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Introduction

Thyroid carcinoma is the most common endocrine neoplasm, of which papillary thyroid carcinoma (PTC) is the most common pathological type, accounting for 80% of thyroid carcinomas (1,2). Cervical lymph node metastasis is a typical clinical feature of PTC and is a risk factor for increased recurrence rates and decreased survival rates (3,4). Therefore, the identification of potential biomarkers that may be used to assess the prognosis and treatment of PTC is required.

Chemokines and their receptors serve critical roles in the development and progression of tumors (5), particularly in promoting cell migration (6). Previous studies have demonstrated that C-X-C chemokine receptor type 7 (CXCR7) expression serves a role in tumor cell proliferation, angiogenesis, invasion and metastasis (7–10). A previous study from our laboratory demonstrated that CXCR7 was overexpressed in PTC tissue compared with peritumoral non-malignant tissue and benign thyroid lesion tissue, and the expression of CXCR7 was positively associated with cervical lymph node metastasis (11). Furthermore, knockdown of CXCR7 in PTC cells has been demonstrated to suppress cell proliferation and invasion, and promote apoptosis (12), which suggests that CXCR7 is involved in the regulation of PTC progression.

In a previous study, to reveal the molecular mechanisms underlying CXCR7-mediated regulation of PTC progression, a gene microarray analysis was performed to detect changes in gene expression between PTC cells and PTC cells transfected with CXCR7. The results demonstrated that CXCR7 promotes the growth and metastasis of PTC via the activation of the phosphoinositide 3-kinase (PI3K)/RAC-α serine/threonine-protein kinase (AKT)/nuclear factor-κB (NF-κB) signaling pathway and regulation of the expression of effector molecules, including fibronectin 1 (FN1), collagen-α-1(I) chain precursor, collagen-α-1(IV) chain precursor, platelet-derived growth factor receptor β, stromelysin-3 precursor and membrane type 1 matrix metalloproteinase 1 (13).

The aim of the present study was to investigate the molecular mechanisms underlying CXCR7-regulated PTC progression and to identify novel biomarkers for PTC. To achieve this, isobaric tag for relative and absolute quantification (iTRAQ)-coupled two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS) was used to detect alterations in protein expression profiles between GLAG-66 and GLAG-66 cells transfected with CXCR7 cDNA (GLAG-66-CXCR7).

Materials and methods

Cell lines and culture conditions

The human PTC cell line GLAG-66 was purchased from the European Collection of Authenticated Cell Cultures (Salisbury, UK). A GLAG-66-CXCR7 cell line (GLAG-66 cells stably transfected with CXCR7 cDNA) was constructed in a previous study (10). Cell lines were cultured in Dulbecco's modified Eagle's medium: Ham's F12: MCDB105 supplemented with 10% fetal bovine serum (FBS) and 2 mmol/l glutamine (all from Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), as described previously (14), at 37°C with 5% CO2.

Cell lysis, protein isolation, digestion and labeling with iTRAQ reagents

Experimental group cells (GLAG-66-CXCR7-1) and the control cells (GLAG-66-1) were collected and lysed with 300 µl cell radioimmunoprecipitation lysis solution with 1% protease inhibitor (both from Beyotime Institute of Biotechnology, Haimen, China) containing 8 M urea on ice with regular vortex-mixing for 30 min. The mixture was centrifuged at 15,000 × g for 1 h at 4°C, the supernatant was removed and a Bradford Protein Assay reagent kit (Beyotime Institute of Biotechnology) was used to quantify the total protein. A 100 µg amount of each sample was precipitated with 4X sample volume of ice-cold acetone at −20°C for ~3 h and centrifuged at 20,000 × g for 20 min at 4°C, carefully decanting the supernatant. Subsequently, according to the iTRAQ protocol (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), as described previously (15), the protein was dissolved and denatured, and cysteine residues were blocked. Each sample was then digested with 20 µl 0.25 µg/µl sequencing grade modified trypsin solution (Promega Corporation, Madison, WI, USA) at 37°C for 12 h and labeled with the iTRAQ tags as follows: GLAG-66-CXCR7-1, iTRAQ 114 (114); GLAG-66-CXCR7-2, iTRAQ 115 (115); GLAG-66-1, iTRAQ 116 (116); and GLAG-66-2, iTRAQ 117 (117), then incubated at room temperature for 2 h and vacuum-dried. The experiments were repeated in duplicate.

iTRAQ-coupled 2D LC-MS/MS analysis

The proteome analysis was performed by the Beijing Proteome Research Center (Beijing, China). In brief, the mixed iTRAQ labeled sample was centrifuged at 13,800 × g for 10 min and the supernatant was eluted with 50% buffer B (98% acetonitrile, 1.9% H2O and 0.1% formic acid) at a flow rate of 0.4 ml/min for 10 min, then with 100% buffer A (1.9% acetonitrile, 98% H2O and 0.1% formic acid) at a flow rate of 0.4 ml/min for 15 min. The mixture was then separated on a C18 reverse-phase pre-column (5 µm, 150 Å, 4.6×250 mm; Agela Technologies, Wilmington, DE, USA) at a flow rate of 1 ml/min. The gradient elution started at 5% buffer B and was followed by an increase from 5 to 18% in buffer B for 17 min, from 18 to 32% buffer B for 26 min, 32 to 95% buffer B for 28 min and finally to 5% buffer B for 40 min. The fractions were collected at 1-min intervals. All 40 fractions were centrifuge-dried at 12,000 × g at 4°C for 15 min. Finally, the sample was resuspended with buffer A, centrifuged at 12,000 × g for 10 min. MS/MS analysis data was acquired from m/z 350 to 1250 with ≤2 precursors selected for MS/MS from m/z 100 to 1500 using dynamic exclusion.

Western blot analysis

Protein was extracted from cells according to the aforementioned protein extraction protocol. Protein concentrations were determined using a Bradford Protein Assay reagent kit (Beyotime Institute of Biotechnology, Nanjing, China). Total protein samples (80 µg/lane) were separated using 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes. Following blocking with 5% non-fat dry milk for 2 h, membranes were incubated with primary antibodies overnight at 4°C. The membranes were incubated for 2 h at room temperature with secondary antibodies. Antibodies used in the present study included the following: rabbit polyclonal anti-FN1 (dilution, 1:400; cat no. BA1771), anti-basigin precursor (BSG; dilution, 1:500; cat no. PB0239) (both from Boster Biological Technology, Pleasanton, CA, USA), rabbit polyclonal anti-periplakin (PPL; dilution, 1:5,000; cat no. 72422), mouse polyclonal anti-AHNAK nucleoprotein 2 (AHNAK2; dilution, 1:1,000; cat no. ab70053) (both from Abcam, Cambridge, UK), rabbit polyclonal anti-transgelin-2 (TAGLN2; dilution, 1:1,000; cat no. 10234-2-AP), β-catenin (dilution, 1:5,000; cat no. 51067-2-AP), anti-serpin family B member 5 (SERPINB5; dilution, 1:5,000; cat no. 11,722-1-AP) and GAPDH (dilution, 1:10,000; cat no. 10,494-1-AP) (all from ProteinTech Group, Inc., Chicago, IL, USA), and goat anti-rabbit and goat anti-mouse immunoglobulin G (dilution, 1:2,000; cat nos. TA130015 and TA130001; OriGene Technologies, Inc., Beijing, China) as secondary antibodies. Binding was detected using BeyoECL Plus kit (Beyotime Institute of Biotechnology). The ratio between the integrated optical density of interest proteins and GAPDH of the same sample was calculated as the relative content of protein detected using ImageJ 1.48v (National Institutes of Health, Bethesda, MD, USA). All experiments were performed three times.

Gene Ontology (GO) analysis

To analyze the functions of the differentially expressed proteins, GO analysis was performed. The GOfact (http://lidong.ncpsb.org/gofact/cgi/gofact2009.cgi) strategy was used to evaluate the biological processes, molecular functions and cell components associated with each differentially expressed protein.

Statistical analysis

For iTRAQ, raw data processing, protein identification, protein quantification, and statistical analyses were performed with ProteinPilot™ Software Beta (version 4.2; SCIEX, Framingham, MA, USA). Protein quantification ratios were log10-transformed. Unpaired Student's t-test was applied to analyze differences in GO and western blot analysis data using SPSS software (version 19.0; IBM Corp., Armonk, NY, USA). All data are expressed as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

Results

Protein quantification ratio distribution trend

Quantification ratios among the protein sample groups (114:116, 115:116 and 117:116) were log10-transformed in order to create a distribution diagram of protein quantification ratios (Fig. 1). The log10-transformed protein quantification ratios were represented by fold change, and log ratios ≥0.3 or ≤-0.3 represented a 2-fold change in expression.

Figure 1. Distribution of protein quantification ratios. Distribution of 114:116 and 115:116 exhibit a high similarity, whereas 117:116 is markedly different.

Proteome analysis by iTRAQ-coupled 2D LC-MS/MS

A total of 24,673 peptides and 2983 proteins were identified by iTRAQ-coupled 2D LC-MS/MS (false discovery rate, <1%). Standard selection criteria to identify differentially expressed proteins were as follows: log ratio of ≥0.3 and P<0.05 for 114:116 and 115:116 ratios (meaning the protein was differentially expressed proteins between GLAG-66 and GLAG-66-CXCR7 cells; log ratios ≥0.3 represented upregulated proteins and log ratios ≤-0.3 represented downregulated proteins) in addition to a log ratio <0.3 and P≥0.05 for 117:116 (meaning the expression was not significantly altered between the control samples). A total of 130 proteins were revealed to be differentially expressed. Among them, 87 and 43 proteins were significantly upregulated and downregulated, respectively. The top 10 differentially expressed proteins are listed in Table I. Proteins selected for further verification and analysis included FN1, TAGLN2, BSG, PPL, AHNAK2, β-catenin and SERPINB5, as they were significantly differentially expressed, and are associated with tumor development (16–22).

Table I. Top upregulated and downregulated proteins from analysis of isobaric tag for relative and absolute quantification-coupled two-dimensional liquid chromatography-tandem mass spectrometry data.

Table I.

Top upregulated and downregulated proteins from analysis of isobaric tag for relative and absolute quantification-coupled two-dimensional liquid chromatography-tandem mass spectrometry data.

Genes	Description	Log ratio (O/N)a	P-valueb (differentially expressed)	Regulation
FN1	Fibronectin 1	1.442	1.02×10−4	Up
ALB	Serum albumin preproprotein	1.414	6.55×10−3	Up
TAGLN2	Transgelin-2	1.398	2.02×10−5	Up
XRCC5	X-ray repair cross-complementing protein 5	1.372	4.40×10−9	Up
HIST3H2BB	Histone H2B type 3-B	1.368	3.38×10−2	Up
BSG	Basigin isoform 2	1.360	1.16×10−2	Up
CAST	Calpastatin isoform f	1.358	2.25×10−2	Up
PPL	Periplakin	1.342	2.64×10−9	Up
TG	Thyroglobulin precursor	1.278	1.30×10−10	Up
AHNAK2	AHNAK nucleoprotein 2	1.272	4.02×10−3	Up
SARDH	Sarcosine dehydrogenase, mitochondrial precursor	−0.824	7.31×10−3	Down
ABCB1	Multidrug resistance protein 1	−0.708	2.92×10−4	Down
ENO3	β-enolase isoform 1	−0.696	6.54×10−4	Down
RPL10	60S ribosomal protein L10	−0.668	8.42×10−4	Down
CTNNB1	Catenin β−1	−0.654	1.50×10−4	Down
SERPINB5	Serpin family B member 5	−0.632	1.41×10−2	Down
SDHA	Succinate dehydrogenase	−0.614	1.16×10−2	Down
PPM1G	Protein phosphatase 1G	−0.614	3.54×10−3	Down
ALDH2	Aldehyde dehydrogenase, mitochondrial isoform 1 precursor	−0.612	4.66×10−4	Down
UBE2L3	Ubiquitin-conjugating enzyme E2 L3 isoform 4	−0.612	7.93×10−3	Down

a Log ratio (O/N) is the logarithm of fluorescence intensity ratio of O and N, where O is the experimental group (GLAG-66 cells transfected with CXCR7) and N is the control group (GLAG-66 cells). Log ratios ≥0.3 and P<0.05 represent differentially expression proteins.

b P-value indicates the significance of log ratio (O/N).

Gene Ontology (GO) enrichment analysis

To analyze function distribution in the differentially expressed proteins, a GO enrichment analysis was performed. GO enrichment analysis revealed that the differentially expressed proteins were primarily enriched in a number of biological processes, including metabolism-associated processes, cellular component organization, transport, cellular development processes and immune response. In molecular function, the differentially expressed proteins were significantly enriched in protein binding, nucleotide binding and ion binding. In cell component organization, the majority of the identified proteins were involved in intracellular part, as illustrated in Table II.

Table II. Top gene ontology enrichment analysis terms.

Table II.

Top gene ontology enrichment analysis terms.

Function category	k/Ka (%)	P-valueb
Biological process
Primary metabolic process	43.30	4.20×10−4
Cellular metabolic process	40.00	1.90×10−4
Macromolecule metabolic process	34.80	2.00×10−5
Cellular component organization	32.60	3.70×10−33
Transport	21.10	1.60×10−2
Cellular developmental process	20.00	1.40×10−22
Anatomical structure morphogenesis	17.80	5.20×10−32
Response to stress	17.40	2.00×10−9
Catabolic process	14.30	4.20×10−3
Immune response	13.70	2.50×10−25
Molecular function
Protein binding	46.20	1.40×10−31
Nucleotide binding	26.70	3.00×10−9
Ion binding	18.90	4.90×10−3
Transferase activity	7.10	8.50×10−8
Transporter activity	5.00	2.00×10−3
Signal transducer activity	4.40	3.00×10−10
Lipid binding	4.40	8.70×10−3
Carbohydrate binding	3.70	2.20×10−4
Transmembrane transporter activity	3.70	3.20×10−3
Transcription regulator activity	1.50	2.50×10−11
Cell component
Intracellular part	80.40	8.00×10−27
Intracellular organelle	58.90	2.30×10−10
Membrane-bounded organelle	54.50	2.80×10−11
Membrane	48.40	4.00×10−13
Intracellular organelle part	41.90	2.10×10−15
Protein complex	34.20	1.30×10−38
Non-membrane-bounded organelle	20.90	1.30×10−5
Cell projection	14.00	4.80×10−31
Vesicle	8.50	3.90×10−15
Intracellular	8.10	9.90×10−7

a k/K is the ratio of k and K, where k is the number of differentially expressed proteins in the corresponding Function category of the Gene Ontology analysis, and K is the total number of differentially expressed proteins within the whole analysis

b p-value indicates the degree of enrichment within the Function category. Smaller P-values indicate more significant category enrichment (compared with the overall distribution).

Verification of differentially expressed proteins

To confirm the differences in protein profile expression between control GLAG-66 and GLAG-66-CXCR7 cells, the upregulated proteins FN1, BSG, PPL, TAGLN2 and AHNAK2, and the downregulated proteins β-catenin and SERPINB5, were selected for further verification. Western blot analysis demonstrated that protein expression levels of FN1, BSG, PPL, TAGLN2 and AHNAK2 were significantly elevated in GLAG-66-CXCR7 cells compared with GLAG-66 cells, whereas β-catenin and SERPINB5 were significantly downregulated (P<0.05; Fig. 2 and Table III), which was consistent with the proteome data.

Figure 2. Protein expression of differentially expression proteins in GLAG-66 and GLAG-66-CXCR7 cells. (A) Western blot analyses of differentially expression proteins. GAPDH protein was used as a control. (B) Quantification of relative protein levels of differentially expression proteins. *P<0.05. FN1, fibronectin 1; BSG, basigin; PPL, periplakin; TAGLN2, transgelin-2; AHNAK2, AHNAK nucleoprotein 2; SERPINB5, serpin family B member 5.

Table III. Protein expression of differentially expression proteins in GLAG-66 cells and GLAG-66-CXCR7 cells.

Table III.

Protein expression of differentially expression proteins in GLAG-66 cells and GLAG-66-CXCR7 cells.

Gene	Protein expression (mean ± standard deviation)	P-value
FN1		<0.001
GLAG-66	0.824±0.031
GLAG-66-CXCR7	0.274±0.022
BSG		0.024
GLAG-66	0.648±0.016
GLAG-66-CXCR7	0.305±0.018
PPL		<0.001
GLAG-66	0.489±0.056
GLAG-66-CXCR7	0.312±0.017
TAGLN2		0.002
GLAG-66	0.534±0.050
GLAG-66-CXCR7	0.248±0.050
AHNAK2		0.001
GLAG-66	0.614±0.045
GLAG-66-CXCR7	0.291±0.010
β-catenin		<0.001
GLAG-66	0.349±0.018
GLAG-66-CXCR7	0.756±0.015
SERPINB5		<0.001
GLAG-66	0.333±0.015
GLAG-66-CXCR7	0.798±0.003

[i] CXCR7, C-X-C chemokine receptor type 7; FN1, fibronectin 1; BSG, basigin; PPL, periplakin; TAGLN2, transgelin-2; AHNAK2, AHNAK nucleoprotein 2; SERPINB5, serpin family B member 5.

Discussion

Previous studies from our group demonstrated that overexpression of CXCR7 in PTC tissue was positively associated with lymph node metastasis, whereas knockdown of CXCR7 in PTC cells suppressed cell proliferation and invasion, and promoted apoptosis (11,12). Furthermore, gene expression profile analysis demonstrated that CXCR7 activates the PI3K/AKT/NF-κB signaling pathway and regulates the expression of effector molecules, thereby regulating PTC progression (13). To further evaluate the molecular mechanisms underlying CXCR7-regulated PTC growth and metastasis, in addition to screening potential biomarkers, iTRAQ-coupled 2D LC-MS/MS was performed to detect protein expression profile alterations between the GLAG-66 and GLAG-66-CXCR7 cell lines. A total of 130 differentially expressed proteins were identified, among which 87 were upregulated and 43 were downregulated.

In the present study, GO enrichment analysis revealed that the differentially expressed proteins were primarily involved in primary metabolic processes, cellular metabolic processes, macromolecule metabolic processes, cellular component organization, transport, cellular developmental processes, biological processes and protein-binding molecular function. The GO enrichment analysis of cell component revealed that the differentially expressed proteins were primarily intracellular. Certain differentially expressed proteins possessed known functions, including FN1, BSG, PPL, SERPINB5 and β-catenin. FN1 is a glycoprotein distributed on the cell surface and in the extracellular matrix, and is involved in endothelium cell invasion, migration and angiogenic processes (15). These processes involve the activation of focal adhesion kinase and the PI3K/AKT/NF-κB signaling pathway (15,23). Previous studies have reported that FN1 is overexpressed in PTC and may serve as a biomarker for PTC diagnosis and treatment (24,25). BSG is a transmembrane protein that is overexpressed in a number of malignant tumor cells, and promotes tumor invasion and metastasis by activating the PI3K/AKT signaling pathway (17,26–29). Previous studies have reported that BSG promotes extra-tumor invasion and lymph metastasis in differentiated thyroid carcinoma (DTC) and is associated with poor prognosis (30,31). PPL, a constituent of desmosomes involved in endothelial cell structure stabilization and considered as a localization signal of AKT, promotes cell growth and survival by binding AKT directly (18). PPL knockdown appeared to decrease the phospho-AKT expression (32,33). Tonoike et al (34) demonstrated that PPL knockdown suppresses cell proliferation, migration and invasion through the PI3K/AKT signaling pathway in pharyngeal cancer cells. In the present study, CXCR7 overexpression upregulated the expression of FN1, BSG and PPL, which suggested that CXCR7 may regulate growth and metastasis of PTC via the activation of the PI3K/AKT signaling pathway.

In addition, the results of the present study demonstrated that SERPINB5, a tumor suppressor that belongs to the serpin family, is downregulated following CXCR7 overexpression. SERPINB5 is a tumor protein p53 (p53)-dependent protein that inhibits tumor angiogenesis by interacting with the p53 signaling pathway (21,35). A number of studies have suggested that SERPINB5 inhibits the migration, invasion, metastasis and angiogenesis of tumor cells (22–37). Boltze et al (38) and Shams et al (39) demonstrated that SERPINB5 is associated with vessel invasion and lymph metastasis in PTC, therefore SERPINB5 can inhibit PTC invasion and metastasis. The results of the present study suggest that CXCR7 promotes PTC growth and metastasis by inhibiting SERPINB5.

The processes of tumor invasion and metastasis include adhesion, degradation and movement (40). The absence of adhesion may induce the degradation of cellular junctions, which is associated with tumor cell invasion and metastasis (41,42). Cellular adhesion comprises cell-cell and cell-matrix contacts, which are distributed in a number of types of tissue, and serve roles in maintaining the integrity of tissues and cells. Adherens junctions are formed by classic cadherins, including epithelial cadherin and neuronal cadherin, which associate with α-/β-catenin, plakoglobin, adherens junction protein p120 and vinculin, and anchor actin microfilaments (43). Calcium-dependent cadherin/catenin/actin cytoskeleton protein complexes serve a vital role in cell morphology maintenance, cell motility and adhesion, cytoskeleton remodeling and regulation of signal transduction (20,44). β-catenin is a multifunctional protein that mediates cell adhesion and signal transduction, and is a component of epithelial cell-cell junctions. However, decreased β-catenin expression and its dysregulation are associated with the initiation of cancer cell invasion and metastasis (20). In the present study, CXCR7 downregulated the expression of β-catenin, which suggests that CXCR7 overexpression may result in the degradation of cellular junctions and the subsequent decrease in cell adhesion, which may eventually lead to the invasion and metastasis of PTC cells.

In addition, the results of the present study identified certain differently expressed proteins with unclear biological functions, including TAGLN2 and AHNAK2. TAGLN2 is a member of the calponin family of actin-binding proteins and is upregulated in lung cancer, colorectal cancer, and head and neck squamous cell carcinoma (16,45,46). Knockdown of TAGLN2 has been reported to inhibit tumor cell invasion and promote apoptosis (47,48). Notably, it has been reported that the overexpression of TAGLN2 is associated with lymph node metastasis, distant metastasis and tumor-node-metastasis stage in colorectal cancer (47). AHNAK2 is a 600 kDa protein that is expressed in the majority of muscle cells and has a similar function to AHNAK1: It regulates the contraction coupling of myocardial cells through its PSD-95/Discs-large/ZO-1 structural domain (49,50). In addition, AHNAK2 is a constituent of costameres in skeletal muscle and may link the extracellular matrix with the cytoskeleton (19,51). Previous studies have identified that PPL and AHNAK1 affect the formation of the ezrin, PPL, periaxin and desmoyokin skeletal protein complex (52). Therefore PPL, AHNAK2, β-catenin and TAGLN2 are associated with cellular adhesion junctions; overexpression of CXCR7 may lead to dysregulation of these proteins and subsequently degrade cellular junctions, inducing invasion and metastasis of PTC.

In conclusion, the results of the present study provide a possible mechanism for the regulation of PTC invasion and metastasis by CXCR7. Proteomic analysis using iTRAQ-coupled 2D LC-MS/MS identified 130 differentially expressed proteins between GLAG-66 and GLAG-66-CXCR7 cells, including a number of proteins associated with cellular junctions. CXCR7 overexpression may result in the degradation of cellular junctions and therefore promote cell invasion and metastasis. In addition, AHNAK2 and TAGLN2 were identified as potential novel biomarkers for PTC. However, further studies on these proteins are required to demonstrate their function and mechanism in PTC generation and development.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (grant no. 81072182) and the Natural Science Foundation of Liaoning Province (grant no. 2013021100; China).

References