Proteomic analysis of differentially expressed proteins in normal human thyroid cells transfected with PPFP

in Endocrine-Related Cancer
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  • 1 Key Laboratory of Cancer Proteomics of Chinese Ministry of Health, Hunan Engineering Laboratory for Structural Biology and Drug Design

The fusion gene encoding the thyroid-specific transcription factor PAX8 and peroxisome proliferator-activated receptor γ (PPARγ (PPARG)) (designated as the PPFP gene) is oncogenic and implicated in the development of follicular thyroid carcinoma (FTC). The effects of PPFP transfection on the biological characteristics of Nthy-ori 3-1 cells were studied by MTT assay, colony formation, soft-agar colony formation, and scratch wound-healing assays as well as by flow cytometry. Furthermore, the differentially expressed proteins were analyzed on 2-DE maps and identified by MALDI-TOF-MS. Validation of five identified proteins (prohibitin, galectin-1, cytokeratin 8 (CK8), CK19, and HSP27) was determined by western blot analysis. PPFP not only significantly increased the viability, proliferation, and mobility of the Nthy-ori 3-1 cells but also markedly inhibited cellular apoptosis. Twenty-eight differentially expressed proteins were identified, among which 19 proteins were upregulated and nine proteins were downregulated in Nthy-ori 3-1PPFP (Nthy-ori 3-1 cells transfected with PPFP). The western blot results, which were consistent with the proteome analysis results, showed that prohibitin was downregulated, whereas galectin-1, CK8, CK19, and HSP27 were upregulated in Nthy-ori 3-1PPFP. Our results suggest that PPFP plays an important role in malignant thyroid transformation. Proteomic analysis of the differentially expressed proteins in PPFP-transfected cells provides important information for further study of the carcinogenic mechanism of PPFP in FTCs.

Abstract

The fusion gene encoding the thyroid-specific transcription factor PAX8 and peroxisome proliferator-activated receptor γ (PPARγ (PPARG)) (designated as the PPFP gene) is oncogenic and implicated in the development of follicular thyroid carcinoma (FTC). The effects of PPFP transfection on the biological characteristics of Nthy-ori 3-1 cells were studied by MTT assay, colony formation, soft-agar colony formation, and scratch wound-healing assays as well as by flow cytometry. Furthermore, the differentially expressed proteins were analyzed on 2-DE maps and identified by MALDI-TOF-MS. Validation of five identified proteins (prohibitin, galectin-1, cytokeratin 8 (CK8), CK19, and HSP27) was determined by western blot analysis. PPFP not only significantly increased the viability, proliferation, and mobility of the Nthy-ori 3-1 cells but also markedly inhibited cellular apoptosis. Twenty-eight differentially expressed proteins were identified, among which 19 proteins were upregulated and nine proteins were downregulated in Nthy-ori 3-1PPFP (Nthy-ori 3-1 cells transfected with PPFP). The western blot results, which were consistent with the proteome analysis results, showed that prohibitin was downregulated, whereas galectin-1, CK8, CK19, and HSP27 were upregulated in Nthy-ori 3-1PPFP. Our results suggest that PPFP plays an important role in malignant thyroid transformation. Proteomic analysis of the differentially expressed proteins in PPFP-transfected cells provides important information for further study of the carcinogenic mechanism of PPFP in FTCs.

Introduction

Thyroid cancer is the most common malignant tumor of the endocrine organs, and its incidence has steadily increased over the past few decades (Davies & Welch 2006, Chen et al. 2009, Wang & Wang 2012). Follicular thyroid carcinoma (FTC), the second most common differentiated thyroid cancer, shows more aggressive clinicopathological features and a greater rate of recurrence than papillary thyroid carcinomas (PTCs). Additionally, FTCs are more prone to metastasize to the lung, bone, brain, and liver in comparison with PTCs (Baloch & LiVolsi 2007, Witt 2008). Moreover, FTC can only be diagnosed if capsule and vascular invasion are found in the surgical resection specimens, making it difficult to diagnose by fine needle aspiration (FNA) biopsy (Baloch & LiVolsi 2007). Understanding the genetic alterations in FTCs and their mechanisms is fundamentally important in developing new molecular markers for earlier diagnosis and novel therapeutic targets.

Kroll et al. (2000) were the first to report the genetic rearrangement of the thyroid-specific transcription factor PAX8 and peroxisome proliferator-activated receptor γ (PPARγ (PPARG)), which results in the overexpression of the PAX8/PPARγ fusion protein (PPFP) in FTC. Later studies showed that this rearrangement was mainly detected in FTC and accounted for about 29–62% of all FTC cases by immunohistochemistry or RT-PCR (Dwight et al. 2003, Nikiforova et al. 2003, Reddi et al. 2007, Algeciras-Schimnich et al. 2010). The PAX8/PPARγ fusion gene has been demonstrated to be an oncogene that is closely related to the occurrence and development of FTC. Research concerning the oncogenic mechanism of PAX8/PPARγ rearrangement has concentrated on its effects on the function of wild-type PPARγ and PAX8 (Gregory Powell et al. 2004, Reddi et al. 2007). Despite some progress, the role of PPFP in the tumorigenesis of thyroid cancer remains unclear. In this study, thyroid cells were transfected with PPFP, and the changes in their biological characteristics were studied to determine the role of PPFP in the malignant transformation of thyroid cells. In order to find proteins that interact with PPFP, the proteins that were differentially expressed in PPFP-transfected cells were identified by two-dimensional electrophoresis and mass spectrometry.

Materials and methods

Construction of the recombinant vector containing the human PPFP gene

The PAX8 and PPARγ genes were isolated from the PAX8-pOTB7 and PPARγ-pCMV-SPORT6 plasmids (Genechem, Shanghai, China) respectively and amplified by PCR. The two genes were then ligated together and subcloned into the eukaryotic expression vector pEGFP-C1. The resulting recombinant vector pEGFP-C1-PAX8/PPARγ was designed to express the PAX8/PPARγ fusion gene. The PCR-amplified PPFP gene was confirmed by endoenzyme digestion and DNA sequencing (Fig. 1A).

Figure 1
Figure 1

Construction and production of the recombinant vector containing the human PPFP gene. (A) Lane 1: marker, lane 2: pEGFP-C1 vector, lane 3: pEGFP-C1 digested with XhoI and KpnI showed a band of about 4.7 kb, which is consistent with the size of the desired fragment. (B) PCR assay to identify PPFP-positive colonies (lane 1: negative control, lane 2: positive control, and lane 3: marker). Lanes 4, 6, and 9 (PPFP-positive colonies) had a 565 bp PCR fragment, which was designed to detect the presence of the PPFP gene. (C and D) Western blot detection of PPFP expression in 293T cells using an anti-mouse FLAG antibody and an anti-mouse PPAR antibody respectively. Lane 1: untransfected 293 T cells, lane 2: 293T cells transfected with empty vector, and lanes 3 and 4: 293T cells transfected with PPFP showed a band with a molecular weight of about 90 kDa, which corresponds to the size of PPFP.

Citation: Endocrine-Related Cancer 19, 5; 10.1530/ERC-12-0156

Cell culture and transfection

To achieve stable and efficient expression, recombinant lentiviral vector was transferred into subjected cells. First, the PAX8/PPARγ fusion gene was isolated from the EGFP-C1-PPFP plasmid and amplified by PCR. The fusion gene was then subcloned into the recombinant lentiviral vector pGC-FU (Genechem), which contains a FLAG tag, to generate the lentiviral expression vector pGC-FU-PPFP. Recombinant lentiviruses, which contained the PPFP gene and FLAG tag, were produced by 293T cells following the co-transfection of pGC-FU-PPFP and the packaging plasmids pHelper1.0 and pHelper2.0 (Genechem). The viral titer was measured after collecting and concentrating the viral supernatant. PCR was used to screen for PPFP-positive colonies (Fig. 1B). In order to verify the expression of the PPFP gene, antibodies against FLAG and PPAR were used to detect PPFP expression in the 293T cells by western blot analysis (Fig. 1C and D). The targeted cells, Nthy-ori 3-1 (HPACC, Salisbury, UK), originate from normal human thyroid cells that have been immortalized by the expression of SV40 large T antigen. Nthy-ori 3-1 cells were transfected by pGC-FU-PPFP to establish a stable Nthy-ori 3-1PPFP cell line. To evaluate the efficacy of transfection, we examined the expression of green fluorescent protein (GFP) and found that the lentiviral supernatants could effectively and stably infect Nthy-ori 3-1 cells, as shown in Fig. 2. Meanwhile, Nthy-ori 3-1vector cells, which were transfected with empty lentiviral vector and untransfected Nthy-ori 3-1 cells, were included as control cell lines. Cells were grown at 37 °C in a humidified 5% CO2 incubator with RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (Gibco). The medium was changed every 2–3 days, and the cells were passaged every 3–5 days.

Figure 2
Figure 2

PPFP expression in Nthy-ori 3-1 cells transfected with PPFP by green fluorescent protein detection: Abundant green fluorescence was observed by fluorescence microscopy, and more than 90% of Nthy-ori 3-1 cells stabilized and highly expressed the targeted gene after 12 days. A, B, and C represent untransfected cells, Nthy-ori 3-1vector, and Nthy-ori 3-1PPFP respectively. Row 1: fields viewed under the light microscope; row 2: fields viewed under the fluorescent microscope.

Citation: Endocrine-Related Cancer 19, 5; 10.1530/ERC-12-0156

MTT, colony formation, soft-agar colony formation, scratch wound-healing assays, and flow cytometry

To study the changes in the biological characteristics of the Nthy-ori 3-1 cells after PPFP gene transfection, we analyzed cell proliferation with the MTT assay, colony number with the plate colony forming assay, colony-forming efficiency with the soft-agar colony formation assay, migration and movement capacity with the scratch wound-healing assay, and apoptosis and cell cycle with flow cytometry. For each of the three groups (Nthy-ori 3-1PPFP, Nthy-ori 3-1vector, and Nthy-ori 3-1), cells in the logarithmic phase were used in all experiments and incubated at 37 °C in 5% CO2. For the MTT assay, cells were incubated with 0.5 mg/ml MTT for 4 h. The formazan crystals produced by the living cells in the culture were dissolved with 100 μl dimethyl sulfoxide, and the absorbance (OD value) was measured at 570 nm using a 96-well plate reader at different time points (0, 24, 48, 72, and 96 h). For the plate colony-forming assay, cells were seeded onto six-well plates at 300 cells/well and incubated with complete medium at 37 °C in 5% CO2 for 14 days. The number of surviving colonies (a colony was defined to contain >50 cells) was counted, and the difference in colony formation between the three experimental groups was determined. For the soft-agar colony formation assay, a two-layer soft-agar culture was prepared with a bottom layer of 0.6% agarose and a top layer of 0.35% agarose. Cells were seeded onto six-well plates at a density of 2×104 cells/well and incubated at 37 °C in 5% CO2 for 2 weeks. The number of colonies (diameter >75 μm or cell count >50) was counted for further analysis. The rate of colony formation was calculated by dividing the number of colonies with the total cell count per well. For the scratch wound-healing assay, cells were seeded at a density of 5×105 cells/well in six-well plates, which were precoated overnight at 4 °C with PBS containing 10 μg/ml fibronectin. Then, the cells were incubated in 5% CO2 at 37 °C until a confluent monolayer formed. A scratch of about 300–500 μm was made manually by scraping the cell monolayer with a 200-μl pipette tip. After washing twice with PBS, the cells were cultured in serum-free medium. Images were captured at different time points (0, 12, 24, 36, and 48 h), and the distance that the cells migrated to the wounded region was measured. For flow cytometry, the prepared cells were collected and digested into single-cell suspensions using 0.25% trypsin. Then, the cells were centrifuged at 500 g for 5 min and washed with PBS (0.01 M, pH 7.4) twice. Seventy percent ethanol stored at 4 °C was used to fix the cells for 24 h before they were fully shaken and dispensed. The plates were incubated with 0.5% Triton X-100 (Sigma) and 0.05% RNase (Sigma) in 1 ml PBS at 37 °C for 30 min and then centrifuged at 1259 g for 5 min. The cells were stained with 50 μg/ml propidium iodide (Sigma) at room temperature for 30 min, and the cell number was adjusted to 1×106/ml. Samples were immediately analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).

IPG-2D PAGE

The three cell lines Nthy-ori 3-1PPFP, Nthy-ori 3-1vector, and Nthy-ori 3-1 were lysed in 100 μl lysis buffer (7 M urea, 2 M thiourea, 100 mM dithiothreitol (DTT), 4% CHAPS, 40 mM Tris, 2% pharmalyte, and 1 mg/ml DNaseI). The lysates were centrifuged at 12 000 g for 30 min at 4 °C, and the supernatant was collected and stored at −80 °C until use. The total protein concentration was determined using a 2D Quantification kit (Amersham Biosciences) before two-dimensional electrophoresis. Triplicate gels were prepared for each cell line, and a total amount of 1000 μg protein per gel was analyzed. Protein samples were diluted to 450 μl with rehydration solution (8 M urea, 4% CHAPS, 40 mM Tris, 40 mM DTT, 0.5% IPG buffer, and trace bromophenol) and applied to IPG strips (pH 3–10L, 240×3×0.5 mm, Amersham Biosciences), which were rehydrated for 12 h at 30 V. The proteins were then focused successively for 1 h at 500 V, 1 h at 1000 V, and 8.5 h at 8000 V for a total of 69 920 Vh on an IPGphor system (Amersham Biosciences). Focused IPG strips were equilibrated for 15 min in 10 ml solution A (50 mM Tris–HCl, pH 8.8, 6 M urea, 30% glycerol, 1% SDS, and 0.2% DTT) and then for an additional 15 min in 10 ml solution B (the same as solution A except that DTT was replaced with 3% iodoacetamide). Protein separation on the second dimension was achieved by SDS–PAGE using 12.5% homogenous gels and the Ettan Dalt II system (Amersham Biosciences). After SDS–PAGE, the protein spots were visualized via Blue Silver stain, a modification of Neuhoff's colloidal Coomassie Blue G-250 stain (Amersham Biosciences), according to Candiano et al. (2004).

Image analysis

The stained 2-DE gels were scanned with LabScan software using ImageScanner (Amersham Biosciences) with the resolution set at 300 dpi. The spot-intensity calibration, spot detection, background subtraction, matching, 1-D calibration, and the establishment of average gel were accomplished with the PDQuest system (Bio-Rad Laboratories). The amount of stained protein within the individual spots was quantified by calculating the spot volume after normalizing the image using the total spot volume normalization method and multiplying by the total area of all spots. The calculation of the theoretical molecular weight and pI value of the identified protein spots was based on algorithms included in the PDQuest analysis software package (Bio-Rad Laboratories). Proteins were classified as being differentially expressed when the spot intensities showed a ≥2-fold difference between the three cell lines. Significant differences in protein expression levels were determined using the Student's t-test with P<0.05.

Protein identification by MALDI-TOF-MS and database analysis

The differentially expressed protein spots were excised from the stained gels using a punch, and in-gel trypsin digestion was performed as described (Chen et al. 2008). After destaining with 100 mM sodium thiosulfate and 30 mM potassium ferricyanide, the protein-containing gel spots were subsequently reduced, alkylated, and dried. Then, the dried gel pieces were incubated in the digestion solution, which consisted of 40 mM ammonium bicarbonate, 9% can, and 20 μg/ml trypsin (Sigma) for 12 h at 37 °C. The purified tryptic peptide mixture was mixed with an α-cyano-4-hydroxycinnamic acid matrix solution and vortexed lightly. Then, 1 μl mixture was analyzed with a Voyager System DE-STR 4307 MALDI-TOF Mass Spectrometer (ABI, Carlsbad, CA, USA) to obtain a peptide mass fingerprint (PMF). To search the PMF map database, Mascot Distiller was used to obtain the monoisotopic peak list from the raw mass spectrometry files. Proteins were identified by searching the Swiss-Prot database via the Mascot search engine (http://www.matrixscience.com/). The search parameters were set as following: the mass tolerance was ±0.5 Da; the number of missed cleavage sites allowed was up to 1; the cysteine residue was modified as carbamidomethyl-cys; variable modifications included oxidation (M); the minimum number of matched peptides was 5; the species was selected as HOMO SAPIENS (HUMAN); the peptide ion was [M+H]+; mass values was set as monoisotopic; the search range was within the experimental pI value±0.5 pH unit and experimental Mr ±20%; isotope masses were used; and protein scores provided by the Mascot program that were >56 were considered significant (P<0.05).

Western blot analysis

Cells were collected from the three groups: Nthy-ori 3-1PPFP, Nthy-ori 3-1vector and Nthy-ori 3-1. Total protein samples were prepared as mentioned earlier, and the concentration was determined using the BCA Protein Assay Kit (Amersham Biosciences). Western blot analysis was performed as described previously (Yang et al. 2006). Briefly, 40 μg lysate was separated by 10% SDS–PAGE and transferred to a PVDF membrane. Blots were blocked with 5% nonfat dry milk for 2 h at room temperature and then incubated with a rabbit polyclonal anti-human prohibitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), a mouse monoclonal anti-human galectin-1 antibody (Santa Cruz), a mouse monoclonal anti-human cytokeratin 8 (CK8) antibody (Santa Cruz), a mouse monoclonal anti-human CK19 antibody (Santa Cruz), or a rabbit monoclonal anti-human HSP27 antibody (Santa Cruz) overnight at room temperature. HRP-conjugated goat anti-rabbit or goat anti-mouse IgG (Santa Cruz) was incubated with the blots in sequence at room temperature for 2 h. The signal was visualized with ECL detection reagent and quantified by densitometry using the ImageQuant image analysis system (Storm Optical Scanner, Molecular Dynamics, Sunnyvale, CA, USA). β-Actin was detected simultaneously using a mouse anti-β-actin antibody (Sigma) as a control.

Statistical analysis

The statistical analyses were performed using SPSS 13.0 software. Single comparisons were performed using the Student's t-test or Mann–Whitney's U test, and multiple comparisons were performed using one-way ANOVA. P values <0.05 were considered statistically significant.

Results

The effect of PPFP on the biological characteristics of Nthy-ori 3-1 cells

In the MTT assay, the absorbance values of Nthy-ori 3-1PPFP at 48, 72, and 96 h were significantly higher than those of Nthy-ori 3-1vector and Nthy-ori 3-1 (P<0.01, Fig. 3A). Compared with the Nthy-ori 3-1vector and Nthy-ori 3-1 cells, the Nthy-ori 3-1PPFP cells formed more colonies after 14 days of culture (P<0.01). However, there was no difference between the Nthy-ori 3-1vector and Nthy-ori 3-1 cells (P>0.05, Fig. 3B). The soft-agar colony formation assay, which was used to examine anchorage-independent growth, showed that all three cell lines were capable of colony formation. However, the colony formation rates for Nthy-ori 3-1vector and Nthy-ori 3-1 were significantly less than that for Nthy-ori 3-1PPFP (P<0.01, Fig. 3C). The migration of Nthy-ori 3-1 cells transfected with pGC-FU-PPFP was greater than that of the Nthy-ori 3-1vector and Nthy-ori 3-1 cells. The results showed a significant increase in cell migration for the Nthy-ori 3-1PPFP cells after 24 h, and the scratch was almost healed after 48 h, while scratches were only partially healed for the Nthy-ori 3-1vector and Nthy-ori 3-1 cells, as shown in Fig. 3D. Flow cytometric analyses demonstrated that the apoptotic percentage of the Nthy-ori 3-1PPFP cells was significantly less than that of the Nthy-ori 3-1vector and Nthy-ori 3-1 cells (P<0.01, Fig. 4). The number of Nthy-ori 3-1PPFP cells in G0/G1 phase decreased significantly (P<0.01) but that in S and G2/M phase increased significantly (P<0.01) in comparison with both the Nthy-ori 3-1vector and the Nthy-ori 3-1 cells. However, there was no significant difference between the Nthy-ori 3-1vector and Nthy-ori 3-1 cells.

Figure 3
Figure 3

Physiological impact of PPFP on thyroid cells. (A) MTT assay. Nthy-ori 3-1PPFP cells had higher absorbance values at 48, 72, and 96 h compared with Nthy-ori 3-1vector and untransfected Nthy-ori 3-1 cells (*P<0.01). (B) Soft-agar colony formation assay. Nthy-ori 3-1PPFP cells formed more colonies compared with Nthy-ori 3-1vector and untransfected Nthy-ori 3-1 cells (*P<0.01). (C) Plate formation assay (1, Nthy-ori 3-1; 2, Nthy-ori 3-1vector; and 3, Nthy-ori 3-1PPFP). Nthy-ori 3-1PPFP cells had a higher colony formation rate compared with Nthy-ori 3-1vector and untransfected Nthy-ori 3-1 cells (*P<0.01). (D) Scratch wound-healing assay (1, Nthy-ori 3-1; 2, Nthy-ori 3-1vector; 3, Nthy-ori 3-1PPFP). The scratch was almost healed in Nthy-ori 3-1PPFP cells after 48 h, while scratches in Nthy-ori 3-1vector and Nthy-ori 3-1 cells were only half-healed.

Citation: Endocrine-Related Cancer 19, 5; 10.1530/ERC-12-0156

Figure 4
Figure 4

Effect of PPFP on the cell cycle and apoptosis of Nthy-ori 3-1 cells. The number of Nthy-ori 3-1PPFP cells in G0/G1 phase decreased significantly but the number in S and G2/M phase increased significantly compared with Nthy-ori 3-1vector and Nthy-ori 3-1 cells (P<0.01). The apoptotic percentage of Nthy-ori 3-1PPFP cells was significantly less than Nthy-ori 3-1vector and Nthy-ori 3-1 cells (*P<0.01).

Citation: Endocrine-Related Cancer 19, 5; 10.1530/ERC-12-0156

Detection of differentially expressed proteins in Nthy-ori 3-1 cells transfected with PPFP by 2-DE

Comparative proteomics analysis was performed to identify the differentially expressed proteins in the Nthy-ori 3-1PPFP, Nthy-ori 3-1vector, and Nthy-ori 3-1 cells. 2-DE of each cell line was performed in triplicate to obtain a reliable map with a clear background, high definition, and repeatability. The protein spots detected in the Nthy-ori 3-1vector and Nthy-ori 3-1 cells were not significantly different after comparing their average 2-DE maps. Compared with the Nthy-ori 3-1vector and Nthy-ori 3-1 cells, 38 protein spots detected in the Nthy-ori 3-1PPFP cells were consistently different (≥2-fold) in triplicate experiments. As shown in Fig. 5, increased expression of 19 protein spots and decreased expression of nine protein spots were found in the 28 differentially expressed protein spots detected in the Nthy-ori 3-1PPFP cells, which were subsequently identified by MS.

Figure 5
Figure 5

(A) Representative 2-DE maps of Nthy-ori 3-1PPFP, Nthy-ori 3-1vector, and Nthy-ori 3-1 cells. The 28 differentially expressed protein spots that were identified by MS are marked with arrows. (B) Close-up image of differentially expressed protein spots in the three groups of cells.

Citation: Endocrine-Related Cancer 19, 5; 10.1530/ERC-12-0156

Identification of the differentially expressed proteins by MS

All the differentially expressed protein spots were excised from stained gels, in situ digested with trypsin, and analyzed by MALDI-TOF-MS. From a total of 28 differentially expressed protein spots identified by MS, the levels of 19 proteins increased and those of nine proteins decreased in Nthy-ori 3-1PPFP cells respectively as shown in Table 1. The MALDI-TOF mass spectrometry map and database query result of a representative spot (protein spot 6) are shown in Fig. 6. Data for 15 monoisotopic peaks were input into the Mascot program to search the Swiss-Prot database, and the query result showed that protein spot 6 was prohibitin.

Table 1

Differential proteins in Nthy-ori 3-1PPFP cells identified by MALDI-TOF MS

Spot no.Swiss-Prot ACProtein nameMW (Da)pIScoreaCoverage (%)Expression in Nthy-ori 3-1PPFPFunctional classification
1P06576ATP synthase subunit β, mitochondrial56 5255.2615554ATP synthesis
2P07237Protein disulfide isomerase57 4804.7613825Metabolism
3P30101Protein disulfide isomerase A357 1465.9822748Protein folding
4P20338Ras-related protein RAB4A24 1415.827433Protein transport
5Q9H239Matrix metalloproteinase-2859 1879.709238Tissue homeostasis and repair
6P35232Prohibitin29 8435.5726264DNA replication, regulating proliferation
7Q997143-Hydroxyacyl-CoA dehydrogenase type-227 1347.6616754tRNA processing
8P68133Actin, α skeletal muscle42 3665.237830Cytoskeleton
9P51530DNA2-like helicase122 0487.956120DNA replication
10Q8N8E3Coiled-coil domain-containing protein 46113 1926.256726
11P05787Keratin, type II cytoskeletal 853 6715.5220043Cytoskeleton
12P67936Tropomyosin α-4 chain28 6194.675936Cytoskeleton
13Q14457Beclin-152 3774.8311136Cell cycle
14P09382Galectin-115 0485.3411861Apoptosis, proliferation, cell differentiation
15P09211Glutathione S-transferase P23 5695.4312748Anti-apoptosis
16P06733α-Enolase47 4817.0117446Transcriptional repressor
17P30084Enoyl-CoA hydratase, mitochondrial31 8238.3411329Metabolism
18P08727Keratin, type I cytoskeletal 1944 0655.0424543Cytoskeleton
19P21964Catechol O-methyltransferase30 4745.268739Metabolism
20P22626Heterogeneous nuclear ribonucleoproteins A2/B137 4648.979338mRNA processing
21Q9Y241HIG1 domain family member 1A10 1369.799868Stress response
22Q562R1β-Actin-like protein 242 3185.296521Cytoskeleton
23P62937Peptidyl-prolyl cis-trans isomerase A18 2297.6814669Protein folding
24P04792Heat-shock protein 2722 8265.9815961Stress resistance and actin organization
25P23528Cofilin-118 7198.229156Cell morphology and cytoskeletal organization
26P04075Fructose-bisphosphate aldolase39 8518.3015255Glycolysis and gluconeogenesis
27P46940Ras GTPase-activating-like protein IQGAP1189 7616.0811835Signal transduction
28P30040Endoplasmic reticulum protein ERp2929 0326.777831Protein secretion

Mascot score is 56. Protein scores by Mascot searching >56 were considered significant (P<0.05).

Figure 6
Figure 6

MALDI-TOF-MS analysis of differential protein spot 6. (A) The mass spectrum of protein spot 6 identified this protein as prohibitin according to the matched peaks. (B) Possible matched proteins to the spectral masses are presented as multiple bars with differential probability-based MOWSE scores.

Citation: Endocrine-Related Cancer 19, 5; 10.1530/ERC-12-0156

Validation of the identified proteins

To confirm the identification of the differentially expressed proteins identified using the 2-DE maps, the expression of five identified proteins (prohibitin, galectin-1, CK8, CK19, and HSP27) in Nthy-ori 3-1, Nthy-ori 3-1vector, and Nthy-ori 3-1PPFP cells was detected by western blot. As shown in Fig. 7, the expression of galectin-1, CK8, CK19, and HSP27 was significantly upregulated in Nthy-ori 3-1PPFP cells compared with Nthy-ori 3-1vector and Nthy-ori 3-1 cells (P<0.05), while that of prohibitin decreased significantly in Nthy-ori 3-1PPFP cells (P<0.05). Also, no obvious difference was observed between the Nthy-ori 3-1vector and Nthy-ori 3-1 cells, which is consistent with the results from the proteomic analysis.

Figure 7
Figure 7

(A) Representative results of western blot analysis for prohibitin, galectin-1, cytokeratin 8, cytokeratin 19, and HSP27 in Nthy-ori 3-1, Nthy-ori 3-1vector, and Nthy-ori 3-1PPFP cells. (B) Histogram of the relative changes in the expression levels of these proteins. The expression of galectin-1, cytokeratin 8, cytokeratin 19, and HSP27 was significantly upregulated in Nthy-ori 3-1PPFP cells in comparison with Nthy-ori 3-1vector and Nthy-ori 3-1 cells, whereas that of prohibitin decreased significantly in Nthy-ori 3-1PPFP cells (P<0.05).

Citation: Endocrine-Related Cancer 19, 5; 10.1530/ERC-12-0156

Discussion

Follicular cancers, which comprise 10–15% of total thyroid cancers, are more difficult to detect at early stages even by FNA. Recently, the detection of somatic mutations has been proposed for the preoperative FNA diagnosis of differentiated thyroid cancer (Cerutti 2011). As seen in most malignant neoplasms, thyroid cancers usually are associated with specific genetic abnormalities; two major genetic alterations (RAS mutation and PAX8/PPARγ rearrangement) are found in FTC. Interestingly, these two genetic backgrounds rarely overlap in the same tumor, suggesting that two independent pathways are involved in the development of FTC (Nikiforova et al. 2003). While the Ras mutation is not restricted to a particular histological type, PAX8/PPARγ rearrangement mainly presents with follicular neoplasms and a small portion of follicular variants of papillary thyroid cancers (Dwight et al. 2003, Nikiforova et al. 2003, Kroll 2004, Lacroix et al. 2004, 2005, Koenig 2010, Nikiforov & Nikiforova 2011). Because the expression of PPFP indicates a favorable clinical presentation and prognosis, FTCs presenting with PAX8/PPARγ rearrangement are thought to be a less aggressive subtype (Sahin et al. 2005). Although it is expressed at a lower frequency in follicular thyroid adenoma, PPFP is believed to play an important role in thyroid tumorigenesis, probably at an early stage (Nikiforova et al. 2002, Gregory Powell et al. 2004, Reddi et al. 2007). However, the role of PPFP in thyroid cancer is not fully understood.

Based on the successful construction of recombinant lentiviral expression vector containing PPFP (pGC-FU-PPFP), we studied the biological characteristics of SV40 large-T-antigen-immortalized normal human-differentiated thyroid cells (Nthy-ori 3-1) after transfection with PPFP. Our results showed that the expression of PPFP in Nthy-ori 3-1 cells could prompt cellular proliferation, inhibit apoptosis, induce cell cycle transition, and spur cellular migration. All these data indicate that PPFP can provide a significant growth advantage and contribute to malignant transformation in thyroid cancers. Consistent with other studies, our results also demonstrated that PPFP plays an important role in stimulating the malignant cellular proliferation, invasion, and metastasis of thyroid cancer (Gregory Powell et al. 2004, Au et al. 2006, Espadinha et al. 2007). Because all these studies are based on cell lines that were already immortalized, such as Nthy-ori 3-1, whether PPFP alone is sufficient to trigger tumorigenesis or whether other molecular events are involved remains unclear. It is essential to develop in vivo animal models with PPFP expression in the thyroid to demonstrate the role of PPFP in tumorigenesis. As shown in our table, 28 differentially expressed proteins, including 19 upregulated and nine downregulated proteins, were identified by a proteomic approach in Nthy-ori 3-1PPFP cells. These differentially expressed proteins indicated that the involvement of PPFP in FTC tumorigenesis is a complicated process that is related to proliferation, apoptosis, differentiation, metabolism, cell cycle regulation, the cytoskeleton, and signal transduction. Moreover, five differentially expressed proteins (prohibitin, galectin-1, CK8, CK19, and HSP27), which were considered to be closely connected with PPFP, were selected to be further analyzed by western blot to validate our 2-DE results. The western results suggest that the proteins identified by the proteomic approach are actually differentially expressed proteins involved with PPFP. These findings lay a foundation for further clarification of the molecular mechanism of the occurrence and development of FTC.

Prohibitin, which is located on chromosome 17q21, is a potential tumor suppressor that is linked to early-onset breast cancer in humans. Prohibitin inhibits cell proliferation by binding with RB and consequently modulating the transcriptional activity of E2F (Wang et al. 1999a,b). Prohibitin can also lead to G1/S arrest, which also favorably influences anti-proliferation. Several studies have revealed the reduced expression of prohibitin in the early stages of prostate, gastric, and liver cancer (Seow et al. 2000, Gamble et al. 2004, Jang et al. 2004). However, conflicting results have reported the overexpression of prohibitin in many other cancers, such as uterine, cervix, bladder, and lung cancer (Tsai et al. 2006, Wu et al. 2007, Guo et al. 2012) and even in the same type of cancers (Wang et al. 2004, Ummanni et al. 2008). Franzoni et al. (2009) demonstrated the high expression of prohibitin in PTCs bearing the BRAFV600E mutation but low expression in follicular adenoma. In our experiments, the expression of prohibitin in thyroid cells expressing PPFP decreased significantly compared with untransfected cells or those transfected with empty vector. These results suggest that PPFP probably accelerates cell proliferation by downregulating the expression of prohibitin in these cells. The relief of cell cycle arrest in G0/G1 phase and the increase in the number of cells in S phase may also attribute to the decrease in prohibitin expression due to PPFP transfection.

Galectin-1 is a developmentally regulated gene that encodes a β-galactoside-binding protein whose overexpression is associated with malignant transformation and loss of differentiation (Salvatore et al. 1995). Further study showed that DNA methylation is involved in the regulation of galectin-1 gene expression in rat thyroid cells (Benvenuto et al. 1996). The function of galectin-1 also involves developmental processes regarding fetal stromal thyroid tissue organization and thyroid epithelium maturation (Savin et al. 2003). High levels of galectin-1 in malignant thyroid tumors have been reported in several studies by immunohistochemical and proteomic analysis and serum detection, suggesting its vital role in discriminating between benign and malignant thyroid lesions (Chiariotti et al. 1995, Xu et al. 1995, Torres-Cabala et al. 2006, Saussez et al. 2008). The detection of galectins in blood and tissue using FNA and large needle aspiration biopsy has been proposed for preoperative diagnosis (Carpi et al. 2010). Our experimental results showed that Nthy-ori 3-1 cells transfected with PPFP had increased expression of galectin-1, suggesting its potent role in the malignant transformation of thyroid cells. However, there are no studies to date that describe the mechanism by which PPFP affects the expression of galectin-1.

CKs are the major structural proteins of epithelial cells, and recent data has shown that they play an important role in cell signaling, the stress response, and apoptosis. Overexpression of some CKs, including CK8, CK18, and CK19, has been reported in several cancers and is believed to be involved in tumor progression based on several studies (Letocha et al. 1993, Silen et al. 1994, Fujita et al. 1999, Fukunaga et al. 2002, Cimpean et al. 2008, Liu et al. 2008, Kabukcuoglu et al. 2010). Another study reported the high expression of pan-CK, CK8, and CK19 in malignant thyroid tumors (Schroder et al. 1996). To date, it is not fully understood why an increase in CK8 is significantly linked to poor clinical characteristics and prognosis. In contrast to CK8, the expression of CK19 and CK19 fragments in the plasma have already been well investigated in thyroid tumors and a variety of other tumors (Flanagan et al. 2008, Krzeslak et al. 2008, Suzuki et al. 2010, Yang et al. 2010, Chung et al. 2011, Stewart et al. 2011). Further studies showed that the expression level of CK19 or CK19 fragments differ in various subtypes of thyroid tumors (Raphael et al. 1994, Miettinen et al. 1997). Most studies demonstrated that overexpression of CK19 is mainly related to papillary thyroid cancer with a positive rate, accounting for 70–100% of all cancers (Flanagan et al. 2008, Krzeslak et al. 2008, Barut et al. 2010, Gong et al. 2012). Furthermore, CK19 has been used in combination with PAX8 and HBME1 to increase the specificity of diagnosis for thyroid tumors by FNA (Schmitt et al. 2010, Cochand-Priollet et al. 2011). The upregulated expression of CK8 and CK19 was detected in Nthy-ori 3-1 cells transfected with PPFP, which may indicate that involvement of CK8 and CK19 is induced by PPFP in the malignant transformation and enhanced migration of thyroid cells.

HSP27 is a member of the small HSP family and acts as a powerful antagonist of programmed cell death in the regulation of epithelial cell growth and differentiation (Sherman & Goldberg 2001, Sherman et al. 2007). Studies have shown that phosphorylated HSP27 enhances cell migration in several cell lines and tumor tissues (Morino et al. 1997, Piotrowicz et al. 1998, Rust et al. 1999, Shin et al. 2005). Reduced expression of HSP27 was reported in PTC, FTC, and multinodular goiters but enhanced expression was demonstrated in diffuse hyperplasia and follicular adenoma by proteomic analyses (Srisomsap et al. 2002, Bian & Tang 2010). The overexpression of HSP27 was observed in PPFP-transformed Nthy-ori 3-1 cells, which suggests that PPFP may involve in the increased cell migration of thyroid cells by HSP27.

Conclusion

In this study, our analysis of Nthy-ori 3-1 cells transfected with PPFP showed that PPFP not only increased cellular proliferation and mobility but also had an anti-apoptotic effect on Nthy-ori 3-1 cells. These results demonstrated that PPFP might play an essential role in the malignant transformation of thyroid follicular cells. A number of differentially expressed proteins were identified using a proteomic approach after thyroid follicular cells were transfected with PPFP, suggesting the involvement of multiple molecular events and signaling pathways in the process of FTC tumorigenesis. These findings not only boost the understanding of the development and progression of PTC but also generate hope for both the early diagnosis and the targeted therapy for FTC. However, further research is required to determine how these differentially expressed proteins act with PPFP with respect to its role in FTC.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the National Natural Science Foundation of China (Grant numbers: 30600601, 30871189, 30972970, and 81171841).

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*

(C Li and C Duan contributed equally to this work)

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    Construction and production of the recombinant vector containing the human PPFP gene. (A) Lane 1: marker, lane 2: pEGFP-C1 vector, lane 3: pEGFP-C1 digested with XhoI and KpnI showed a band of about 4.7 kb, which is consistent with the size of the desired fragment. (B) PCR assay to identify PPFP-positive colonies (lane 1: negative control, lane 2: positive control, and lane 3: marker). Lanes 4, 6, and 9 (PPFP-positive colonies) had a 565 bp PCR fragment, which was designed to detect the presence of the PPFP gene. (C and D) Western blot detection of PPFP expression in 293T cells using an anti-mouse FLAG antibody and an anti-mouse PPAR antibody respectively. Lane 1: untransfected 293 T cells, lane 2: 293T cells transfected with empty vector, and lanes 3 and 4: 293T cells transfected with PPFP showed a band with a molecular weight of about 90 kDa, which corresponds to the size of PPFP.

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    PPFP expression in Nthy-ori 3-1 cells transfected with PPFP by green fluorescent protein detection: Abundant green fluorescence was observed by fluorescence microscopy, and more than 90% of Nthy-ori 3-1 cells stabilized and highly expressed the targeted gene after 12 days. A, B, and C represent untransfected cells, Nthy-ori 3-1vector, and Nthy-ori 3-1PPFP respectively. Row 1: fields viewed under the light microscope; row 2: fields viewed under the fluorescent microscope.

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    Physiological impact of PPFP on thyroid cells. (A) MTT assay. Nthy-ori 3-1PPFP cells had higher absorbance values at 48, 72, and 96 h compared with Nthy-ori 3-1vector and untransfected Nthy-ori 3-1 cells (*P<0.01). (B) Soft-agar colony formation assay. Nthy-ori 3-1PPFP cells formed more colonies compared with Nthy-ori 3-1vector and untransfected Nthy-ori 3-1 cells (*P<0.01). (C) Plate formation assay (1, Nthy-ori 3-1; 2, Nthy-ori 3-1vector; and 3, Nthy-ori 3-1PPFP). Nthy-ori 3-1PPFP cells had a higher colony formation rate compared with Nthy-ori 3-1vector and untransfected Nthy-ori 3-1 cells (*P<0.01). (D) Scratch wound-healing assay (1, Nthy-ori 3-1; 2, Nthy-ori 3-1vector; 3, Nthy-ori 3-1PPFP). The scratch was almost healed in Nthy-ori 3-1PPFP cells after 48 h, while scratches in Nthy-ori 3-1vector and Nthy-ori 3-1 cells were only half-healed.

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    Effect of PPFP on the cell cycle and apoptosis of Nthy-ori 3-1 cells. The number of Nthy-ori 3-1PPFP cells in G0/G1 phase decreased significantly but the number in S and G2/M phase increased significantly compared with Nthy-ori 3-1vector and Nthy-ori 3-1 cells (P<0.01). The apoptotic percentage of Nthy-ori 3-1PPFP cells was significantly less than Nthy-ori 3-1vector and Nthy-ori 3-1 cells (*P<0.01).

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    (A) Representative 2-DE maps of Nthy-ori 3-1PPFP, Nthy-ori 3-1vector, and Nthy-ori 3-1 cells. The 28 differentially expressed protein spots that were identified by MS are marked with arrows. (B) Close-up image of differentially expressed protein spots in the three groups of cells.

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    MALDI-TOF-MS analysis of differential protein spot 6. (A) The mass spectrum of protein spot 6 identified this protein as prohibitin according to the matched peaks. (B) Possible matched proteins to the spectral masses are presented as multiple bars with differential probability-based MOWSE scores.

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    (A) Representative results of western blot analysis for prohibitin, galectin-1, cytokeratin 8, cytokeratin 19, and HSP27 in Nthy-ori 3-1, Nthy-ori 3-1vector, and Nthy-ori 3-1PPFP cells. (B) Histogram of the relative changes in the expression levels of these proteins. The expression of galectin-1, cytokeratin 8, cytokeratin 19, and HSP27 was significantly upregulated in Nthy-ori 3-1PPFP cells in comparison with Nthy-ori 3-1vector and Nthy-ori 3-1 cells, whereas that of prohibitin decreased significantly in Nthy-ori 3-1PPFP cells (P<0.05).

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