In this study, NSCLC tumor tissues and paired adjacent normal tissues were obtained from 35 patients who underwent percutaneous lung puncture or biopsy of lung cancer tissue at The Second Affiliated Hospital of Xi'an Jiaotong University (Xi'an, China) between October 2013 and May 2014. All NSCLC cases were clinically and pathologically confirmed by two independent experts. All tissues were frozen at −80°C for further analysis. The follow-up data for 60 months were recorded by communicating with the patients or their relatives, the median duration of follow-up was 59 months (range, 1–60 months). Informed consent was obtained from each patient, and this study was approved by the Human Ethics Committee of Second Affiliated Hospital of Xi'an Jiaotong University.

The human lung adenocarcinoma cell lines A549 and H1299 and normal human bronchial epithelial cell line (NHBE) were obtained from the Shanghai GeneChem Co., Bank (Shanghai, China). All cell culture reagents were purchased from Invitrogen and supplied with 1% penicillin/streptomycin (Sigma-Aldrich, ON, Canada). Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (Hyclone, Logan, UT, USA), and maintained in monolayer culture at 37°C in an incubator of humidified air with 5% CO₂.

For NACK, the sequences for siRNA were design as follows: sense strand, 5′-UCG CAU UGA CCA UUC AAA CUG GUG G-3′ and antisense, 5′-CCA CCA GUU UGA AUG GUC AAU GCG A-3′. The scramble siRNA was random sequenced by Blast website. The cells were harvested at 48 h post-transfection. A549 and H1299 cells were used in this experiment. Twenty-four hours before transfection, 1×10⁴ cells/well, in 5 ml medium, were plated in 60 mm dish (Nunc; Thermo Fisher Scientific, Waltham, MA, USA), and then transfected with plasmids of NACK-siRNA or scramble siRNA as negative control using Lipofectamine 2000 as described in the manufacturer's instruction.

For reporter assay, A549 and H1299 cells were cultured in 24-well plates. Hes1-luciferase reporter plasmids were constructed by subcloning the 5-upsteam elements of human Hes1 into the pGL3-enhancer vector (Promega, Madison, WI, USA). Hes1 promoter-specific primers (−747−+66): Forward, 5′-CGA GCT CAG CGG CAA CTT TAG ATG TG-3′; and reverse, 5′-CCC AAG CTT GTT GAC ACT GGC TGG GGT A-3′. After 24 h, the cells were co-transfected with the siRNA expression plasmid targeting NACK (NACK siRNA). Fourty-eight hours later, luciferase activities were measured using Dual-Luciferase Reporter Assay system (Promega) according to manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity.

Total RNA was isolated from cells or tissues by TRIzol reagent (15596-026; Life Technologies) following the manufacturer's instructions. Quantitative real-time reverse transcriptase-PCR assay (qRT-PCR) was performed using a SYBR-Green method with a Master Mix buffer system. Gene expression in human was normalized to GAPDH. The primers sequences were as follows: GAPDH forward, 5′-CCG ATT TCT CCT CCG GGT G-3′ and reverse, 5′-TGG TCA TGA GTC CTT CCA CG-3′; NACK forward, 5′-TCT CTT GTG AAG GAA CCG GC-3′ and reverse, 5′-CCG GCT TGT AAG TCC TGG TT-3′; Notch1 forward, 5′-GGG CCT CAA GTG AGC GGA C-3′ and reverse, 5′-GGT GAG GGG TCG AGA AGT GA-3′; Hes1 forward, 5′-TTT CTT CCA GAC TTC CGC CC-3′ and reverse, 5′-GGA CAA TGC CTC CCA ATC CA-3′; and HeyL forward, 5′-AGA CCG CAT CAA CAG TAG CC-3′ and reverse, 5′-TCA GGC AGC TGC TAC CAA TC-3′. The PCR conditions were as follows: 95°C for 2 min, 95°C for 15 sec, 60°C for 30 sec for 40 cycles. The relative expression levels were calculated by the value of 2^−ΔΔCt. All experiments were repeated at least three times.

The tissues and cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (Solarbio, Beijing, China). The proteins were separated on SDS-polyacrylamide gels (SDS-PAGE) and electrophoretically transferred to PVDF membranes (Roche Diagnostics). Then the membranes were blocked in 5% milk, and incubated with the appropriate primary antibody overnight. The primary antibodies NACK (1:1,000; AbMax Company, China), Notch1 (1:1,000), Hes1 (1:1,000), and HeyL (1:1,000) (all from Abcam, UK), were incubated with HRP-conjugated secondary antibodies (Abcam) at 1:1,000 for 1 h, then the protein bands were detected using the enhanced chemiluminescence detection system (Pierce; Thermo Scientific, Rockford, IL, USA).

The proliferation of NSCLC cells was evaluated by Cell Counting Kit-8 (CCK-8) assay (Beyotime, Shanghai, China) according to manufacturer's instruction. Briefly, cells transfected with negative-siRNA or NACK-siRNA were seeded into a 96-well plate (100 μl/well) and incubated at 37°C, 5% CO₂ for 5 days. CCK-8 (10%) was added to the well at 24 h intervals and maintained for 2 h according to the manufacturer's protocol, and then the absorbance was measured at 450 nm by a microplate reader (BioTek ELx800; BioTek, USA).

Non-small cell lung cancer cells (A549 and H1299) were transfected with negative-siRNA or NACK-siRNA for 24 h, then the transfected cells were resuspended, counted and seeded at a density of 600 cells/well in a 6-well plate. The cells were continually cultured for 10 days with a change of media every other day. On day 10, cells were fixed with 3.7% methanol, stained with 0.1% crystal violet the number of colonies were counted under a microscope (Olympus, Beijing, China).

Cell motility was assessed by migration and invasion assays. A 24-well Transwell insert containing a pore size of 8 μm polycarbonate membrane (Corning, Inc., Corning, NY, USA) was used to determine the effect of NACK on A549 and H1299 migration and invasion in vitro. Briefly, the transfected cells were cultured in serum-free medium for 12 h, then cells were resuspended in serum-free medium and placed in the upper chambers at the density of 2×10⁴. The lower chamber was filled with medium containing 10% fetal bovine serum as the chemoattractant and incubated 24 h for migration assay and 48 h for the invasion assay, respectively. For migration assay, the number of cells on the lower surface of the membrane was counted in 5 fields under a microscope. For invasion assay, the upper chambers of the inserts were covered with Matrigel. After 48 h incubation, cells on the lower surface of the member was stained with 0.1% crystal violet. The number of cells on the lower surface of the membrane was counted in 5 fields under a microscope.

NSCLC cells transfected as negative-siRNA or NACK-siRNA were collected and washed with PBS twice. After centrifuge, the cells were resuspended with 1X staining buffer at the dose of 1×10⁶ cell/ml, then cells were dyed with 5 μl Annexin V-APC in the dark, at room temperature for 15 min. Flow cytometry was applied with FACSCalibur flow cytometer and analysis was performed with FlowJo software (Tree Star).

Four weeks old female nude mice were purchased from the Animal Center of The Fourth Military Medical University (Xi'an, China) and randomly divided into three groups with 10 per group, receiving subcutaneous injection of NACK-siRNA, negative-siRNA, or control cells, respectively. of Tumor volume was measured using calipers every 4 days for 28 days and calculated as follows: Tumor size [volume (mm³) = width² (mm)/2 × length (mm)]. Following 28-day post-inoculation, the animals were euthanized and the tumors were collected for further analyzing. The animal experiment was reviewed and approved by the Animal Care and Use Committee of the Second Affiliated Xi'an Jiaotong University (Xi'an, China).

All assays were repeated three times to insure reproducibility. The significance of results obtained from the control and treated groups were analyzed using the Student's t-test. The results are given as mean ± SD. p<0.05 was considered statistically significant.

The expression level of NACK in 35 NSCLC tissue samples and corresponding adjacent non-cancerous tissues were detected by the quantitative RT-PCR (qRT-PCR) and western blot assay. As shown in Fig. 1A and B, NACK was discovered highly expressed both at mRNA and protein levels in the NSCLC tissue samples compared with the paired adjacent normal tissues. Furthermore, the expression level of NACK was assessed in NSCLC cell lines (A549 and H1299) and NHBE, the result showed that NACK was greatly higher in A549 and H1299 cell lines than the normal cell line NHBE (Fig. 1A and C), indicating that NACK may play a positive role in NSCLC progression.

To determine the potential role of NACK in the progress of NSCLC, the relationship between NACK expression and clinicopathological parameters in NSCLC were analyzed by Pearson's Chi-square. NSCLC cases were divided into two groups according to the mRNA level of NACK (low < median; high > median). The high expression of NACK was observed in 26/35 (74.29%) of NSCLC samples, and the expression of NACK was significantly correlated with the degree of differentiation (p=0.034), lymphatic metastasis (N stage) (p=0.007) and clinical stage (p=0.014) (Table I). However, the high level of NACK expression was not associated with gender, age, smoking, tumor size or histological type (p>0.05). Hence, NACK may be used as a potential unfavorable prognostic biomarker in NSCLC.

To investigate the correlation between NACK expression and the survival rates of NSCLC patients, Kaplan-Meier survival analysis was performed until the patients died or end of the research. Patients were divided into two groups depending on NACK expression level (low and high). We observed that the cumulative 4-year overall survival rate (OS) and decreased survival rate (DFS) of patients with high level of NACK detection were 30.7 and 34.6%, respectively, when compared to low NACK expression patients (77.8 and 77.8%, respectively) (Fig. 2). Hence, the level of NACK expression was an unfavorable predictive factor for prognosis of NSCLC patients.

The efficient inhibition of NACK expression was examined by qRT-PCR and western blotting, the result showed that after transfected with NACK-siRNA, the expression of NACK was significantly decreased at mRNA level (Fig. 3A) and hardly observed at protein level (Fig. 3B) either in A549 or H1299 cells. No significant difference was observed between negative-siRNA and control groups.

Cell proliferation assay and cell invasion assay were performed in NSCLC cell lines A549 and H1299 with NACK knockdown by siRNA. CCK-8 assay verified that the cells transfected with NACK-siRNA showed a significant decrease in cell proliferation when compared with control group both in A549 (p<0.001) and H1299 (p<0.001) cells at the 5th day (Fig. 4A). The colony formation assay was used to evaluate the tumor growth of a transfected cells. Our data indicated that when A549 and H1299 cells were treated with NACK-siRNA, the ability of colony formation was remarkably decreased in A549 (p<0.05) and H1299 (p<0.05) cells (Fig. 4B and C).

To investigate whether NACK regulates the migration and invasion of NSCLC cell, NSCLC cells were transfected with either NACK-siRNA or negative-siRNA. Cell migration and invasion were evaluated by using a Transwell insert. The results demonstrated that knockdown of NACK significantly reduced the number of migration at 24 h (p<0.01) and invasion at 48 h (p<0.01) (Fig. 4D and E), suggesting that NACK is necessary for NSCLC cell metastasis during the tumor progress.

We further investigated whether interference NACK could induce apoptosis in A549 and H1299 cells. FITC assay results indicated that the apoptosis rates were significantly increased both in A549 and H1299 cells after knocking down NACK (Fig. 5). These results suggest the interference of NACK inhibits NSCLC progression through apoptosis induction in vitro.

NACK was identified as a direct target gene of Notch pathway in human esophageal adenocarcinoma cells (15). Considering the specific action of the Notch pathway in different types of tumors, we investigated the role of NACK on the regulation of Notch pathway in NSCLC by transfecting NACK-siRNA into A549 and H1299 cells to knock down NACK expression. The results showed that the expression of the Notch target genes Hes1 and HeyL were reduced significantly at mRNA level (Fig. 6A). Western blot results also showed that transfection with NACK-siRNA reduced expression of Notch1, Hes1 and HeyL protein both in A549 and H1299 cells (Fig. 6B). Furthermore, as luciferase reporter assay result showed, co-transfection of NACK-siRNA with Hes1 reporter construct strongly reduced luciferase activity in both A549 and H1299 cells (Fig. 6C) when compared to the cells co-transfected with negative-siRNA. The data demonstrated that the transcriptional activity of Notch1 signaling pathway could be inhibited by knocking down the expression of NACK in NSCLC.

To address whether changes of NACK expression could influence the growth of tumor in vivo, A549 and H1299 cells infected with negative-siRNA or NACK-siRNA were injected subcutaneously into the nude mice, and tumor size was measured every 4 days for 28 days. The results revealed that the knockdown of NACK inhibited tumorigenesis in vivo. The average tumor volume (Fig. 7A) and weight of mice (Fig. 7B and C) in NCAK-siRNA group at day 28 was significantly decreased (p<0.01) compared to that of mice in control group. These results demonstrated that the interference of NACK inhibits NSCLC progression in vivo.