PTC and adjacent non-tumor thyroid tissue specimens were collected immediately following surgical removal and stored at −80°C until use. A total of 30 pairs of fresh-frozen PTC tissues and adjacent non-tumor tissues were used for the analysis of FOXE1 gene expression by quantitative real-time PCR (qPCR) and western blotting. An additional 81 paraffin-embedded tissue blocks of PTC were obtained from the Department of Pathology, Affiliated Sixth People’s Hospital, Shanghai Jiaotong University (Shanghai, China) between 2010 and 2012, and were randomly selected for IHC analysis.

The average age of the patients from which the 81 PTC cases were derived was 49.6 years (range, 25–73 years) and included 27 males and 54 females. The study protocol and consent form were approved by the Institutional Ethics Committee of the Affiliated Sixth People’s Hospital, Shanghai Jiaotong University. All patients were informed of the aims of the study and provided written informed consent.

Total RNA was extracted from PTC and adjacent non-tumor tissue specimens using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. First strand cDNA was then synthesized using the Reverse Transcription Reagent kit (Takara Bio, Inc., Dalian, China). The qPCR assay was conducted in a 10-μl reaction mixture according to the SYBR-Green PCR kit (Takara Bio, Inc.) and analyzed in a 96-well plate using the Applied Biosystems 7500 Real Time PCR system (Applied Biosystems, Foster City, CA, USA). The primer sequences used in this study are shown in Table I. The PCR conditions were as follows: 95°C for 15 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 34 sec. β-actin was used as an internal control. Relative values of transcripts were calculated using the formula: 2^−ΔΔCt. Experiments were performed in triplicate.

To evaluate the level of FOXE1 protein expression, tissue lysates were prepared from fresh-frozen PTC and adjacent non-tumor tissue specimens by centrifugation at 12,000 × g for 20 min at 4°C. The concentration of the protein lysate was then determined using BCA reagent (Beyotime, Shanghai, China). For each sample, a volume equivalent to 20-μg protein lysate was separated by SDS-PAGE followed by transfer onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). The membrane was blocked with 5% non-fat milk for 1 h and then incubated with rabbit anti-FOXE1 monoclonal antibody (1:1000; Abcam, Cambridge, MA, USA) overnight at 4°C. After washing in Tris-buffered saline with Tween-20 to remove excess primary antibody, the blots were incubated for 1 h with a specific secondary antibody (goat anti-rabbit IgG, 1:5,000; Santa Cruz Biotechnology, Inc., CA, USA). Antibody binding was detected using the enhanced chemiluminescent reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) and measured using Kodak Scientific Imaging Systems (New Haven, CT, USA). A goat-specific monoclonal GAPDH antibody (1:5,000; Bioworld Technology, Minneapolis, MN, USA) was used as a control.

In total, 81 paraffin-embedded tissue blocks of PTC were retrieved for IHC analysis. The blocks were cut into 4-μm sections, deparaffinized with xylene and rehydrated in a graded ethanol series. Antigen retrieval was performed by boiling tissue sections in EDTA solution (1:50) for 20 min. The sections were incubated with the primary antibody, rabbit anti-FOXE1 monoclonal antibody (1:250; Abcam) overnight at 4°C. The slides were then incubated with a biotinylated secondary antibody (goat anti-rabbit IgG) at 37°C for 30 min. The slides were then stained with hematoxylin and eosin (H&E) for 2 min, dehydrated, mounted and imaged using the Microscope ScanScope (Olympus, Tokyo, Japan).

Two pathologists blinded to the identity of the specimens, examined the percentage of positively stained cells in contrast to the total section area (TSA = 100%). They also assessed the intensity of the immunostained slides and scored them as previously described (12). Based on the percentage of positive cells, the level of staining was defined as follows: 0%, negative (−); 1–33%, weak (+); 34–66%, moderate (++) and 67–100%, strong (+++). The intensity of the immunoreactions was scored as follows: 0, negative (−); 1, weak (+); 2, moderate (++) and 3, strong (+++). The total scores in the tumor and non-tumor regions were determined as the sum of the products of the positivity and intensity grades. A total score of >2.0 was considered to represent a strong positive score.

Clinicopathological parameters, including age, gender, tumor size, extra-capsular invasion, multifocality, lymph node metastasis, distant metastasis and tumor stage (TNM classification) were analyzed (13,14). Correlations between the IHC results and the clinicopathological parameters were evaluated.

The differences between the FOXE1 expression levels of PTC and adjacent non-tumor tissue specimens were determined statistically using the Mann-Whitney U test or the independent samples t-test. Correlations between FOXE1 expression and the clinicopathological parameters of PTC patients were analyzed using the Pearson’s Chi-squared test. Statistical analyses were performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant result.

Out of the 30 pairs of specimens investigated by qPCR, 26 paired results were analyzed. The average mRNA expression levels of FOXE1 were significantly higher in PTC tissues compared with adjacent non-tumor thyroid tissues (0.1575±0.0566 versus 0.0598±0.0350, P<0.01), equating to an ~2.6-fold increase in the expression of FOXE1 in PTC tissues (Fig. 1). The results also demonstrated that 65% (17 out of 26) of PTC tissues expressed higher levels of FOXE1 compared with the matched adjacent non-tumor thyroid tissues.

FOXE1 protein expression levels were also analyzed by western blotting. FOXE1 protein levels in PTC tissues were significantly higher compared with the matched adjacent non-tumor thyroid tissues. FOXE1 was more markedly expressed in 58% (15 out of 26) of PTC tissues compared with the paired non-tumor tissues (Fig. 2). Thus, these results are consistent with the data obtained by the qPCR assay.

IHC staining was performed to analyze FOXE1 expression in paraffin-embedded tissue samples of PTC. The results demonstrated that FOXE1 staining was widely expressed in all tumor regions of the 81 PTC tissue sections (100%); however, it was present in only 31 out of 81 samples in the non-tumor regions (38.3%). A higher degree of FOXE1 staining was evident in the tumor regions compared with the corresponding non-tumor tissues (Fig. 3). Additionally, FOXE1 staining was predominantly present in cell nuclei. However, in non-tumor thyroid follicular epithelial cells, FOXE1 staining was negative or exhibited a weak level of staining. Thus, FOXE1 expression is significantly elevated at the RNA and protein levels in PTC.

To examine the clinical significance of FOXE1 in PTC, samples were divided into the high and low expression groups, according to the mean value of the FOXE1 staining scores in the PTC sections (Table II). We then examined the correlation between FOXE1 and the clinicopathological parameters of PTC. Our data demonstrated that FOXE1 expression in PTC significantly correlated with the extra-capsular invasion of tumor cells, lymph node metastasis and tumor stage (P=0.048, P=0.036 and P=0.009, respectively). No significant correlation was identified between FOXE1 expression and other clinical parameters, including age, gender, tumor size, multifocality and thyroid-stimulating hormone (TSH) level. However, we demonstrated that FOXE1 expression was significantly higher in stages III/IV compared with stages I/II of PTC (P<0.01). Taken together, these data suggest that FOXE1 is useful as a prognostic index for PTC.