Serum samples were obtained from 54 individuals (mean age, 49.48 years; age range, 31–66 years), including 26 patients with benign thyroid nodules and 28 patients with DTC at Beijing Luhe Hospital (Beijing, China) between December 2016 and December 2017. The present study was approved by the Research Ethics Board of Beijing Luhe Hospital and was performed according to the World Medical Association Declaration of Helsinki. All individuals provided written informed consent. Serum samples were stored at −80°C until further analysis.

mRNA expression data (RNA Seq v2) and clinical information of patients in The Cancer Genome Atlas (TCGA) thyroid cancer data set were downloaded from the Synapse (https://www.synapse.org) and cBioPortal (www.cbioportal.org) databases and used for differential mRNA expression and prognosis analyses. 501 thyroid cancer (492 cases of DTC and 9 cases of other histological type) and 58 adjacent thyroid tissues were included in TCGA thyroid cancer dataset. In TCGA dataset, ‘01’ representeda primary solid tumor, and ‘11’ represented paracancerous tissue. The Gene Expression Omnibus (GEO) datasets GSE3678 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3678) (21) and GSE3467 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE3467) (22) were also used for ANGPTL1 expression analysis. The data from TCGA dataset were divided into high (n=250) and low (n=251) ANGPTL1 expression groups based on the median values of ANGPTL1 RNA-seq quantification results and further analyzed using GSEA.

Cells were purchased from the China Infrastructure of Cell Line Resources, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences. TPC-1 DTC cells were cultured in RPMI-1640 medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (all from Gibco; Thermo Fisher Scientific, Inc.), in a 37°C/5% CO₂ incubator. Cell transfection was performed with Lipofectamine^® 2000 (Invitrogen; Thermo FisherScientific, Inc.) according to the manufacturer's instructions. Small interfering RNA duplexes, siRNA-ANGPTL1 (cat. no. sc-88171) and siRNA-negative control (cat. no. sc-37007) were purchased from SantaCruz Biotechnology, Inc. Full-length cDNA encoding human ANGPTL1 were cloned into the vector GV366 plasmid (Shanghai Genechem Co.) with a HA-tag. And the vector GV366 plasmid with a HA-tag was used as vector control (Shanghai GeneChem Co., Ltd.). The cells were treated with 10% FBS at 37°C for 48 h, then the medium was removed, and the cells were harvested in sodium dodecyl sulfate (SDS) sample buffer and analyzed by western blotting.

Cells were lysed in ice-cold RIPA buffer (150 mM sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, protease and phosphatase inhibitor cocktail; Roche Diagnostics). Protein concentrations were measured using a BCA assay kit (Thermo Fisher Scientific, Inc.). Cell lysates were heated for 10 min at 95°C. Equal amount of protein lysates (30 µg) were separated on 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (0.22 µm). The blots were blocked in 5% non-fat dry milk in TBS + 0.05% Tween-20 (TBST) buffer for 1–2 h at room temperature and then incubated with primary antibodies against ANGPTL1 (1:1,000; cat. no. sc-365146, Santa Cruz Biotechnology), hemagglutinin tag (HA) (1:5,000; cat. no. 561, MBL International Co.) and GAPDH (1:2,000; cat. no. TA-08; OriGene Technologies, Inc.) overnight at 4°C. The blots were washed four times for 5 min with TBST buffer and incubated with a horseradish peroxidase (HRP)-conjugated anti-mouse IgG or anti-rabbit IgG secondary antibody (1:3,000; cat. nos. TA130001 and TA130015, OriGene Technologies, Inc.) for 1 h at room temperature. The blots were washed four times for 5 min with TBST buffer and visualized using an electrochemiluminescence (ECL) kit (Applygen Technologies, Inc.).

The Cell Counting Kit-8 (CCK-8) assay (Dojindo Molecular Technologies, Inc.) was used to assess the cell proliferation, according to the manufacturer's protocol. The cells were seeded at a density of 2×10³ cells/well in 96-well plates and incubated for 0, 24, 48, 72, 96 and 120 h. Cell medium with CCK-8 (10%; 100 ul) was added per well, then incubated for 1 h in 37°C in 5% CO₂. Viable cells were analyzed by CCK-8 assay using an Enspire microplate reader (PerkinElmer, Inc.) at 450 nm.

A total of 3×10⁵ cells were seeded in 6-well plates and allowed to reach 90% confluence. Cell monolayers were carefully scratched through the center of the well with a sterile 200 µl pipette tip. The detached cells were removed by washing twice with PBS, and the monolayers were maintained in RPMI-1640 medium with 5% FBS and 1% penicillin/streptomycin for 6 h at 37°C. Images of the wound were captured under a light microscope (magnification, ×40). The width of wound surfaces for each group was noted and measured using NIH Image 1.62 (National Institutes of Health). The relative migration distance at the final time point relative to the starting time point was quantified and analyzed (n=4).

The cell invasion assay was performed using modified Boyden chambers in 24-well plates with 8-mm pore inserts (BD Biosciences) coated with 1 mg/ml Matrigel for 5 h at 37°C in 5% CO₂. A total of 5×10⁴ cells were plated in 100 µl serum-free RPMI-1640 medium in the upper chamber. The lower chamber contained 600 ml complete medium (RPMI-1640 medium with 5% FBS and 1% penicillin/streptomycin). After 24-h incubation at 37°C, the invaded cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.5% crystal violet for 15 min at room temperature. For each experiment, the numbers of cells in five randomly selected fields were counted under light microscopy at ×200 magnification.

ANGPTL1 expression levels were determined by Gene Set Enrichment Analysis (GSEA) in gene sets were obtained from the Molecular Signatures Database of the Broad Institute (http://software.broadinstitute.org/gsea/msigdb). Tests were performed using the default settings with permutation numbers set at 1,000. A false discovery rate (FDR) <0.25 was considered to indicate a statistically significant difference.

Serum ANGPTL1 levels were measured using a commercially available human ELISA kit (cat. no. LS-F5677; Lifespan BioSciences, Inc.) according to the manufacturer's instructions with an intra-assay coefficient of variation (CV) ≤10% and an inter-assay CV ≤12%.

Statistical analyses were performed using SPSS 18.0 (SPSS, Inc). Data are presented as the mean ± standard deviation. Two-tailed unpaired student's t-test was used to compare differences between two groups, while one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to compare differences between multiple groups. χ² test was used to determine the association between ANGPTL1 expression and tumor stages. One-way ANOVA followed by Sidak's multiple comparisons test were used to assess the differences in cells proliferation. Receiver operator characteristic (ROC) curve and area under the curve (AUC) analyses were performed to assess the diagnostic ability of serum ANGPTL1 to DTC. P<0.05 was considered to indicate a statistically significant difference.

The mRNA expression of ANGPTL1 in DTC and adjacent tissues was analyzed using TCGA RNA-seq data. ANGPTL1 expression was downregulated in DTC tissue compared with that in adjacent non-cancerous tissue (Fig. 1A and B). To verify this result, ANGPTL1 expression levels were assessed in the GEO thyroid cancer datasets. These results also demonstrated that ANGPTL1 mRNA expression levels were downregulated in DTC tissues compared with those in adjacent non-cancerous thyroid tissues (Fig. 1C and D).

To explore the clinical relevance of ANGPTL1 in DTC, the association between ANGPTL1 expression levels and tumor stage was assessed in patients with different stages of DTC. Thyroid cancer staging was determined using the 7th edition of the American Joint Committee on Cancer Tumor-Node-Metastasis (AJCC-TNM) staging system (23). The results demonstrated that low ANGPTL1 expression was associated with advanced tumor stages (Fig. 1E and F).

Tumor cell proliferation is a major representative indicator of a malignant phenotype. RNA-seq data analytical results demonstrated that ANGPTL1 mRNA levels were negatively associated with the T stage of the primary tumor (Fig. 2A). The 7th edition of the TNM staging system was used in the T stage of thyroid cancer (24). To determine the role of ANGPTL1 in thyroid cancer cell proliferation, TPC-1 thyroid cancer cells were transfected with the HA-ANGPTL1 plasmid. Overexpression of ANGPTL1 was confirmed by western blotting, and cell viability was determined by CCK-8 assay. The results demonstrated that ANGPTL1 overexpression in TPC-1 cells reduced cell viability compared with that of the vector group (Fig. 2B). In addition, knockdown of ANGPTL1 in TPC-1 cells transfected with siRNA significantly decreased ANGPTL1 expression and cell proliferation compared with that of cells transfected with si-NC (Fig. 2C). To determine the relationship between the level of ANGPTL1 and signaling pathways associated with proliferation in DTC, data from TCGA dataset were divided into high and low ANGPTL1 expression groups as described in the method. The results demonstrated that gene signatures for proliferation were enriched in patients with low levels of ANGPTL1, which suggested a negative association between ANGPTL1 levels and the activation of proliferation signaling (Fig. 2D). Taken together, these results suggested that ANGPTL1 expression may inhibit DTC cell proliferation.

Cell migration and invasion are important factors in tumor metastasis that are associated with the prognosis for patients with thyroid cancer (25,26). The ANGPTL1 mRNA levels were compared between DTC patients with N0 (no lymphatic metastasis) and N1 (lymph node metastasis) stage using TCGA data. The results demonstrated that the levels of ANGPTL1 mRNA were downregulated in patients with N1 stage tumors compared with those in patients with N0 stage tumors (Fig. 3A). DTC cell migration was evaluated by wound healing assay to assess the role of ANGPTL2 in thyroid cancer metastasis. The migration distance was significantly lower in ANGPTL1-overexpressing cells compared with that in the vector group (Fig. 3B). The effect of ANGPTL1 on cell invasion was assessed by the Transwell invasion assay. As displayed with staining of cells in Matrigel-coated Boyden chambers, overexpression of ANGPTL1 significantly inhibited the invasive ability of TCP-1 cells compared with that of the control group (Fig. 3C). To investigate the association between the level of ANGPTL1 mRNA and activation of migration/invasion signaling in thyroid cancer specimens, the high and low ANGPTL1 expression groups from TCGA dataset were used. The results demonstrated that gene signatures for migration/invasion were enriched in patients with low levels of ANGPTL1, suggesting a negative association between ANGPTL1 levels and the activation of migration/invasion signaling (Fig. 3D and E). These results suggested that ANGPTL1 may inhibit the migration and invasion of DTC cells.

The association between ANGPTL1 expression and the recurrence status of patients with DTC was examined using TCGA dataset. Patients with recurrence exhibited significantly lower ANGPTL1 expression compared with that in patients without recurrence (Fig. 4A). The mitogen-activated protein kinase (MAPK)/ERK1/2 and PI3K/Akt signaling pathways have been reported to be associated with thyroid cancer recurrence (27,28); thus, the relationship between ANGPLT1 levels and the activation of these signaling pathways was assessed by GSEA. The results demonstrated that the gene signatures for the MAPK/ERK1/2 and PI3K/Akt signaling pathways were enriched in patients with low levels of ANGPTL1, suggesting a negative association between ANGPTL1 levels and the activation of MAPK/ERK1/2 and PI3K/AKT signaling (Fig. 4B and C). These results suggested that low ANGPTL1 levels may promote the recurrence of DTC.

Serum ANGTPL1 levels were evaluated in patients with DTC, the results demonstrated that serum ANGTPL1 was lower in patients with DTC compared with that in patients with benign thyroid nodules (Fig. 5A). The results demonstrated that serum ANGPTL1 levels were a potential biomarker for patients with DTC, with an AUC of 0.696 (95% confidence interval: 0.556–0.814), a sensitivity value of 60.71% and a specificity value of 80.77% (Fig. 5B).