All samples used for paraffin-embedded sections were collected from the First Hospital of China Medical University (Shenyang, China) between January 2003 and December 2004, and consisted of a total number of 257 patients with surgically resected NSCLC and lung tissue adjacent to carcinoma tissues. All paracancerous lung tissues were at least 5 cm from the tumor edge. Frozen tissue samples (35 pairs) were obtained between July and December 2013 and kept in liquid nitrogen immediately following surgical resection and stored in a −70°C refrigerator. None of the patients received any preoperative anticancer treatment. Relevant clinical data including gender, age, tumor size, location, histological type, differentiation degree and lymph node metastasis were collected. The sampling procedures in all cases were reviewed and approved by the Ethics Committee of the Taizhou Hospital (Taizhou, China). The pathological diagnosis was confirmed by ≥2 experienced pathologists.

Paraffin-embedded tissue sections were deparaffinized, rehydrated using xylene and a descending ethanol series, and washed with PBS. Antigen retrieval was performed by heating to 93°C for 15 min. Following 30 min of endogenous peroxidase quenching and 30 min of blocking at 37°C (both UltraSensitive™ SP kit; Maxim Biotech, Inc., Rockville, MD, USA), samples were incubated with the primary anti-AXL rabbit polyclonal antibody (1:100; cat. no. ab37861; Abcam, Cambridge, UK) overnight at 4°C. Samples were subsequently washed with PBS and incubated with a biotin-labeled secondary antibody for 30 min at 37°C, prior to incubation with streptavidin-peroxidase (both UltraSensitive™ SP kit) at 37°C for 30 min, according to the manufacturer's protocol. 3,3-Diaminobenzidine (DAB) reagent (Maxvision™ DAB kit; Maxim Biotech, Inc.) was added for 45 sec to stain the samples. Images were captured using an inverted microscope (IX53; Olympus Corporation, Tokyo, Japan). The results were reported as the product of staining density score and staining intensity score. To determine the staining density score, which was defined as the percentage of the positive staining area, samples with a staining density <30% scored 1 point, samples with a staining density of between 30 and 60% scored 2 points, and samples with a staining density >60% scored 3 points. To determine the staining intensity score, samples with no color or yellowish color scored 1 point, samples with brown staining scored 2 points, and samples with dark brown staining scored 3 points. The results were determined by 2 experienced pathologists, and the mean of the three observations was taken to be the final score.

The 3 NSCLC cell lines used in the present study, adenocarcinoma A549, adenocarcinoma H1299 and squamous cell carcinomas SK-MES-1 were all obtained from the Cell Culture Center of the Forth Hospital of China Medical University (Shenyang, China). The H1299 cell line was cultured in RPMI-1640 medium (Hyclone: GE Healthcare Life Sciences, Logan, UT, USA), and the SK-MES-1 and A549 cell lines were cultured in Dulbecco's modified Eagle's medium (Hyclone: GE Healthcare Life Sciences). The media were supplied with 10% fetal bovine serum (Hyclone: GE Healthcare Life Sciences) without antibiotics. Cells were cultured in a 37°C incubator containing 5% CO₂. The culture media were changed every 2–3 days. Cells with 80% confluency were passaged. Cells at 60–70% confluency were transfected with AXL-siRNA using Lipofectamine 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), Lipofectamine 2000 treatment alone was taken as the mock control. The target sequences were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China) and are shown as follows: AXL-siRNA sense, 5′-GGAGACCCGUUAUGGAGAATT-3′ and antisense, 5′-UUCUCCAUAACGGGUCUCCTT-3′; and negative control siRNA sense, 5′-GCGACGAUCUGCCUAAGAUdTdT-3′ and antisense 5′-AUCUUAGGCAGAUCGUCGCdTdT-3′.

The protein from the fresh tissue was isolated and quantified using radioimmunoprecipitation assay buffer and a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China), respectively. The protein was denatured with loading buffer at 100°C. The western blot analysis was conducted using a SDS-PAGE Gel kit (cat. no. P0012A) and the DAB Horseradish Peroxidase Color Development kit (cat. no. P0203) (both Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The anti-AXL antibody was used at a dilution of 1:300. The mouse monoclonal antibodies against β-actin (cat. no. sc-47778; 1:500) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The results were analyzed using the Gel-Pro Analyzer software (version 4.0; Media Cybernetics, Inc., Rockville, MD, USA).

Quantitative analysis of AXL mRNA expression was achieved by RT-qPCR; the total RNA of the samples was isolated by RNAiso Plus (Takara Biotechnology Co., Ltd., Dalian, China), the first strand of cDNA was synthesized by PrimeScript^® RT Master Mix Perfect Real Time (Takara Biotechnology Co., Ltd.), and the amplification was assessed using SYBR^® Premix Ex Taq™ (Takara Biotechnology Co., Ltd.). All were completed according the manufacturer's protocols. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. The relative expression was calculated by the 2⁻ΔΔ^Cq method. The primers were synthesized by Takara, and their sequences were as follows: AXL, forward, 5′-GCAACCTTCACCTACCGAGTTC-3′ and reverse, 5′-GGCCAACATGGTGAAACCCT-3′; GAPDH, forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse 5′-TCCACCACCCTGTTGCTGTA-3′. The PCR amplification and data analysis were assessed using a ThermoCycler Dice Real Time System (Takara Biotechnology Co., Ltd.). The conditions were as follows: 1 cycle of 95°C for 30 sec; 40 cycles of 95°C for 5 sec and 60°C for 30 sec; and 1 cycle 95°C for 30 sec (Table I). Experiments were performed in triplicate and the mean ΔCq values were used for subsequent analysis.

Cells were plated into 24-well plates at a density of 3×10⁴ cells/well 1 day prior to transfection. Cell culture medium was replaced with fresh RPMI-1640 medium containing 10% fetal bovine serum without antibiotics one day following AXL-siRNA transfection. Following another 24, 48 and 72 h of culture, the MTT Cell Proliferation Assay kit (American Type Culture Collection, Manassas, VA, USA) was used to perform the MTT assay, according to the manufacturer's protocol. Each group was detected at a wavelength of 490 nm using a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA). Experiments were performed in triplicate and the mean was used in the statistical analysis. The Transwell invasion assay was performed using a 24-well, 8 µm pore size Costar Transwell chamber system (Corning Life Sciences, Lowell, MA, USA), according to the manufacturer's protocol. A total of 1×10⁵ cells/well were seeded in the upper chamber. The number of cells in the lower chamber was counted in the lower chamber following 36 h of culture for the transfection and control groups. Experiments were performed in triplicate and the mean was used in the statistical analysis.

The data were analyzed by SPSS 19.0 software (IBM SPSS, Armonk, NY, USA); the means between two groups were compared by paired t-test and Mann-Whitney U test, and the analysis of the associations between AXL expression and clinical characteristics was assessed by χ² test or Fisher's exact test. A two tailed P<0.05 was considered to indicate a statistically significant difference.

The results of the immunohistochemistry analysis are shown in Fig. 1. The present study found that the AXL staining was mainly located in the cytoplasm, and a small portion of nuclear staining was also observed. Out of the total 257 patients 147 showed positive staining for AXL, and the average positive rate was 55.25%, which is significantly higher than that of the adjacent lung tissue (26.85%; P=0.009; Table II).

The clinical characteristics and the statistical analysis are shown in Table III. The present study found that the expression level of AXL was significantly associated with the degree of tumor differentiation. The lower the differentiation of the tumor, the higher level of AXL expressed (P=0.001). Patients with a stage higher than stage I showed significantly higher expression level of AXL than those with stage I (P=0.005). By contrast, no association between AXL expression and age (P=0.722), gender (P=0.238), histological type (P=1.000), lymph node metastasis (P=0.091) or tumor size (P=0.166) was found.

Next, the expression of AXL in fresh tissues was detected by western blot analysis and qPCR. Consistent with the immunohistochemistry results, the mRNA (P=0.037; Fig. 3) and protein (P=0.044; Fig. 2) levels of AXL were significantly increased in NSCLC tissues compared with those in their adjacent lung tissues.

To investigate the function of AXL in vitro, the current study first detected its expression in lung cancer cell lines A549, H1299 and SK-MES-1 by western blot analysis and qPCR. As shown in Fig. 4, H1299 demonstrated markedly higher level of AXL protein expression compared with the other two cell lines. In addition, H1299 cells demonstrated significantly increased AXL mRNA expression (P=0.003 vs. A549 cells; P=0.005 vs. SK-MES-1 cells; Fig. 5).

Due to the relative high expression of H1299, a methyl thiazolyl tetrazolium (MTT) assay was performed with a Transwell migration assay to determine whether inhibition of AXL expression could reduce its proliferation and invasion activity. Western blotting and qPCR analysis confirmed the knockdown efficiency of the AXL-siRNA. AXL protein expression was markedly decreased (Fig. 6) and AXL mRNA expression was significantly decreased (P=0.019 vs. the negative control; P=0.013 vs. the mock transfection; Fig. 7) in H1299 cells following the silencing of AXL by its specific siRNA.

Subsequent to transfection, cells were incubated in complete medium for another 24, 48 or 72 h. MTT results showed that transfection with AXL-siRNA resulted in a significant decrease of cell viability compared with the negative control at each time point (P=0.007; Fig. 8). In addition, the number of cells in the bottom layer of the culture was significantly decreased following AXL-siRNA transfection compared with the negative control (P=0.018). The number of cells that migrated to the underside of the membranes was calculated by counting 5 random separate fields, as shown by Transwell analysis (Fig. 9), indicating the decreased invasion activity subsequent to AXL inhibition.