A total of 36 thyroid cancer and corresponding adjacent (distance, ~5 cm) normal tissue (ANT) samples were obtained from patients with thyroid cancer (mean age, 52.1 years; range, 24-72 years; 12 male; 24 female) at Ningbo Medical Centre Lihuili Hospital (Ningbo, China). All procedures were performed with the approval of the Ethics Committee of the Ningbo Medical Centre Lihuili Hospital (approval no. KY2022SL277-01) and written informed consent was obtained from all patients. All experiments were performed in accordance with the Declaration of Helsinki. None of the patients received any radiotherapy, chemotherapy or other therapy before undergoing surgery. All tissue samples were immediately snap frozen and stored at -80˚C until further use. Clinical information of patients is displayed in Table I.

TPC-1 cells were purchased from the Chinese Academy of Sciences and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator at 37˚C with 5% CO₂.

miR-384 mimi cs (miR-384; 5'-AUUCCUAGAAAUUGUUCAUA-3'), inhibitors (anti-miR-384; 5'-UAUGAACAAUUUCUAGGAAU-3'), scrambled negative control (NC; 5'-UUCUCCGAACGUGUCACGU-3') oligos miR-NC and anti-miR-NC (5'-CAGUACUUUUGUGUAGUACAA-3'), pGPH1 plasmid containing short hairpin (sh)RNA targeting circPVT1 (sh-circPVT1; 5'-UGGGCUUGAGGCCUGAUCU-3') and pGPH1 plasmid containing scrambled control shRNA (sh-NC; 5'-UUCUCCGAACGUGUCACGUTT-3') were purchased from Shanghai GenePharma Co., Ltd. A total of 5x10⁵ TPC-1 cells was plated in 96-well plates and incubated for 24 h at 37˚C. The plasmids (1 µg/µl), mimics (100 nM) and inhibitors (100 nM) were transfected into TPC-1 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. After 48, the transfection efficiency was assessed.

Stable TPC-1 cells with sh-NC or sh-circPVT1 were selected using 400 ug/ml Geneticin (G418,Thermo Fisher Scientific, Inc.) and 200 µg/ml G418 as the maintenance concentration.

Total RNA was isolated from thyroid cancer and ANT samples and cultured cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Nucleus and cytoplasm of TPC-1 cells were isolated using a PARIS extraction kit (cat no. AM1556; Thermo Fisher Scientific, Inc.). Complementary DNA was synthesized from 1 µg total RNA using PrimeScript RT (Takara Biotechnology Co., Ltd.) or All-in-one^™ miRNA FirstStrand cDNA Synthesis kit (GeneCopoeia, Inc.). RT-qPCR was performed using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.) and SYBR PrimeScript miRNA RT-PCR kit (Takara Biotechnology Co., Ltd.) for detecting circPVT1 and miR-384, respectively. Thermocycling conditions were as follows: Initial denaturation of 40 sec at 95˚C; followed by 40 cycles of denaturation at 95˚C for 5 sec and annealing and extension at 58˚C for 40 sec. GAPDH and U6 were chosen as the internal control for detection of circPVT1 and miR-384, respectively.

The primer sequences were as follows: circPVT1 forward, 5'-ATCGGTGCCTCAGCGTTCGG-3' and reverse, 5'-CTGTCCTCGCCGTCACACCG-3'; human GAPDH forward, 5'-ATCAATGGAAATCCCATCACCA-3' and reverse, 5'-GACTCCACGACGTACTCAGCG-3'; miR-384 forward, 5'-TGTTAAATCAGGAATTTTAA-3' and reverse, 5'-TGTTACAGGCATTATGAA-3' and U6 forward, 5'-CTCGTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'. The relative expression levels were calculated using 2^-ΔΔCq method (23).

CCK-8 assay was performed to assess cell proliferation. Briefly, transfected cells (5x10³ cells/well) were seeded into 96-well plates at 37˚C for 24-72 h. A total of 10 µl CCK-8 regent (Dojindo Molecular Technologies, Inc.) was added into each well at indicated time and incubated for 2 h at 37˚C. Finally, optical density at 450 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.).

Exponentially growing cells (1,000 cells/well) were seeded into a 6-well plate and cultured in DMEM at 37˚C with 5% CO₂ for 10 days to form colonies. Subsequently, cells were stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) for 15 min at 25˚C after being fixed with 4% paraformaldehyde for 30 min at 25˚C. Finally, the number of clones (>50 cells) was counted under a light microscope (X71; Olympus Corporation; magnification, x40).

Transwell plates (Corning, Inc.) were employed to determine cell invasion and migration. The upper chambers were pre-coated with 50 µl 2.5 mg/ml Matrigel at 37˚C for 2 h (Corning, Inc.) for the invasion assays or uncoated for the migration assays. A total of 4x10⁵ transfected cells resuspended in serum-free DMEM were plated into the upper chamber DMEM supplemented with 20% FBS was added to the lower chamber. Following incubation for 24 h at 37˚C with 5% CO₂, the invaded and migrated cells were fixed with 4% paraformaldehyde at 25˚C for 30 min. and stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA) at 25˚C for 10 min. The stained cells were counted in five randomly selected fields of view using an inverted light microscope (Olympus Corporation).

The potential target miRs of circPVT1 were predicted using The Encyclopedia of RNA Interactomes (ENCORI; starbase.sysu.edu.cn).

Wild-type (WT; 5'-UUUCUAGACUUGCUAGGAAA-3') or mutant type (MT, 5'-UUUCUUGACAAGGAUCCUUA-3') circPVT1 3' untranslated regions containing the predicted binding site for miR-384 were synthesized by Shanghai GenePharma Co., Ltd, and were cloned into the pmirGLO luciferase reporter vector (Promega Corporation) to generate circPVT1-WT or circPVT1-MT, respectively. The recombinant vectors circPVT1-WT or circPVT1-MUT and miR-384 mimics or miR-NC mimics were co-transfected into the TPC-1 cells using Lipofectamine^® 2000 and cultured for 48 h. Dual-Luciferase Reporter Assay System (Promega Corporation) was used to detect luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.

biotinylated WT miR-384 (WT-bio-miR-384), biotinylated MT miR-384 (MT-bio-miR-384) or control probe Bio-NC were purchased from Sangon Biotech Co. The biotinylated RNA was transfected into TPC-1 cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and cultured at 37˚C with 5% CO₂ for 48 h according to the manufacturer's instructions. A total of ~1x10⁷ cells were dissolved in the soft lysis buffer with 80 U/ml RNasin (Promega Corporation). The cell lysate were precipitated with M-280 streptavidin beads (Sigma-Aldrich; Merck KGaA) at 4˚C for 12 h. The beads were collected by centrifugation at 13,000 g for 10 min at 4˚C and cell debris was discarded. The bound RNAs were purified using RNeasy Mini kit (Qiagen GmbH). The relative expression of circPVT1 was measured via RT-qPCR.

At least three independent replicates were performed and data are presented as the mean ± SD. Data were analyzed using SPSS v 22.0 software (IBM Corp.). Differences between two or three groups were analyzed using parametric paired Student's t-test (two groups) or one-way analysis of variance followed by Bonferroni's post hoc test (three groups). The correlation between circPVT1 and miR-384 in thyroid cancer tissue was determined by Pearson's correlation coefficient analysis. P<0.05 was considered to indicate a statistically significant difference.

Relative expression of circPVT1 in 36 thyroid cancer and corresponding ANT samples was detected using RT-qPCR. The results revealed that the relative expression level of circPVT1 was significantly increased in thyroid cancer tissues compared with ANT (Fig. 1). Further analysis of the association between circPVT1 expression and clinicopathological features of patients with thyroid cancer demonstrated that high circPVT1 expression was positively associated with lymph node metastasis and advanced tumor-node-metastasis stage (TNM; Table I) but not with sex, age or tumor size (Table I).

As increased circPVT1 expression found in thyroid cancer tissue, the biological role of circPVT1 in thyroid cancer was investigated. TPC-1 cells were transfected with sh-NC vector or sh-circPVT1 and expression of circPVT1 was measured using RT-qPCR. Transfection with sh-circPVT1 decreased circPVT1 expression in TPC-1 cells compared with that in cells transfected with sh-NC, suggesting that circPVT1 was successfully knocked down in TPC-1 cells (Fig. 2A). CCK-8 assay demonstrated that circPVT1-knockdown significantly decreased cell proliferation in a time-dependent manner compared with sh-NC group (Fig. 2B). In addition, circPVT1 depletion decreased colony formation in TPC-1 cells compared with the sh-NC group (Fig. 2C).

The biological effects of circPVT1 on thyroid cancer cell migration and invasion were evaluated. Transwell assay indicated that circPVT1-knockdown significantly reduced the number of migrated and invaded cells compared with sh-NC (Fig. 3A and B), suggesting that circPVT1-knockdown resulted in decreased cell migration and invasion.

circPVT1 was primarily distributed in the cytoplasm of TPC-1 cells (Fig. 4A). It was hypothesized that circPVT1 interacted with miRs; miRs that bind to circPVT1 were selected using ENCORI. Among these miRs, miR-384, a tumor suppressive miR (24-26), was selected as a target of circPVT1 (Fig. 4B). To test this prediction, dual-luciferase reporter assay was performed; luciferase activity was decreased in TPC-1 cells co-transfected with circ-PVT1-WT and miR-384 mimics compared with that in TPC-1 cells co-transfected with circ-PVT1-WT and miR-NC (Fig. 4C) but remained unchanged following co-transfection with circ-PVT1-MT and miR-384 mimics or miR-NC (Fig. 4C). Compared with that wt-bio-miR-NC, RNA pull-down assay using wt-bio-miR-284 indicated that circPVT1 was significantly enriched in miR-384 mimics-precipitated RNA transcripts in TPC-1 cells (Fig. 4D). Moreover, RT-qPCR showed that relative expression of miR-384 was significantly upregulated in TPC-1 cells transfected with sh-circPVT1 compared with that in in TPC-1 cells transfected with sh-NC (Fig. 4E). Furthermore, miR-384 expression was downregulated in thyroid cancer tissue (Fig. 4F), and its expression was negatively correlated with circPVT1 in thyroid cancer tissue (r=-0.5406 Fig. 4G). These results suggested that circPVT1 bound to miR-384 in thyroid cancer cells.

Transfection of sh-circPVT1 significantly increased expression of miR-384 in TPC-1 cells compared with that in TPC-1 cells transfected with sh-NC, whereas transfection of miR-384 inhibitor partially reversed this trend (Fig. 5A). Moreover, inhibition of miR-384 partly reversed circPVT1 depletion-mediated inhibitory effect on thyroid cancer cell proliferation, colony formation, migration and invasion (Fig. 5B-E). Collectively, the present findings suggest that miR-384 contributed to circPVT1-mediated thyroid cancercell proliferation and migration.