Primary tumors and corresponding adjacent healthy tissues were obtained from 65 patients, including 40 men and 25 women, with a mean age 61.7 years, who were diagnosed in Department of Thoracic Surgery, Hubei Cancer Hospital (Wuhan, China) between March 2010 and July 2011. All the tumors used in this study were squamous cell carcinoma, and tumor stages were confirmed by pathologists according to the criterion of Union for International Cancer Control. The clinical characteristics were obtained from medical records. This study was approved by the ethical committees of Hubei Cancer Hospital and written informed consent was obtained prior to surgery. All tissue specimens were surgically resected and immediately flash-frozen in liquid nitrogen, and stored at −80°C.

The H520 and SK-MES-1 LSCC cell lines and the BEAS-2B healthy human bronchial epithelial cell line were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in the conditions as recommended (22). The cells were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum and 100 U/ml penicillin sodium at 37°C in an humidified atmosphere of 5% CO₂. To construct a vector stably expressing PTPRO, a pcDNA 3.1-Hemagglutinin A (HA)-tagged vector (Invitrogen; Thermo Fisher Scientific, Inc.) was purchased and used in this study. The cDNA encoding the complete coding region of human PTPRO cDNA was obtained from GeneBank (NM_030667.2). The HA label was introduced to protein C in the vector, and the E.coli strain of DH5a was also preserved in a laboratory at Hubei Cancer Hospital. A pcDNA-PTPRO-HA expression vector was established using a traditional method (23).

Total amounts of DNA (2 µg) were extracted from cells and tissues using a DNeasy Blood & Tissue kit (Qiagen GmbH, Germany) according to manufacturer's protocol. The quantity of DNA was tested by a NanoDrop spectrophotometer (Thermo Fisher Scientific, Inc.), and stored at −80°C until use. All DNA samples were treated using an EpiTect Bisulfite kit (Qiagen GmbH), and converted-DNA was used as a template in next step analysis. For bisulfite sequencing-polymerase chain reaction (BSP-PCR) analysis, the PCR reaction was conducted in 50 µl solution containing converted-DNA (200 ng), dNTP (200 nM for each), forward and reverse primers (50 pM), and Taq DNA Polymerase (2.5 U; Thermo Fisher Scientific, Inc.). The 4 µl PCR products (0.1 µg/µl) were ligated into the PMD18T vector. Recombinant vectors were then transformed to E. coli and the positive colonies were selected for sequencing. As inactivation of tumor suppressor genes may occur via hypermethylation of CpG islands upstream of the transcription start site, the present study selected a target region spanning from −405 to −74 (containing 23 CpG sites) in the BSP analysis, and the primers for PTPRO (forward: 5′-GAGGTTGTTGTTATTTTATGGG-3′; reverse: 5′-TAAAACTACAACCTCAAACCCT-3′) were used. Methylation specific PCR (MSP) assays were performed using a Techne-512 system (Techne, Staffordshire, UK) and included an initial incubation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 56°C for 20 sec, extension at 72°C for 20 sec and a final extension step at 72°C for 10 min. One pair of primers for methylated PTPRO (forward: 5′-TGTTGTTAGAGGATTACGGC-3′; reverse: 5′-CAAAAACGTACCAAACGCTA-3′) and unmethylated PTPRO (forward: 5′-TTTTGTTGTTAGAGGATTATGGT-3′; reverse: 5′-TCCAAAAACATACCAAACACTAC-3′) were used to amplify methylation and unmethylation alleles of PTPRO, respectively. Quantitative (Q) MSP was performed to detect the methylation levels of PTPRO in tumors and matched healthy tissue, and the quantity of methylated PTPRO was normalized to β-actin. Briefly, 10 ng bisulfite-coverted DNA was used in the QMSP assay in 384-well plates with a LightCycler480 system (Roche Diagnostics, Basel, Switzerland). The PCR reaction included an initial incubation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 30 sec, 58°C for 10 sec, 72°C for 20 sec and 80°C for 1 sec. Each plate consisted of clinical samples, water blanks and a positive control. Serial dilutions of the H520 PTPRO methylation-positive cell line were used for constructing the calibration curve. QMSP analyses yield values are expressed as ratios between two absolute measurements (PTPRO:β-actin ×100) (24). Each sample was analyzed in duplicate.

For the demethylation assay, 1×10⁵ H520 and SK-MES-1 cells were seeded into 6-well plates, cultured for 24 h and treated with 0, 2.5 or 5 µM 5-AZA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). Fresh medium containing 5-AZA was changed every 24 h for 3 days and the treated cells were harvested for reverse transcription-quantitative (RT-q) PCR analysis.

Total RNA was isolated from cells using TRIzol^® regent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Total RNA was reverse transcribed into cDNA, and this procedure was performed once using the PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan), which was subsequently used for RT-qPCR analysis using an ABI 7500 fast Sequence Detector (ABI, Carlsbad, CA, USA). The reaction conditions were as follows: an initial predenaturation step at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 72°C for 30 sec. β-actin served as the endogenous control for detection of mRNA expression levels (25). Relative quantification analysis was performed using the 2^−ΔΔCq method (25). The following primers for PTPRO (NM_030667.2, transcript variant 1) were used: Forward, 5′-ACTGCCCCTTATCCACCTCA-3′ and reverse, 5′-TGTTGCCCGAGGGAATTTCA-3′.

Cell proliferation was assessed by MTT assay. H520 and SK-MES-1 cells were seeded into 96-well culture plates at a density of 1.5×10³ per well. after 1–6 days, cells were incubated with 20 µl MTT (5 mg/ml, Sigma-Aldrich; Merck KGaA) for 4 h at 37°C. The cell medium was removed and 150 µl dimethyl sulfoxide was added to each well. The absorbance was measured at a wavelength of 490 nm using a microtiter plate reader (Tecan Schweiz AG, Männedorf, Switzerland). To investigate clonogenic ability, cells were transfected with PTRPO or an empty vector, and subsequently seeded into 6-well (200 cells per well) plates. The culture medium was replaced every 3 days. After 2 weeks, the medium was removed and the plates were washed twice using PBS. The colonies were fixed in methanol at −20°C for 5 min, stained with 0.1% crystal violet (Sigma-Aldrich; Merck KGaA), and counted using an inverted microscope (Nikon Corporation, Tokyo, Japan) in five random fields.

Xenograft experiments were performed to evaluate the tumorigenicity of H520 cells transfected with PTPRO or an empty vector. Briefly, 1×10⁷ H520 cells resuspended in 200 µl PBS were subcutaneously injected into the flanks of athymic nude male mice (n=5; age, 4 weeks), which were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Animal experiments were conducted in accordance with guidelines approved by the Institutional Animal Care and Use Committee of Hubei Cancer Hospital. The mice were maintained at a temperature of 18–22°C and humidity of 50–60% under 12:12 h light-dark cycle with had free access food and water. Each mouse was injected in left flank with the PTPRO vector, and in the right flank with the empty vector. A total of 28 days after injection, tumors were harvested, weighed and assayed for mRNA expression.

Equal amount of protein extracts were lysed using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA). Cells were centrifuged in a microcentrifuge at 12,000 × g for 15 min at 4°C to collect the supernatant. Protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). A total of 30 µg protein were separated by 10% SDS-PAGE and then transferred onto polyvinyl fluoride membranes (Merck KGaA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline for 1 h, and incubated with anti-PTPRO (1:1,000; catalog no. sc-365354; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-β-actin (1:2,000; catalog no. 4970, Cell Signaling Technology, Inc., Danvers, MA, USA) primary antibodies at 4°C overnight. The membranes were washed three times with Tris-buffered saline containing 0.1% Tween and incubated for 2 h at room temperature with a horseradish peroxidase-conjugated goat anti-rabbit (catalog no. 7074; 1:1000; Cell Signaling Technology, Inc.) or anti-mouse secondary antibody (catalog no. sc-516102, 1:2000, Santa Cruz Biotechnology). Proteins were visualized using a Bio-Rad ChemiDoc Imaging system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Statistical analysis was performed using Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS version 16.0 (SPSS, Inc., Chicago, IL, USA). Data are expressed as the mean ± standard error of three independent experiments. Two-way ANOVA followed by Bonferroni correction was used to determine the statistical significance when the number of groups was more than three. The methylation and expression levels of PTPRO in tumors and healthy controls were compared using paired-samples t-test. The overall survival of LSCC patients were analyzed using the Log-rank test. All tests were two sided and P<0.05 was considered to indicate a statistically significant difference.

As the methylation status of PTPRO in LSCC cells is unclear, a BSP assay was performed in H520 and SK-MES-1 cells, and BEAS-2B cells served as a healthy control. As presented in Fig. 1A and B, the CpG island in the 1st exon was hypermethylated in H520 and SK-MES-1 cells, while partially methylated in BEAS-2B cells. Following this, the methylation status of PTPRO in LSCC tissues was assessed using the MSP method. The intensity of methylated alleles was noticeably increased compared with unmethylated alleles (Fig. 1C). To assess if a demethylation agent could restore transcriptional activity, LSCC cells were treated with 0, 2.5 or 5 µM 5-AZA for 72 h. The mRNA expression levels of PTPRO were significantly increased following 5-AZA treatment in all groups except the 2.5 µM treatment group in SK-MES-1 cells (Fig. 1D). These data demonstrated that the CpG island of PTPRO exon 1 was hypermethylated in LSCC cells and tissues, suggesting that the epigenetic regulation of PTPRO may serve a role in LSCC tumorigenesis.

To understand the methylation and mRNA levels of PTPRO, QMSP and RT-qPCR analyses were performed in LSCC and matched healthy tissues. The methylation levels of PTPRO were significantly increased in LSCC tissues compared with healthy controls (0.0438±0.0263 vs. 0.0381±0.0264; P=0.0005; Fig. 2A). The mean methylation level of PTPRO in tumors was used as cut-off to divide cases into two groups (high- or low-methylation). A low level of PTPRO methylation was significantly associated with high overall survival probability in LSCC patients (P=0.027; Fig. 2B). Furthermore, the mRNA expression levels of PTPRO were markedly reduced in tumors compared with healthy tissues (66.87±12.11 vs. 68.24±11.81; P=0.005; Fig. 2C), and low expression of PTPRO (<the mean of mRNA levels of PTPRO in tumors) was associated with poor prognosis of patients (P=0.002; Fig. 2D). Pearson correlation coefficient analysis identified an inverse correlation between methylation and mRNA levels of PTPRO in tumors (Pearson r=−0.409; P=0.0007; Fig. 2E).

Subsequently, the present study verified whether the methylation or expression of PTPRO was associated with clinicopathological features of patients. As presented Table I, mRNA expression levels of PTPRO were significantly reduced in advanced tumors compared with early-stage tumors (P=0.042). The methylation or mRNA levels of PTPRO were not associated with other clinical parameters (Table I). Univariate analysis demonstrated that the high methylation of PTPRO, low PTPRO mRNA, smoking, advanced tumor stage, higher T stage and lymph node metastasis were predictors of poor prognosis for patients, whereas only mRNA expression levels of PTPRO (P=0.005) and higher TNM stage (P=0.001) were identified as significantly independent prognostic factors in multivariate analysis, with relative risks of 2.826 and 3.714, respectively (Table II). Taken together, these data suggested that epigenetically downregulated PTPRO may be involved in LSCC development and may be a potential prognostic marker. Detailed clinical and molecular data of the patients are presented in Table III.

The expression levels of PTPRO in transfected cells and control cells were detected. The mRNA (Fig. 3A) and protein (Fig. 3B) expression levels of PTPRO were upregulated in H520 and SK-MES-1 cells transfected with stably expressing PTPRO vectors in comparison with control cells and non-transfected cells. The results of MTT demonstrated that when compared with the empty vector group (control) and non-transfected group (untreated), the proliferation of LSCC cells was significantly inhibited in overexpressing PTPRO cells (Fig. 3C). Colony formation assay was performed to evaluate the effect of PTPRO on LSCC cells. As a result, the number of colonies were significantly reduced in H520 and SK-MES-1 cells transfected with PTPRO expressing vectors, compared with control and untreated cells (Fig. 3D). These in vitro analyses demonstrated the inhibitory effect of PTPRO on cell viability.

The inhibitory role of PTPRO was further confirmed using a xenograft model of H520 cells in nude mice. As expected, there was a significant reduction in tumor volume and weight in the PTPRO overexpression group compared with the empty vector group (Fig. 4A and B). In addition, mRNA expression levels of PTPRO were upregulated in tumors injected with PTPRO vectors (Fig. 4C).