The present study included 159 NSCLC patients who had consented and enrolled before surgery (30 women and 121 men). Patient ages ranged from 39–83 years (median, 63.2 years). A total of 151 NSCLC sections for immunohistochemical analysis were obtained from patients with newly diagnosed NSCLC between 2005–2009, along with corresponding clinicopathological data. Eight fresh NSCLC samples and corresponding para-cancerous tissues from patients who underwent surgery but had received no chemotherapy or radiation therapy before sample collection were also collected for western blotting analysis between 2015–2016. All samples were collected at the Affiliated Hospital of Nantong University, Jiangsu Province, China. The study protocol was approved by the Human Research Ethics Committee of the Affiliated Hospital of Nantong University (Nantong, China). The collection of fresh-frozen human NSCLC tissue samples met the requirement of an institutional review board protocol approved by the Partners Human Research Committee Affiliated Hospital of Nantong University, Nantong, China). All tissue sections used for immunohistochemistry were formalin-fixed, and paraffin-embedded. Tumor staging was in accordance with the guidelines of the 7th edition of TNM staging in lung cancer (16). The 5-year actual overall survival time was calculated from the date of surgery until the date of death or last follow-up appointment. Representative 2.0-mm tissue core samples from each patient were used to conduct TMA analysis (Shanghai Outdo Biotech Co., Ltd.). All tissues for immunoblot analysis were frozen immediately after surgery, then manually homogenized with a homogenizer in RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology, Nantong, China). After centrifugation at 10,000 × g, 4°C for 15 min, the supernatant was extracted immediately for western blot analysis or stored at −80°C.

Immunostaining was performed using the avidin-biotin-peroxidase complex. Tissue sections were deparaffinized using a graded ethanol series, and endogenous peroxidase activity was blocked by soaking in 3% hydrogen peroxide (H₂O₂) for 10 min. The sections were then processed in 10 mmol/l citrate buffer (pH 6.0) and heated to 121°C in an autoclave for 20 min to retrieve the antigen. After rinsing in PBS (pH 7.2), the sections were incubated with anti-NLK (diluted 1:1,00; cat. no. ab116715) and anti-vimentin antibodies (diluted 1:1,00; cat. no. ab92547; both from Abcam) for 1 h at room temperature. All slides were processed using the peroxidase-anti-peroxidase method (Dako, Hamburg, Germany). After being rinsed in PBS, the peroxidase reaction was visualized by incubating the sections with a liquid mixture (0.02% diaminobenzidine tetrahydrochloride, 0.1% phosphate buffer solution, and 3% H₂O₂). After rinsing in water, the sections were counterstained with hematoxylin, dehydrated, and cover-slipped. Stained sections were observed under a microscope. The extent of immunostaining was evaluated and scored separately by two independent investigators with no prior knowledge of the clinical or pathological parameters of the patients. This TMA followed the Tissue Microarray System Quick-Ray manual (UT06; Unitma Co., Ltd.). For the semi-quantification of positive staining, both the intensity (0,1,2 or 3) and proportion of positive cells (0 to 100) were noted. Thus, the score ranged from 0 (no. staining) to 300 (all cells strongly stained). X-tile software (Rimm Laboratory, Yale University; http://www.tissuearray.org/rimmlab) (17) was used to divide the protein expression into two categories (low expression and high expression); high expression represented a microscopic score of 70–300, and low expression was from 0–70.

Tissues and cell protein were promptly homogenized after collection in a homogenization buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40, 5 mM EDTA, 60 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM NaF, and complete protease inhibitor cocktail (Roche Diagnostics), then centrifuged at 12,000 × g for 15 min to collect the supernatant. Protein concentrations were measured with a BioRad protein assay (BioRad Laboratories, Inc.). The supernatant was diluted in 2X SDS loading buffer and boiled for 15 min. Proteins were then separated with 10% SDS-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride filter membranes (EMD Millipore). Then, the mass of protein loaded per lane was initially set at 200 µg, and the loading amount was adjusted according to the measured protein concentration.

The membranes were blocked with 5% non-fat milk in TBST (150 mM NaCl, 20 mM Tris, 0.05% Tween-20) for 2 h at room temperature. They were then washed three times with TBST and incubated overnight with primary antibodies anti-β-actin (dilution 1:500, cat. no. 4970, Cell Signaling Technology, Inc.), anti-NLK (dilution 1:500; cat. no. ab116715), anti-β-catenin (dilution 1:500; cat. no. ab22656), anti-E-cadherin (dilution 1:500; cat. no. ab1416), and anti-vimentin (dilution 1:500; cat. no. ab92547; all from Abcam). Horseradish peroxidase-linked IgG secondary antibodies (cat. no. A0208; Beyotime Institute of Biotechnology) at 1:5,000 dilution were added for 1 h incubation at room temperature. The band intensity was measured by the ImageJ analysis system (National Institutes of Health, USA) (18) and normalized against β-actin levels. Experiments were carried out on three separate occasions.

The human NSCLC cell lines NCI-H1975, NCI-H1299, NCI-H1650, and A549 were purchased from the Shanghai Institute of Cell Biology Academia Sinica. All cancer cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, and 100 U/ml penicillin-streptomycin (Gibco-BRL; Thermo Fsiher Scientific, Inc.) at 37°C and 5% CO₂. The medium was changed after 24 h and replaced with fresh medium for transfection. Full-length NLK (Gene ID: 51701) was isolated from the human cDNA library. NLK-shRNA and control-shRNA lentiviruses were obtained from GeneChem Technologies. The shRNA target sequences were: 5′-GAATATCCGCTAAGGATGC-3′ and 5′-CAGATCCAAGAGATGGAAA-3′. A549 cells were infected with control-shRNA or NLK-shRNA lentiviruses according to the manufacturer's protocol.

A549 cells were seeded in 6-well plates (Corning, Inc.) and transfected with NLK-shRNA, control-shRNA and full-length NLK according to the manufacturer's instructions (19). When they reached ~80% confluency 48 h post-transfection, cells were serum-starved for 12 h, then scratched using the fine end of a 100-µl pipette tip. Wound healing was observed at different time-points within the scrape line. Representative scrape line images were captured using an inverted Leica phase contrast microscope (Leica DFC 300 FX) under a 20× objective lens every 24 h. Image Processing and Analysis in Java was used to measure the wound healing assays. Duplicate wells for each condition were examined, and each experiment was repeated three times.

The cell invasion assay was performed in a Transwell chamber (24-well type, 8 µm pore size; Corning, Inc.) with the BD Matrigel Basement Membrane Matrix according to the manufacturer's recommended protocol. Serum-free RPMI-1640 medium (0.5 ml) was placed in the upper chamber, and DMEM with 10% FBS was added to the bottom chambers. An equal amount (1×10⁵) of cells were plated in the upper chambers of the quadruplicate wells and incubated at 37°C for 72 h. Cells were then fixed with methanol and stained with 3% crystal violet at 37°C to visualize the nuclei. The results of three independent experiments were averaged. Image Processing and Analysis in Java was used to assess the Transwell assay.

Cells were seeded on coverslips in 24-well plates and cultured overnight. They were then washed with PBS, fixed with 4% paraformaldehyde for 1 h, and incubated at 4°C overnight with anti-β-catenin (dilution 1:100; cat. no. ab32572) and anti-vimentin (dilution 1:100; cat. no. ab92547; both from Abcam) antibodies. After three washes with PBS, the cells were incubated with Alexa Fluor-conjugated secondary antibodies (Alexa Fluor 594 (cat. no. R37117; dilution 1:200), Alexa Fluor 488 (cat. no. R37114; dilution 1:500; Molecular Probe, Inc.), and counterstained with DAPI. Fluorescence was detected using a Leica fluorescence microscope (Leica Microsystems GmbH). All assays were performed three times.

Immunoprecipitation was performed as previously described (20). Briefly, total cell lysates were incubated with an anti-NLK antibody, anti-β-catenin antibody or control IgG at 4°C overnight. Protein A/G (Sigma-Aldrich; Merck KGaA) was then added for 2 h at 4°C with gentle shaking. The precipitates were washed three times with homogenization buffer and boiled for another 15 min with SDS sample buffer followed by western blotting.

Values are presented as the means ± standard deviations (SDs) (21). Each experiment was repeated at least three times per condition. The Chi-square method was used to determine statistical significance. One-way analysis of variance (ANOVA) followed by Bonferroni post hoc test was used for the comparison of multiple groups. And SPSS 20.0 statistical software (IBM Corp.) was used for statistical analysis. Survival analysis was performed using the Kaplan-Meier method with the log-rank test. Univariate and multivariate analyses were based on the Cox proportional hazards regression model. A P-value <0.05 was considered statistically significant for all analyses.

To investigate the expression of NLK, E-cadherin and vimentin protein in NSCLC tissues, western blotting was performed in 8 pairs of fresh NSCLC and non-tumorous adjacent tissues. As presented in Fig. 1A and B, the expression levels of NLK and E-cadherin protein were significantly decreased in tumor tissues, while vimentin was upregulated compared with matched healthy tissues (P<0.05).

To further determine the association between NLK, E-cadherin and vimentin in NSCLC tissues, IHC analysis of 151 NSCLC patient samples was conducted. NLK was observed to be expressed mainly in the membrane and cytoplasm, E-cadherin was located in the membrane and vimentin was detected in the cytoplasm. NLK and E-cadherin were notably upregulated, while vimentin expression was decreased in healthy lung tissues compared with NSCLC tissues (Fig. 1C).

Statistical analysis was performed to determine the association between NLK and the clinicopathological features of NSCLC (Table I) in 151 NSCLC specimens. A total of 89 (58.9%) patients exhibited low or undetectable expression of NLK, while 62 (41.1%) presented NLK upregulation. NLK was significantly associated with TNM stage grouping (χ²=7.885, P=0.019), primary tumor (χ²=8.966, P=0.011), lymph node metastasis (χ²=19.085, P<0.001), and E-cadherin (χ²=47.111, P=0.001) and vimentin expression (χ²=52.847, P<0.001). A notable association was observed between NLK expression and patient age, sex, smoking status, tumor size and histological type.

A Cox proportional hazards model was constructed to perform univariate and multivariate analyses of all prognostic variables for the 5-year survival rate of patients with NSCLC (Table II). Univariate Cox regression analyses revealed that low NLK expression [hazard ratio (HR) 0.341, 95% confidence interval (CI) 0.219–0.531, P<0.001], lymph node metastasis (HR=5.732, 95% CI=3.732–8.803, P<0.001), advanced TNM stage (HR=3.214, 95% CI=2.341–3.941, P<0.001), and E-cadherin (HR=0.239, 95% CI=0.158–0.363, P<0.001) and vimentin expression (HR=5.820, 95% CI=3.644–9.295, P<0.001) were significantly associated with the survival of patients with NSCLC for all variables. In addition, multivariate analysis indicated NLK expression (HR=0.387, 95% CI=0.248–0.605, P<0.001) and TNM (HR=3.037, 95% CI=2.341–3.941, P<0.001) as independent prognostic factors in patients with NSCLC. Collectively, the results inidcated that reduced expression of NLK could be a potential indicator of poor prognosis in NSCLC.

Kaplan-Meier survival curves further confirmed that the reduction of NLK protein expression was significantly associated with poor prognosis in patients with NSCLC (log-rank test, P<0.001; Fig. 2A). In accordance with NLK expression, the survival curve demonstrated a high degree of discrimination, indicating that the score standard for assessing NLK staining was suitable and that NLK was a reliable prognostic factor. Kaplan-Meier analysis also revealed that patients with lymph node metastasis had lower overall survival rates than patients without metastasis (log-rank test, P<0.001; Fig. 2B). Additionally, patients with lymph node metastasis and upregulated NLK expression demonstrated significantly better prognosis than those with lymph node metastasis and downregulated NLK expression (P<0.05; Fig. 2B). Patients with increased NLK expression and no metastasis had a significantly higher overall survival rate than those with downregulated NLK expression and no metastasis (P=0.007; Fig. 2D).

To investigate the function of NLK in the carcinogenesis of NSCLC, NLK expression was determined in four NSCLC cell lines. Western blotting demonstrated upregulated NLK expression in A549 cells and downregulated NLK expression in H1650 cells compared with H1975 and H1299 and (Fig. 3A and C). Subsequently, cells were transfected with NLK-shRNAs to downregulate the expression of NLK. As presented in Fig. 3B and D, western blotting revealed notable reductions in NLK protein expression in cells transfected with NLK-shRNAs compared with non-transfected or control-shRNA-transfected cells. In addition, a Flag-NLK vector was constructed to overexpress NLK in A549 cells.

In advanced stages of cancer or postoperative recurrence, NSCLC can be characterized by increased metastatic potential. Therefore, wound healing and Transwell assays were performed to investigate whether NLK affects NSCLC cell migration and invasion. In the wound healing assay, it was observed that the migration ability of the NLK-Flag group was decreased compared with the control group. Conversely, cells transfected with NLK-shRNA exhibited increased metastatic potential compared with the negative group; the migration ability of the NLK-shRNA group was promoted by reducing the expression of NLK (Fig. 4A). Similarly, the Transwell assay revealed that upregulating the expression of NLK suppressed cell migration compared with the control and negative groups (Fig. 4B).

To determine whether NLK affects EMT in NSCLC cells, immunofluorescence staining was conducted. A549 cells were transfected with NLK-shRNA and Flag-NLK; alterations in the expression of EMT markers were then analyzed. As presented in Fig. 5A, upregulating NLK expression via a Flag-NLK vector resulted in increased expression of the epithelial marker E-cadherin in the cell membrane, while the expression of the mesenchymal marker vimentin was decreased in the cytoplasm. Following NLK downregulation by NLK-shRNA, E-cadherin expression was notably undetected in the cell membrane, while vimentin expression was upregulated in the cytoplasm.

Western blotting revealed that upregulation of NLK led to increased E-cadherin expression, while that of vimentin and β-catenin was downregulated. Similarly, the expression of vimentin and β-catenin was increased, while that of E-cadherin was decreased following NLK downregulation (Fig. 5B and C). Immunoprecipitation of A549 cell protein revealed that NLK could co-immunoprecipitate with an anti-β-catenin antibody, but not under control conditions, indicating a naturally occurring interaction between endogenous NLK and β-catenin in vivo and vice versa. Thus, this could be a novel mechanism underlying the role of the tumor suppressor gene NLK in the progression of NSCLC (Fig. 5D and E).