Paired NSCLC samples and adjacent normal tissues were obtained from 28 patients who underwent primary surgical resection to treat NSCLC in the Department of Thoracic Surgery, Jinling Hospital, Nanjing University School of Medicine (Nanjing, China) between July 2014 and September 2014. The specimens were collected during surgery and were kept frozen in liquid nitrogen until RNA and protein extraction. In addition, 62 patients with NSCLC with clinicopathological data and follow-up information (61/62) who underwent surgical resection between June 2007 and November 2008 were enrolled, and their paraffin-embedded specimens were collected from the Department of Pathology, Jinling Hospital, for immunohistochemical staining. Follow-up lasted until February 2015, with a median follow-up period of 84.5 months for living patients (range, 75–94 months). None of these patients received chemotherapy or radiotherapy prior to surgery. The present study was reviewed and approved by the Institutional Review Board of Jinling Hospital, and all patients signed an informed consent form prior to undergoing surgery.

The gene search function of Oncomine^® Platform (www.oncomine.org) was used to analyze the mRNA expression of PYCR1 in NSCLC tissues relative to their normal controls (14). Gene lists based on fold change were obtained from six NSCLC datasets which were named by the first author and numbers of patient as follows: Selamat (116), Su (66), Stearman (39), Hou (156), Beer (96) and Bhattacharjee (203).

All NSCLC cell lines (A549, SPC-A1, H1703, H1299, PC9, H1915, SK-MES-1) were obtained from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) except for SPC-A1, which was cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (Hyclone; GE Healthcare, Chicago, IL, USA), 100 U/ml penicillin and 100 mg/ml streptomycin at 37.8°C in a humidified atmosphere containing 5% CO₂.

Total RNA was extracted from frozen tissues or cultured cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Total RNA was reverse transcribed to cDNA using a PrimeScript^™ RT Master Mix (Perfect Real Time) cDNA synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. Amplifications were performed in a quantitative real-time PCR machine (Agilent Technologies, Inc., Santa Clara, CA, USA) using SYBR Premix Ex Taq (Takara Biotechnology Co., Ltd.). The PCR cycling conditions were conducted as follows: 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 31 sec. The primer sequences used were as follows: Human β-actin forward, 5′-AGCGAGCATCCCCCAAAGTT-3′; and reverse, 5′-GGGCACGAAGGCTCATCATT-3′ (15); human PYCR1 forward, 5′-ACACCCCACAACAAGGAGAC-3′; and reverse, 5′-CTGGAGTGTTGGTCATGCAG-3′ (16). All samples were loaded in triplicates. Relative mRNA expression levels were compared via the 2^−ΔΔCq method or log-transformed (17). The data were analyzed using MxPro qPCR software v1.2 (Agilent Technologies, Inc.).

Total protein was extracted from frozen tissues or cells using RIPA lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a protease inhibitor cocktail (Roche, Basel, Switzerland), at 4°C for 20 min, and centrifuged at 14,000 × g, for 10 min at 4°C. A total of 50 mg of protein (5 mg/µl sourced from the tissues and 2.5 mg/µl sourced from the cells) were separated on 12% SDS-PAGE and blotted onto polyvinylidene fluoride Immobilon-P membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 5% milk solution for 2 h at room temperature, the membranes were incubated with primary antibodies against PYCR1 (cat. no. ab150347; dilution, 1:1,000; Abcam, Cambridge, MA, USA), β-tubulin (cat. no. 2128; dilution, 1:1,000), cyclin D1 (cat. no. 2978; dilution, 1:1,000), B-cell lymphoma-2 (Bcl-2; cat. no. 2870; dilution, 1:1,000), B-cell lymphoma-extra large (Bcl-xl; cat. no. 2764; dilution, 1:1,000) and BCL2 associated X, apoptosis regulator (Bax; cat. no. 5023; dilution, 1:1,000), all purchased from Cell Signaling Technology, Inc., Danvers, MA, USA, overnight at 4°C. The following day, membranes were probed with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (dilution, 1:10,000; cat. no. 7074; Cell Signaling Technology, Inc., Danvers, MA, USA), for 2 h at room temperature. The bands were detected using an Immobilon Western enhanced chemiluminescence HRP substrate kit (cat. no. WBKLS0500; EMD Millipore).

Sections of formalin-fixed and paraffin-embedded specimens (5-µm thick) were deparaffinized with xylene and rehydrated in a descending ethanol series, 100, 95, 70, 50 and 30%. Heat-induced antigen retrieval was performed by submerging slides in 0.01 mol/l sodium citrate buffer (pH 6.0), and boiling in a microwave oven for 10 min. The sections were treated with 10% normal goat serum (cat. no. ZLI-9021; ZSJQ-BIO, Beijing, China) to block non-specific binding, for 30 min at room temperature. Subsequently, the sections were incubated at 4°C overnight with the aforementioned primary rabbit anti-PYCR1 antibody (1:200; Abcam). The following day, slides were incubated with HRP-conjugated anti-rabbit IgG secondary antibody (cat no. PV-9001; ZSJQ-BIO; dilution, 1:50), at room temperature for 30 min. Finally, the sections were stained using 3,3′-diaminobenzidine for 10 sec-1 min at room temperature, and counterstained with 0.5% hematoxylin for 1 sec at room temperature. Subsequently, slides were dehydrated, and mounted.

IHC staining was independently scored by two pathologists from the Department of Pathology, Jinling Hospital, without any prior knowledge of patient characteristics, and any discrepancy was solved by consensus review. Immunostaining was assessed in five fields of view for each sample, under a light microscope (Carl Zeiss AG, Oberkochen, Germany), at ×200 magnification. The presence of brown staining within cells was considered to be positive staining for PYCR1. Scores representing the percentage of positive cells were as follows: 0, ≤5%; 1, 6–20%; 2, 21–50% and 3, ≥50%. Staining intensity was scored as follows: 0, negative staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The product of the two scores was used as the total staining score. In the following analysis, the expression levels were divided into two groups based on the final scores: Low expression (<3.5) and high expression (≥3).

A549 (2×10⁵ per well) and H1703 (1.5×10⁵ per well) cells were cultured for 1 day until they reached 70–90% confluence. Cells were transfected with PYCR1-specific small interfering (si)RNA or control siRNA using the Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The sequences of the siRNAs used were as follows: Control siRNA sense, 5′-UUCUCCGAACGUGUCACGUTT-3′; and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′; siRNA-PYCR1 sense, 5′-GCCACAGUUUCUGCUCUCATT-3′; and antisense, 5′-UGAGAGCAGAAACUGUGGCTT-3′.

Cell proliferation was measured using the CCK-8 cell proliferation kit (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) and 5-ethynyl-2′deoxyuridine (EdU) assay kit (Guangzhou Ribobio Co., Ltd., Guangzhou, China), respectively. For the CCK-8 assay, siRNA-PYCR1 or control siRNA-transfected cells were cultured in a 96-well plate at a density of 3,000 cells per well. CCK-8 solution (20 µl) was added to each well after 0, 24, 48, 72 and 96 h. Cells were incubated at 37°C for 2 h, and the absorbance of samples was recorded at 450 nm using an epoch microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). All the experiments were performed in triplicate.

For the EdU incorporation assay, dissociated cells were exposed to 50 mM EdU (Guangzhou RiboBio Co., Ltd.) for 2 h at 37°C. Following fixation with 4% formaldehyde for 15 min and permeabilization with 0.5% Triton X-100 for 10 min at room temperature, the cells were incubated with 1X Apollo reaction cocktail (Guangzhou RiboBio Co., Ltd.), 100 µl/well for 30 min. Then, the cells were stained with Hoechst 33342 for 30 min at room temperature and visualized in 3 fields of view/well under a fluorescence microscope (Carl Zeiss). The EdU incorporation rate was expressed as the ratio of EdU-positive cells to total Hoechst 33342-positive cells.

SiRNA-PYCR1 or control siRNA-transfected cells were seeded into a 6-well culture plate at a density of 1,500 cells per dish. Cells were maintained in DMEM containing 10% FBS for 2 weeks and stained with crystal violet for 20 min at room temperature, for colony counting. The visible colonies were manually counted with the naked eye. All experiments were done in triplicate.

Cells, cultured at a density of 1×10⁵/assay, were trypsinized into single cell suspensions and fixed with 70% ethanol for 30 min on ice. Then, the cells were stained with propidium iodide (PI) using the CycleTEST TM PLUS DNA reagent kit (BD Biosciences, San Jose, CA, USA) and analyzed using a FACS Calibur flow cytometer (BD Biosciences) equipped with BD CellQuest software v6.1 (BD Biosciences). For apoptosis analysis, cultured cells were harvested by trypsinization and were double stained with Annexin V-fluorescein isothiocyanate/PI apoptosis detection (BD Biosciences) according to the manufacturer's protocol, and analyzed with the aforementioned flow cytometer and software.

Statistical evaluation was performed using SPSS 19.0 software (IBM Corp., Armonk, NY, USA). Two-group comparisons were performed using the Student's t-test. χ² and Fisher's exact tests were used to analyze the associations between PYCR1 expression and clinicopathological features. Kaplan-Meier survival analyses and Cox's proportional hazards models were utilized to determine the association between PYCR1 expression and survival. P<0.05 was considered to indicate a statistically significant difference.

To investigate the differences between PYCR1 transcript levels between NSCLC tumor tissues and normal tissues, 28 paired samples of fresh frozen normal tissues and tumor tissues were randomly selected, and the mRNA expression levels examined using RT-qPCR. The results revealed that 16/28 patients (57.1%) demonstrated higher PYCR1 mRNA expression levels in NSCLC specimens compared with normal tissue specimens (≥2-fold change; Fig. 1A). Overexpression of PYCR1 in lung cancers was further supported by data analysis from the Oncomine platform, which revealed higher PYCR1 mRNA expression levels in lung adenocarcinoma tissues, large cell lung carcinoma tissues and squamous cell lung carcinoma tissues than in normal lung tissues. In 4 paired specimens, PYCR1 was overexpressed in all lung tumor samples compared with normal tissues, as detected by western blot (Fig. 1B).

To further study whether PYCR1 protein levels were associated with clinicopathological features and prognosis of patients with NSCLC, 62 paraffin-embedded archived NSCLC tissues were examined using immunohistochemical staining. Representative examples of tumor tissues with either strong or weak staining of PYCR1 are presented in Fig. 1C. The associations between PYCR1 cytoplasmic staining in tumor cells and the clinicopathological features of the 62 patients with NSCLC were further analyzed (Table I). There was no significant association between PYCR1 expression and any clinicopathological characteristic. However, there was a trend towards higher PYCR1 protein expression in tumor specimens with a larger size (>4 cm). Clinicopathological characteristics and PYCR1 protein expression were also analyzed using Cox's univariate and multivariate hazard regression models to evaluate whether high PYCR1 expression was an independent risk factor of poor prognosis (Table II). Kaplan-Meier analysis demonstrated that poor overall survival was associated with lymph node metastasis (P=0.010), tumor-node-metastasis (TNM) stage (P=0.010) and PYCR1 expression (Fig. 1D; P=0.01). For multivariate survival analysis, TNM stage was associated with overall patient survival [hazard ratio (HR): 1.926, 95% confidence interval (CI): 1.149–3.227, P=0.013]. High PYCR1 protein expression was also revealed to be an independent risk factor for poor prognosis (HR: 2.48, 95% CI: 1.164–5.286, P=0.019).

In addition, the mRNA and protein levels of PYCR1 were examined in seven NSCLC cell lines by qPCR and western blotting, respectively (Fig. 1E and F). The results demonstrated that PYCR1 expression level is highly variable in NSCLC cell lines. According to the results, two NSCLC cell lines with high PYCR1 expression (SPC-A1 and H1703) were selected to perform further in vitro investigations.

As demonstrated by the aforementioned results, tumors with a larger size tended to have relatively high PYCR1 expression, and overexpression of PYCR1 may be involved in survival. Therefore, the influence of PYCR1 expression on the proliferation of human NSCLC cells was studied. SiRNA was used to specifically knock down PYCR1 expression in SPC-A1 and H1703 cells (Fig. 2A and B). PYCR1 knockdown reduced SPC-A1 and H1703 cell proliferation, as assessed using a CCK-8 cell proliferation kit (Fig. 2C and D). The colony-forming ability of the population was also significantly attenuated following knockdown of PYCR1 (Fig. 2E and F). In addition, EdU incorporation assays were performed to explore the effect of PYCR1 knockdown on DNA replication. Following transfection with siRNA-PYCR1, the percentage of EdU-positive cells was decreased by 21% in SPC-A1 and 71% in H1703 cells (Fig. 2G and H). These data revealed that PYCR1 is involved in NSCLC cell proliferation.

To further examine the function of PYCR1 knockdown in cell cycle and apoptosis, the cell cycle and apoptosis were analyzed in siRNA-PYCR1 or control siRNA-transfected SPC-A1 and H1703 cells using flow cytometry. In si-PYCR1-transfected SPC-A1 cells, the proportion of cells in the G1 phase increased by 14.6% and the proportion of cells in S phase decreased by 21.6% (Fig. 3A and B). The altered proportion of G1 and S phase cells was more apparent in siRNA-PYCR1-transfected H1703 cells (Fig. 3A and C). PYCR1 knockdown also induced apoptosis of SPC-A1 cells, in particular late apoptosis (Fig. 3D and E). In H1703 cells, although there was no significant difference in the total apoptosis rate between si-PYCR1-transfected cells and control cells, the late apoptosis rate was increased following PYCR1 silencing (Fig. 3D and F). Taken together, these results demonstrated that the loss of PYCR1 results in the arrest of the cell cycle in the G1 phase and the induction of apoptosis in NSCLC cells.

In order to investigate the molecular mechanism underlying the influence of PYCR1 on the cell cycle and apoptosis in NSCLC, key associated regulators were focused on. The cyclin D family are a group of closely related G1 cyclins, and of these cyclin D1 exhibits a more widespread function in human cancers compared with others (18). Thus, it was hypothesized that PYCR1 may promote the cell cycle from the G1 phase into the S phase by regulating the expression of cyclin D1. To test this hypothesis, the expression levels of cyclin D1 following PYCR1 silencing were examined by western blot. The results revealed a significant decrease of cyclin D1 expression in siRNA-PYCR1-transfected SPC-A1 and H1703 cells (Fig. 4A and B).

As for apoptosis, the Bcl-2 family is essential to mitochondrial-controlled apoptosis. As PYCR1 is located on the surface of mitochondrion (19), it was hypothesized that PYCR1 may inhibit apoptosis through its connection to the Bcl-2 family. The results of the present study demonstrated that, following PYCR1 knockdown, Bcl-2 expression levels were significantly decreased in SPC-A1 and H1703 cells (Fig. 4A and C). There was also a decline in Bcl-xl expression in the two cell lines (Fig. 4A and D). The Bcl-2/Bax ratio was also decreased, although Bax expression was not affected by the knockdown of PYCR1 (Fig. 4A, E and F).