NSCLC tissue samples were collected from 123 patients from The First Affiliated Hospital of Nanchang University (Nanchang, China) in 2010 and 2013, and formalin fixed (Sigma-Aldrich, St. Louis, MO, USA) for immunohistochemical staining. Age-matched normal tissue samples from 18 patients with primary NSCLC were used as a control. Tissue samples were stored and ground in liquid nitrogen to isolate total RNA and protein. Written informed consent was provided by all patients who participated in the study. The study was approved by the ethics committee of The First Affiliated Hospital of Nanchang University (Nanchang, China) and protocols were performed according to their ethical guidelines.

The lung squamous cell line YTMLC-9, carcinoma cell lines A549, SPC-A-1, LTEP-a-2, GLC82, A2 and PC-9 as well as large-cell lung carcinoma cell lines NCI-H460, 95C and 95D were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium (Invitrogen Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Gibco-BRL, Invitrogen Life Technologies), 100 IU/ml penicillin and 100 mg/ml streptomycin (Life Technologies, Carlsbad, CA, USA), maintained at 37°C in 5% CO₂ atmospheric air. The cells were cultured to 80% confluence prior to transfection with recombined eukaryotic or empty vectors using Lipofectamine^® 2000 (Invitrogen Life Technologies) according to the manufacturer’s instructions. For the transfection of cells, the pZERO-mcs mammalian expression vector was used (Invitrogen Life Technologies). This vector contains a hybrid EF1a/HTLV promoter, allowing efficient transcription of the recombinant DKK1-cDNA, and the puromycin resistance gene for easy selection of the transfected cells. The full-length sequence of DKK1 cDNA was isolated from the recombinant TA plasmid used for subcloning (Invitrogen Life Technologies) with the following primers: Forward, 5′-CAAGGGGATCCCCCTGCAGTCAGGACTCTGGGAC-3′ and reverse, 5-GTGTTCTGCTAGCTAGGTATTATTAATTTATTGGAAAC-3 (including BamHI and NheI sites). The amplified DKK1 full-length cDNA was subsequently subcloned into the pZERO-mcs expression vector. The PCR product was gel-purified and digested with BamHI and NheI, then ligated into the plasmid pZERO-mcs, resulting in the recombinant plasmid pZERO-mcs-Dkk1. Transfection of the cells with either the DKK1-cDNA or an empty vector was performed using Lipofectamine 2000 (Invitrogen Life Technologies), according to the manufacturer’s instructions.

The protein expression levels of DKK1 were evaluated by western blot analysis. Proteins extracted from NSCLC specimens and cell lines were used for this analysis. Tissue samples and cells were lysed in radioimmunoprecipitation buffer (Cell Signaling Technology, Danvers, MA, USA) at 4°C for 30 min. The lysate was incubated on ice for 20 min, followed by sonication for 30 sec, after which the lysate was incubated on ice for a further 15 min. The lysate was then centrifuged at 10,000 x g for 10 min in a microcentrifuge tube, and the supernatant was maintained at −80°C. The protein concentration was quantified using the Bradford method (16). The soluble proteins (10–20 µg) were separated by 10% SDS-PAGE and blotted onto polyvinylidene fluoride membranes (Sigma-Aldrich). Subsequently, the membranes were blocked with 5% nonfat dry milk in 1X Tris-buffered saline and Tween 20 buffer (TBST) at room temperature for 1 h, prior to incubation with rabbit anti-human DKK1 polyclonal antibody (1:1,000; cat. no. LS-A2867; LifeSpan Biosciences, Inc., Seattle, WA, USA) and rabbit polyclonal anti-β-actin (1:1,000; cat. no. A2066; Sigma-Aldrich) at 25°C for 90 min. Following rinsing with TBST containing 3% bovine serum albmin (BSA; Bio-Rad Laboratories, Inc., Hercules, CA, USA), the membranes were incubated with secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature and the immunostained bands were subsequently visualized using enhanced chemiluminescence (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). The intensity of each band was normalized to β-actin, the internal control, and the relative intensities were analyzed with ImageJ 1.0 software (National Institutes of Health, Bethesda, MD, USA).

Total RNA was extracted from the NSCLC cells using Quick-RNA™ MicroPrep solution (Zymo Research Corp., Orange, CA, USA) according to the manufacturer’s instructions. Subsequently, the purified total RNA was reverse transcribed with the iScript™ Reverse Transcription supermix for PCR (Bio-Rad Laboratories, Inc.) according to the manufacturer’s instructions. The reverse transcribed RNA was subjected to PCR using the SsoFast™ EvaGreen^® supermix (Bio-Rad Laboratories, Inc.). Primer sequences were designed using the OligoPerfect™ Designer 1.0 software (Invitrogen Life Technologies), according to the manufacturer’s instructions for optimal primer design, and were synthesized commercially (Generay Biotech Co., Ltd., Shanghai, China). The primer sequences used in the present study are listed in Table I. The PCR cycling conditions were as follows: Initial densturation at 35°C for 30 sec, followed by 30 cycles at 95°C for 30 sec, 55°C for 60 sec and 68°C for 2 min, and final extension at 68°C for 10 min. Each reaction was performed in triplicate and three independent experiments were conducted. A standard curve was constructed using serial dilutions of a reference sample and was included in each run to correct potential variations in amplification efficiency. The relative copy numbers were obtained from the standard curve and normalized to the values for β-actin. The fold-change in expression was calculated using the 2^−∆∆CT method.

Histopathological analyses were performed using immunohistochemical and immunofluorescent staining methods. The NSCLC samples collected from patients had previously been embedded in paraffin blocks. The paraffin-embedded sections were deparaffinized with xylene (Sigma-Aldrich) and rehydrated in graded ethanol solutions. Endogenous peroxidase activity was blocked by incubation with 3% H₂O₂ (Sigma-Aldrich) for 15 min at room temperature. Sections were subsequently heated with 0.01 M citrate (pH 6.0; Sigma-Aldrich) at 95°C for 15 min in a microwave (220 watts) for antigen retrieval. Following incubation with rabbit polyclonal anti-DKK1 antibody (Abcam Inc., Cambridge, MA, USA) for 2 h at room temperature the sections were incubated with horseradish peroxidase-labeled anti-rabbit immunoglobulin G secondary antibody. The primary antibody was omitted in the negative controls. The intensity of DKK1 staining was evaluated in tumor cells in the cytoplasm, thereby determining the immunoreactivity of DKK1 in the tumor cells.

TE13 cells were cultured on glass coverslips, fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 10 min at room temperature. Cells were subsequently blocked with 3% BSAfor 30 min at room temperature, and incubated with primary antibodies diluted in PBS supplemented with 3% BSA for 60 min at room temperature. Following washing with PBS, the cells were stained with fluorescein isothiocyanate-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) for 30 min at 37°C. Finally, the coverslips were washed with PBS and the nuclei were stained with DAPI (Sigma-Aldrich) or rhodamine B (Sigma-Aldrich) and visualized with a confocal laser scanning microscope (Leica SP5; Leica Microsystems, Wetzlar, Germany). The images were analyzed using Leica LAS AF software (Leica Microsystems).

Statistical analyses were performed using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA) software. Values are presented as the mean ± standard deviation. Student’s t-test, one-way analysis of variance and χ² test were performed. P<0.05 was considered to indicate a statistically significant difference. All experiments were performed at least in triplicate.

DKK1 expression levels in the NSCLC samples collected from 123 patients were evaluated and compared with those of age-matched normal tissue from patients with low-grade NSCLC. Evaluation included quantification of DKK1 gene and protein expression levels and immunohistochemical analyses. For the analysis of protein and gene expression, 25 samples of cancerous tissue and 15 samples of age-matched normal tissue were evaluated (17). The mRNA expression levels of DKK1 in cancer tissues were found to be two- to six-fold greater than those in normal tissues (Fig. 1A). Similarly, DKK1 protein expression levels were found to be significantly greater in cancer tissue samples than those of the normal tissue samples (Fig. 1B). These results suggested a potential role for DKK1 in cancer progression. Clinical samples were obtained from 98 patients were subjected to immunohistochemical analysis. Of the 98 samples analyzed, 62 (63.3%) were positively stained for DKK1 and 36 (37%) demonstrated negative staining (Fig. 1C). Furthermore, no immunohistopathological staining for DKK1 was observed in normal tissues. These results indicated that increased expression of DKK1 is common in human NSCLC, which may potentially be associated with the metastasis of NSCLC.

DKK1 expression was evaluated in ten human NSCLC cell lines. Western blot analysis revealed that DKK1 protein was detected in all ten cell lines, but that the expression levels varied amongst the cell lines. A significantly increased level of DKK1 protein was observed in the LTEP-a-2, GLC-82 and PC-9 cell lines. The lowest levels of DKK1 protein were observed in the A2 and 95C cell lines (Fig. 2A). The mRNA expression levels of DKK1 were quantified using RT-qPCR, and the results were concurrent with those of the protein expression levels of corresponding cell lines (Fig. 2B). The LTEP-a-2 cell line demonstrated significantly (six-fold) increased mRNA expression levels. However, the minimum mRNA expression levels of DKK1 were observed in 95D cells, compared with those of the other cell lines. Immunofluorescent staining with rhodamine B revealed the sub-cellular localization of DKK1 protein, which was determined by the detection of rhodamine B-stained, fine-grained particles in the cytoplasm (Fig. 2C). The corresponding cell nuclei were stained with DAPI.

The involvement of DKK1 in the cell cycle (cyclin D1), apoptosis (Bcl-2, BAX), signaling pathways (Akt-1), as well as invasion and metastasis (MMP2, VEGC) in selected cell lines with high (LTEP-a-2) and low (95D) mRNA expression of DKK1, were evaluated. A relative differential gene expression profile with respect to the expression of DKK1 was constructed for the two cell lines (Fig. 3). The gene expression profile in LYEP-a-2 cells revealed increased mRNA levels of cyclin D1, MMP2 and VEGC. However, the levels of Bcl-2, BAX and Akt-1 were found to be significantly decreased (Fig. 3A). By contrast, the gene expression profile in 95D cells revealed a contrary expression with respect to the low levels of DKK1 expression. The mRNA expression levels of cyclin D1, MMP2 and VEGC were decreased concurrently with the decreased levels of DKK1. However, the levels of Bcl-2, BAX and Akt1 were found to be increased (Fig. 3B). These results demonstrated a regulatory effect of the DKK1 gene in the suppression of cell cycle, signaling pathways and apoptosis in NSCLC cells. In addition, enhanced DKK1 gene expression facilitates the cell cycle, invasion and metastasis (6).