Normal thyroid follicular epithelial cell line Nthy-ori 3-1 and Human thyroid cancer cell lines FTC133 and TPC-1 cells were cultured in DMEM, comprising 10% FBS and 100U/ml penicillin/streptomycin at the humidified chamber of 37°C and 5% CO₂. The shRNAs of circ_0067934 and SLC7A11 (LV-12 (pGLVH6-CMV-LUC-2A-Puro-U6-shRNA), pCMV-3tga-1A carrying SLC7A11 CDS (2μg) and circ_0067934 (2μg), miR-545-3p mimic (100 nM) and inhibitor (100 nM) were purchased (GenePharma/Genscript, China). The cells were treated with lentivirus at a multiplicity of infection (MOI) of 10 in the presence of Polybrene (8 ng/ml) or transfected with the specific plasmid by using Lipofectamine 3000 (ThermoFisher, USA, L3000001).

RNAs were isolated from tissues and cells by applying TRIZOL reagent (Biosntech, China, Lot B131905). Total RNAs were subjected into cDNA reverse transcription (Thermo, USA) and RT-qPCR assays were performed SYBR-Green (Vazyme, China, #P122). The data was normalized by GAPDH expression and the relative expression was calculated by 2^-ΔΔCt.

The primer sequences:

circ_0067934 forward: 5′- TAGCAGTTCCCCAATCCTTG-3′

circ_0067934 reverse: 5′- CACAAATTCCCATCATTCCC-3′

miR-545-3p forward: 5′-TGGCTCAGTTCAGCAGGAAC-3′

miR-545-3p reverse: 5′-TGGTGTCGTGGAGTCG-3′

SLC7A11 forward: 5′-TGCTGGGCTGATTTTATCTTCG-3′

SLC7A11 reverse: 5′-GAAAGGGCAACCATGAAGAGG-3′

GAPDH forward: 5′- GAAGGTGAAGGTCGGAGTC-3′.

GAPDH reverse: 5′- GAAGATGGTGATGGGATTTC-3′

U6 forward: 5′-CTCGCTTCGGCAGCACA-3′

U6 reverse: 5′-AACGCTTCACGAATTTGCGT-3′

Cells were placed into 96-well plates for 24 hours after treatment, and were added with MTT reagent (Sigma, USA, M2128) for the incubation of 4 hours. The samples were treated with DMSO and observed in the POLARstar Omega system (BMG, Australia). The cells were treated with erastin (5 mmol/L, Sigma, USA, 571203-78-6) for the investigation of ferroptosis.

The iron and reactive oxygen species (ROS) levels were detected as previously reported (18, 19). The ROS levels were analyzed by DCFH-DA staining (Sigma, USA, 4091-99-0. 287810). The of iron and Fe²⁺ levels were measured by Iron Assay Kit (Sigma, USA, MAK025).

For colony formation experiment, FTC133 and TPC-1 cells with the indicated treatment were placed in 6-well plate (1,000 cells per well), and incubated in complete medium for 2 weeks to form visible colonies. Subsequently, the colonies were washed, stained with crystal violet, and counted by a microscope (Carl Zeiss, German) and counted using ImageJ software.

The transfected FTC-133 and TPC-1 cells were digested and washed, suspended in 100 μL binding buffer and stained with Annexin V-FITC and PI staining reagents under the instruction of detection kit (CST, USA, #6592) in dark condition for 20 minutes. Afterward, the cells were washed and resuspended in binding buffer and measured by a flow cytometry (BD Biosciences, USA) immediately.

To conduct dual-luciferase assay, cells were placed in a 24-well plate at a density of 5 × 10⁴ cells per well and transfected with wild type (WT) or mutated (MUT) luciferase reporter plasmids along with miR-186-5p mimics. After 48-hours transfection, luciferase reporter assay kit (Promega, USA, E1910) was adopted to measure luciferase activities. The WT and MUT plasmids containing the sequences of circ_0067934 and the 3’UTR of SLC7A11 were established by inserting the sequences into pmirGLO plasmids.

Proteins with equal concentration were subjected into the sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred in PVDF membrane (Bio-Rad, USA, 162-0177). The sample was blocked by non-fat milk (5%) and cultured by the antibodies of SLC7A11 (Abcam, USA, ab37185) and β-actin (Abcam, USA, ab8227) overnight at 4°C, followed by the secondary antibody incubation for 2 hours at 25°C. The expression was detected by ECL reagent (Sigma, USA, WBULS0100).

BALB/c nude mice (18-20 g, 4-week-old, male) (n = 6) were purchased and applied for the tumor growth analysis of FTC133 cells in vivo. About 1×106 FTC133 cells were transfected with control shRNA or circ_0067934 shRNA and were subcutaneously injected into the left fat pad of mice. After 5 days of injection, we measured tumor growth every 5 days. We sacrificed the mice after 30 days of injection. The tumor volume was calculated by (length × width²)/2. And the tumor weight was calculated at day 30. The experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Harbin Medical University.

Data was quantified by means ± SD and compared using Student’s t-test or ANOVA (P < 0.05 as statistically significant) based on SPSS 16.0 software.

The expression of circ_0067934 was significantly enhanced in the FTC133 and TPC-1 cells compared with that in the normal thyroid follicular epithelial Nthy-ori 3-1 cells ( Figure 1A ). We validated that the circ_0067934 expression was depleted by shRNAs in FTC133 and TPC-1 cells ( Figure 1B ). The erastin reduced FTC133 and TPC-1 cell viabilities, while the circ_0067934 shRNA further repressed cell viabilities in erastin-treated FTC133 and TPC-1 cells ( Figures 1C, D ). The silencing of circ_0067934 increased the levels of ferroptosis-related markers, including Fe²⁺, iron, and ROS, in FTC133 and TPC-1 cells ( Figures 1E–G ), and the treatment of erastin served as a positive control, suggesting that circ_0067934 inhibits the thyroid cancer cell ferroptosis.

Moreover, the overexpression efficiency of circ_0067934 was validated in the FTC133 and TPC-1 cells ( Figure S1A ). The erastin repressed FTC133 and TPC-1 cell viabilities, while the circ_0067934 overexpression rescued cell viabilities in erastin-treated FTC133 and TPC-1 cells ( Figures S1B, C ). The overexpression of circ_0067934 reduced the levels of ferroptosis-related markers, including Fe²⁺, iron, and ROS, in FTC133 and TPC-1 cells ( Figures S1D–F ), and the treatment of erastin served as a positive control, confirming that circ_0067934 inhibits the thyroid cancer cell ferroptosis.

We then observed that circ_0067934 depletion reduced FTC133 and TPC-1 cell viabilities ( Figures 2A, B ), implying that circ_0067934 contributes to the survival of thyroid cancer cells. The circ_0067934 silencing repressed FTC133 and TPC-1 cell colony formations as well ( Figures 2C, D ), indicating that circ_0067934 promotes the proliferation of thyroid cancer cells. The knockdown of circ_0067934 enhanced FTC133 and TPC-1 cell apoptosis in vitro ( Figures 2E, F ). Moreover, the inhibition of circ_0067934 by shRNA attenuated the growth of FTC133 cells in vivo ( Figures 2G–I ), suggesting that circ_0067934 enhances cell growth of thyroid cancer in vivo.

In the circ_0067934-interacted miRNAs in bioinformatics analysis (CircInteractome online database), we found that miR-545-3p has presented tumor suppressor function in various cancers, such as liver cancer, lung cancer, and ovarian cancer (15–17), but the function of miR-545-3p has not been reported. Meanwhile, we identified that circ_0067934 inhibited ferroptosis of thyroid cancer cells and miR-545-3p potentially targeted ferroptosis-regulator SLC7A11 in a bioinformatics analysis (ENCORI online database). Consequently, we selected miR-545-3p for further investigation. The binding site of miR-545-3p with circ_0067934 was identified by bioinformatics analysis (CircInteractome online database) ( Figure 3A ). Circ_0067934 silencing enhanced miR-545-3p expression in FTC133 and TPC-1 cells ( Figures 3B, C ), implying that circ_0067934 may targeting miR-545-3p in thyroid cancer cells. The miR-545-3p mimic significantly enhanced miR-545-3p expression in FTC133 and TPC-1 cells ( Figures 3D, E ). The luciferase activity of circ_0067934, but not circ_0067934 containing miR-545-3p-binding site mutant, was attenuated by miR-545-3p mimic in FTC133 and TPC-1 cells ( Figures 3F, G ), suggesting that circ_0067934 serves as a sponge of miR-545-3p.

Moreover, our data showed that the depletion of circ_0067934 by shRNA repressed cell viabilities in erastin-treated FTC133 and TPC-1 cells, and miR-545-3p inhibitor could rescue the phenotype ( Figure 4A ). In addition, the levels of Fe²⁺, iron, and ROS were enhanced by circ_0067934 knockdown, while miR-545-3p inhibitor reversed this effect in FTC133 and TPC-1 cells ( Figures 4B–D ), indicating that circ_0067934 represses ferroptosis by targeting miR-545-3p.

We found that circ_0067934 inhibited ferroptosis of thyroid cancer cells and circ_0067934 sponged miR-545-3p in thyroid cancer cells. We also identified the miR-545-3p-targeted binding site of SLC7A11 by a bioinformatics analysis (ENCORI online database) ( Figure 5A ). The luciferase activity of SLC7A11, but not SLC7A11 containing miR-545-3p-binding site mutant, was repressed by miR-545-3p mimic in FTC133 and TPC-1 cells ( Figure 5B ), implying that miR-545-3p may affect mRNA 3’UTR of SLC7A11. The miR-545-3p mimic reduced SLC7A11 mRNA and protein levels in FTC133 and TPC-1 cells ( Figures 5C, D ), suggesting that miR-545-3p targets SLC7A11 in thyroid cancer cells. The silencing of circ_0067934 decreased SLC7A11 expression and miR-545-3p inhibitor rescued the expression in FTC133 and TPC-1 cells ( Figures 5E, F ), indicating that circ_0067934 up-regulating SLC7A11 by sponging miR-545-3p.

Furthermore, we found that miR-545-3p mimic repressed cell viabilities in erastin-treated FTC133 and TPC-1 cells, and SLC7A11 overexpression could rescue the phenotype ( Figure 6A ). In addition, the levels of Fe²⁺, iron, and ROS were enhanced by miR-545-3p mimic, while SLC7A11 overexpression reversed this effect in FTC133 and TPC-1 cells ( Figures 6B–D ), suggesting that miR-545-3p contributes to ferroptosis of thyroid cancer cells by targeting SLC7A11.

Meanwhile, the depletion of circ_0067934 by shRNA repressed cell viabilities in erastin-treated FTC133 and TPC-1 cells, and SLC7A11 overexpression could rescue the phenotype ( Figure 7A ). In addition, the levels of Fe²⁺, iron, and ROS were enhanced by circ_0067934 knockdown, while SLC7A11 overexpression reversed this effect in FTC133 and TPC-1 cells ( Figures 7B–D ), indicating that circ_0067934 represses ferroptosis of thyroid cancer cells by inducing SLC7A11.

Next, we found that the silencing of circ_0067934 attenuated FTC133 and TPC-1 cell viabilities, and miR-545-3p inhibitor or SLC7A11 overexpression was able to rescue the cell viabilities ( Figures 8A, B ). The FTC133 and TPC-1 cell apoptosis was enhanced by circ_0067934 depletion, while miR-545-3p inhibitor or SLC7A11 overexpression could block this phenotype in the cells ( Figures 8C–E ). Together these data imply that circ_0067934 regulates cell growth and ferroptosis of thyroid cancer cells by miR-545-3p/SLC7A11 axis.