTH W-3 and PTC-uc3 cells, while depletion of miR-214 resulted in a significant increase in cell migration, compared with the respective NC group (Fig. 3A). Similarly, miR-214 mimics significantly decreased CGTH W-3 and PTC-uc3 cell invasion, while inhibition of miR-214 decreased cell invasion, compared with the respective NC group (Fig. 3B).

Furthermore, the expression of proteins associated with tumor cell metastasis was examined in CGTH W-3 and PTC-uc3 cells by western blotting. The results demonstrated that miR-214 mimics significantly decreased the expression levels of matrix metallopeptidase (MMP)-2 and MMP-9 (Fig. 4A). However, inhibition of miR-214 promoted the upregulation of MMP-2 and MMP-9 in both CGTH W-3 and PTC-uc3 cells (Fig. 4A). In addition, the results revealed that overexpression of miR-214 reversed EMT-associated protein expression in CGTH W-3 and PTC-uc3 cells; miR-214 mimics increased the expression of E-cadherin and inhibited the expression of N-cadherin and vimentin compared with the mimics NC group (Fig. 4B). However, the EMT-associated expression displayed opposite patterns in the miR-214 inhibitor-transfected groups (Fig. 4B).

The TargetScan software was utilized to analyze potential gene targets for miR-214 (23), and PSMD10 was predicted to be a target gene of miR-214 (Fig. 5A). Dual luciferase reporter assays were then employed to test the hypothesis that miR-214 directly targets PSMD10. The results indicated that miR-214 mimics reduced the luciferase activity of a PSMD10-3′-UTR wild-type reporter plasmid (Fig. 5B), while the luciferase activity of a PSMD10-3′-UTR mutant reporter was not affected. In addition, miR-214 mimics downregulated the expression of PSMD10 in CGTH W-3 and PTC-uc3 cells at the mRNA and protein levels, while miR-214 inhibitors markedly increased the expression of PSMD10 compared with the inhibitors NC group (Fig. 5C and D). These results demonstrated that PSMD10 was a direct target of miR-214.

First, RT-qPCR was used to verify that PSMD10 levels were successfully silenced by siRNA transfection compared with control, in both CGTH W-3 and PTC-uc3 cells (Fig. 6A). Then, the effects of PSMD10 silencing were evaluated in the PTC cells. Proliferation capacity was determined by colony formation assay. The results demonstrated that colony formation in the PSMD10-siRNA transfection group was markedly decreased compared with the control group, in both CGTH W-3 and PTC-uc3 cells (Fig. 6B). In addition, cell migration was significantly decreased in CGTH W-3 and PTC-uc3 cells transfected with PSMD10-siRNA (Fig. 6C). Finally, the cell invasion rate was significantly decreased in CGTH W-3 and PTC-uc3 cells in the PSMD10-siRNA group compared with the control group (Fig. 6D).

To further investigate the molecular basis of PSMD10-mediated antitumor effects, the role of PSMD10 was examined in regulating glycogen synthase kinase (GSK)-3β/β-catenin signaling in CGTH W-3 and PTC-uc3 cells. The results demonstrated that PSMD10 downregulation significantly inhibited phosphorylation of GSK-3β and β-catenin (Fig. 7A). Furthermore, the activity of AKT was markedly clearly decreased in the PSMD10-siRNA group. These data indicated that PSMD10 repressed GSK-3β/β-catenin and AKT signaling in PTC cells.

Next, the correlation between the expression levels of miR-214 and PSMD10 was evaluated in PTC tissues. The results revealed that the expression of miR-214 was inversely correlated with the expression of PSMD10 in PTC tissues (Fig. 7B).