mRNA expression and DNA CN of CDH16 in TC were investigated using the Oncomine 4.5 database (oncomine.org), an integrated data mining platform for collecting, analyzing and delivering cancer transcriptome data (22). The present study analysis focused on four TC studies, including He (accession no. GSE3467), Vasko (accession no. GSE6004), Giordano (accession no. GSE27155) and TCGA thyroid (23–25). The CDH16 mRNA expression and DNA CN were assessed between TC and normal thyroid tissue. P<0.05 was considered to indicate a statistically significant difference.

LinkedOmics database (linkedomics.org/login.php), a publicly available portal analyzing multi-omics data from 32 types of cancer in TCGA (26), was used to visualize genes that were co-expressed with CDH16 and perform KEGG pathway analysis of these genes by Gene Set Enrichment Analysis (GSEA).

A total of 35 paired PTC surgical and corresponding adjacent normal thyroid specimens were obtained from Tangshan Gongren Hospital and Tangshan Renmin Hospital between January 2017 and November 2021. The inclusion criteria were: i) histologically confirmed PTC; ii) voluntary participation in the research; iii) no preoperative radiotherapy, chemotherapy or other treatment for TC. The exclusion criteria were: i) presence of tumor other than TC; ii) heart, lung, liver, kidney or hematopoietic system disease; iii) pregnant or lactating. Patient characteristics are presented in Table I. The samples were stored in liquid nitrogen for reverse transcription-quantitative (RT-q)PCR or fixed in 4% paraformaldehyde solution for 48 h at room temperature and embedded in paraffin for immunohistochemistry (IHC). The present study was approved by the Ethical Committee of Tangshan Gongren Hospital (Hebei, China; approval no. GRYY-LL-2020-104) and all patients provided written informed consent.

Human TC BCPAP and TPC1 cells were obtained from American Type Culture Collection and the human thyroid follicular cell line Nthy-ori3-1 was purchased from Shanghai Institute of Cell Biology of the Chinese Academy of Sciences (Shanghai, China). BCPAP, TPC1 and Nthy-ori3-1 cells were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.). All cell lines were cultured in a 5% CO₂ humidified incubator at 37°C.

Tissue fixation, paraffin embedding, sectioning and dewaxing were performed as previously described (27). Tissue samples were incubated with mouse anti-CDH16 (1:100; cat. no. ab215769; Abcam) overnight at 4°C. Subsequently, sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (1:2,000; cat. no. ab205719; Abcam) for 30 min at 37°C. The slides were examined by two blinded senior pathologists from Tangshan Gongren Hospital. Cells within five randomly selected fields of view were counted under a light microscope (magnification, ×20) and those with brown or yellow staining on the cell membrane were considered CDH16-positive. The percentage of positive tumor cells was calculated. A threshold of >25% positively stained tumor cells was used to define CDH16 positivity.

RNA was isolated from thyroid tissue samples or cells using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). RNA was reverse transcribed into cDNA using cDNA Synthesis kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The prepared cDNA was subjected to RT-qPCR using a SYBR Green PCR Supermix kit (Invitrogen; Thermo Fisher Scientific, Inc.) with the Rotor Gene-3000 instrument (Corbett Research Ltd.). Reactions were conducted in a 20 µl volume with 1 µl cDNA according to the manufacturer's protocol of the SYBR Green PCR Supermix kit. Primer sequences for CDH16 were forward, 5′-CCCTGAGTTCATCACTTCCC-3′ and reverse, 5′-AGAGTCTGGCTCCCAATCC-3′. Primer sequences for GAPDH were forward, 5′- GAAAGCCTGCCGGTGACTAA-3′ and reverse, 5′-AGGAAAAGCATCACCCGGAG-3′. The PCR protocol was as follows: Initial denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 15 sec and 60°C for 30 sec. Relative expression was calculated using the 2^−ΔΔCq method (28) with GAPDH as a reference gene for normalization.

Protein was isolated from cells using RIPA buffer (Beyotime Institute of Biotechnology) with protease inhibitor phenylmethylsulfonyl fluoride (1:200; Beyotime Institute of Biotechnology). Total protein concentration was determined using a BCA kit (Beyotime Institute of Biotechnology), according to the manufacturer's protocol. Aliquots of 25 µg protein were electrophoresed on 10% polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Membranes were blocked in 5% non-fat milk in TBS containing 0.05% Tween-20 (TBST) for 2 h at room temperature. The membranes were incubated overnight at 4°C with primary antibodies against CDH16 (1:1,000, cat. no. 15107-1-AP, ProteinTech Group, Inc.), DNA polymerase (POL)D1 (1:1,000, cat. no. 15646-1-AP, ProteinTech Group, Inc), minichromosome maintenance (MCM)6 (1:2,000, cat. no. 13347-2-AP; ProteinTech Group, Inc), claudin (CLDN)1 (1:1,500, cat. no. 13050-1-AP, ProteinTech Group, Inc), intercellular adhesion molecule (ICAM)1 (1:2,000, cat. no. 60299-1-Ig, ProteinTech Group, Inc), syndecan (SDC)4 (1:1,000, cat. no. 11820-1-AP, ProteinTech Group, Inc) or β-actin (1:1,000, cat. no. TA-09; OriGene Technologies, Inc.). The membranes were washed with PBS three times and subsequently probed with anti-rabbit (cat. no. ZB-2301) or anti-mouse horseradish peroxidase-linked secondary antibody (both 1:2500; cat. no. ZB-2305) for 60 min at room temperature and visualized with ECL reagent (cat. no. sc-2048; all OriGene Technologies, Inc.). The protein bands were scanned and quantified by Tanon Gis software v4.2 (Tanon Science and Technology Co., Ltd.). Expression of β-actin served as the loading control.

A full length CDH16 cDNA clone was chemically synthesized by Sino Biological, Inc. and ligated into pCMV3 vector (Sino Biological, Inc.). Empty pCMV3 vector was used as the negative control. Untransfected cells were used as the blank control group. To introduce the vector, BCPAP cells were seeded in 6-well plates at a density of 5×10⁵ cells per well. After incubation at 37°C overnight, the degree of cell fusion was 50–70%. The cells were transiently transfected with plasmid carrying CDH16 or negative control plasmid (both 4 µg/well) using TransIntro EL (TransGen Biotech Co., Ltd.) according to the manufacturer's instructions. The effect of pCMV3-CDH16 on CDH16 mRNA and protein expression was analyzed by RT-qPCR and western blotting, as aforementioned, at 48 h post-transfection.

Cells (1×10⁴ per well) were seeded in 96-well plates. At 0, 24, 48, 72, and 96 h post-transfection, cell viability was determined by adding 10 µl 5 mg/ml MTT solution (Sigma-Aldrich; Merck KGaA) to each well and incubating the samples for 4 h at 37°C. The cell culture medium was removed and 150 µl DMSO (Sigma-Aldrich; Merck KGaA) was added to dissolve the formazan crystals. The absorbance at 490 nm was measured using a microplate reader (Bio-Rad Laboratories, Inc.). The experiment was repeated three times.

Effect of CDH16 on BCPAP cell apoptosis was determined by flow cytometry. Cells (5×10⁵ per well) were seeded in 6-well plates. At 48 h post-transfection, the cells were trypsinized using 0.25% trypsin (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1 min and collected by centrifugation at 800 × g for 5 min at room temperature. The cells were incubated with 0.5 ml binding buffer and 1.0 µl Annexin V-FITC (Merck KGaA) at room temperature for 15 min and resuspended in fresh 0.5 ml binding buffer containing 5 µl PI. Early apoptosis was measured using a Beckman Coulter FC500 flow cytometer (Beckman Coulter, Inc.) and MXP 2.2 software (Beckman Coulter, Inc.).

Effect of CDH16 on BCPAP cell migration was determined by scratch test. The cells were inoculated on 6-well plates at a density of 5×10⁵ cells/well and incubated in RPMI-1640 (Gibco) containing 10% FBS (Gibco) at 37°C for 24 h. When confluency reached 80%, the medium was replaced with serum-free RPMI-1640 and the cells were starved for 24 h. A scratch was then drawn with a 200-µl plastic pipette tip and the plates were rinsed with PBS to remove any cells suspended in culture medium after scratching. The gap distance of each scratch wound was photographed at 0 and 48 h after scratching using an light microscope (Olympus Corporation IX71; ×40 magnification) and scratch area was measured with ImageJ v1.50 (National Institutes of Health). Wound healing was calculated as a percentage.

Pre-coated Matrigel chambers (BD Biosciences) were used for the invasion assays according to the manufacturer's instructions. After transfection of pCMV3-CDH16 plasmid and empty pCMV3 plasmid at 37°C for 48 h, the cells were collected. A total of 1×10⁴ BCPAP cells were seeded into the upper chamber of the Transwell apparatus in serum-free RPMI-1640, while RPMI-1640 supplemented with 10% FBS was added to the lower chamber. After incubation at 37°C for 24 h, cells adhering to the lower surface were fixed with 100% methanol for 20 min at room temperature and stained with 5% Giemsa solution for 20 min at room temperature. The number of infiltrated cells was calculated under a light microscope (Olympus Corporation IX71) on five randomly selected fields (×100 magnification).

All experiments were performed ≥3 times. Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc.). The χ² or Fisher's exact test was used to analyze differences in CDH16 levels between PTC and normal thyroid tissue in IHC staining. Data are presented as the mean ± standard deviation. The paired t-test was used to compare CDH16 mRNA expression between 35 paired thyroid tissue samples. Pearson correlation test was used in correlation analysis. Single factor analysis of variance (one-way ANOVA) followed by post hoc Fisher's least significant difference test was used to compare >2 samples. P<0.05 was considered to indicate a statistically significant difference.

To verify that CDH16 is downregulated in TC, CDH16 transcript levels were evaluated in three independent TC studies from GEO. Data from all three studies in the Oncomine database showed that mRNA expression of CDH16 was significantly lower in PTC than in normal thyroid tissue (P<0.05; Fig. 1A-C). CDH16 expression was also significantly lower in FTC (P<0.05; Fig. 1D) and ATC (P<0.05; Fig. 1E) than in normal thyroid tissue. Notably, CDH16 ranked in the top 5% of all genes downregulated in each of these studies and the fold difference for PTC was >2. To determine whether decreased expression of CDH16 in PTC may be accounted for by DNA CNV, a dataset from TCGA that has previously been shown to have lower CDH16 expression in PTC (21) compared with matched normal tissue was used. The DNA CN of CDH16 showed no significant difference in blood, normal thyroid or TC tissue (P>0.05; Fig. 1F). Collectively, these findings indicated that CDH16 expression was decreased in TC in three PTC datasets as well as FTC and ATC, and also demonstrated that lower expression of CDH16 in TC was not accounted the result of CNV.

To support these findings, RT-qPCR and IHC were performed for 35 paired thyroid tissue samples collected from patients with PTC. CDH16 mRNA expression was significantly decreased in PTC compared with normal tissue (P<0.05; Fig. 2A). Furthermore, IHC showed that CDH16 was localized to the cell membrane of PTC cells. Of 35 PTC tissue samples, 17 (48.6%) stained negative for CDH16, while only two (5.7%) of the normal thyroid tissue samples stained negative for CDH16 (P<0.05; Fig. 2B). Therefore, decreased expression of CDH16 in PTC was observed at both the mRNA and protein level.

Next, to determine whether downregulation of CDH16 was correlated with the outcome of PTC, the association between CDH16 expression and clinicopathological characteristics of 35 patients with PTC was assessed. CDH16 protein expression was associated with tumor size, lymph node metastasis and pathological stage of PTC but there was no significant association with sex or age (Table I).

To confirm the downregulation of CDH16 in TC, CDH16 levels in TC-derived cell lines (BCPAP and TPC1) and the human thyroid follicular cell line Nthy-ori3-1 were measured. CDH16 expression was lower in TC cell lines, particularly BCPAP cells, both at the mRNA and protein level (P<0.05; Fig. 2C and D). These results were consistent with the aforementioned downregulation of CDH16 in patients with advanced PTC and support the use of BCPAP cells as a model for evaluating CDH16 biological activity.

To determine the role of CDH16 in TC, genes co-expressed with CDH16 in TC samples from TCGA were investigated using the LinkedOmics database. Volcano plot suggested that the most highly co-expressed genes were polypeptide N-acetylgalactosaminyltransferase 7 (GALNT7), NGFI-A binding protein 2 (NAB2) and UDP-galactose-4-epimerase (GALE), and the most highly inversely co-expressed genes are trefoil factor 3 (TFF3), odontogenic, ameloblast associated (ODAM) and dipeptidyl peptidase like 6 (DPP6) (P<0.05; Fig. 3A). KEGG pathway analysis by GSEA showed that negatively CDH16-associated genes were enriched in DNA replication and cell adhesion pathways (Fig. 3B; Table II). Correlation between expression levels of POLD1, MCM6, CLDN1, ICAM1 and SDC4 and those of CDH16 in 503 patients with PTC in TCGA was analyzed. The expression of each of these genes was significantly negatively correlated with expression of CDH16 in PTC (r=−0.290, −0.395, −0.594, −0.552, −0.583, respectively; P<0.001; Fig. 4).

To verify the function of CDH16 in TC cells, BCPAP cells were transfected with CDH16 expression vector. RT-qPCR and western blot assay verified higher mRNA and protein CDH16 levels in CDH16 overexpression compared with negative control BCPAP cells (P<0.05; Fig. 5A and B). To evaluate the effect of CDH16 on proliferation, MTT assay was performed. The proliferation of BCPAP cells was significantly decreased in overexpression compared with negative control cells (P<0.05; Fig. 5C).

To determine whether CDH16 expression affects tumor-associated functions, we the effect of CDH16 overexpression on cell apoptosis, migration and invasion was assessed. Compared with negative control, the CDH16 overexpression group exhibited a significantly higher apoptosis rate, as evaluated by flow cytometry with Annexin V and PI staining (P<0.05; Fig. 5D and E). Cell scratch test indicated that overexpression of CDH16 significantly inhibited migration of BCPAP cells (P<0.05; Fig. 6A and B). Furthermore, Transwell invasion assay indicated that overexpression of CDH16 significantly inhibited invasion capacity of BCPAP cells (P<0.05; Fig. 6C and D). Overall, these results demonstrated that CDH16 promoted apoptosis and inhibited TC cell proliferation, migration and invasion in vitro, which is consistent with the pattern of expression in patients with PTC.

To verify that CDH16 inhibited cell proliferation, migration and invasion via DNA replication and cell adhesion molecular pathways, western blot assay was used to detect protein expression levels. Following overexpression of CDH16 in TC cells, expression of POLD1, MCM6, CLDN1, ICAM1 and SDC4 was significantly downregulated (53.42±6.38, 37.67±14.27, 42.17±3.36, 37.04±9.34 and 47.83±9.23% decrease, respectively; P<0.05; Fig. 7) compared with negative control. These data indicated that CDH16 may regulate proliferation, migration and invasion of TC cells via DNA replication and cell adhesion pathways.