NSCLC cell lines, including H1975, H1299, H520, A549, PC-9 and GLC82, were obtained from American Type Culture Collection (Manassas, VA, USA). The H520 cell line was derived from lung squamous cell carcinoma, while the other 5 cell lines were all derived from adenocarcinoma. Among the 5 lung adenocarcinoma cell lines, H1650 was derived from bronchoalveolar carcinoma cells from the pleural effusion of a Caucasian male; H1299 was derived from the lymph nodes of a male patient with lung adenocarcinoma; A549 was also derived from a Caucasian male with lung adenocarcinoma; PC9 was established by Tokyo University (Tokyo, Japan) and derived from adenocarcinoma in pleural effusion; and GLC82 was derived from a female Chinese patient with lung adenocarcinoma.

All 6 cell lines were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C with 5% CO₂ in a humidified atmosphere. Monoclonal mouse anti-human CD44 antibodies were purchased from Abcam (cat no. ab6124; Cambridge, UK).

There were 94 cases of NSCLC tissue specimens excluding adjacent normal tissues acquired from patients who underwent surgery at Peking University Cancer Hospital and Institute (Beijing, China) from July 2013 to October 2015 without any other treatments prior to surgery. Clinicopathological characteristics of these patients are presented in Table I with 72 cases of squamous carcinoma, 19 cases of adenocarcinoma, 2 cases of adenosquamous carcinoma and one carcinoid. Written informed consent was obtained from all patients included in the present study, and this study was approved by the Ethics Committee of Peking University Cancer Hospital and Institute.

Tumor tissues were fixed with 10% formaldehyde solution overnight at room temperature and then paraffin-embedded tumors were cut into 4-µm thick sections. Sections were routinely stained, as described in our previous study (20). Briefly, the sections were immersed in xylene to remove the paraffin, washed in a graded series of ethanol, immersed in citrate buffer and then incubated in a high-pressure sterilization oven for antigen retrieval with citrate buffer at pH 6.0 for 3 min at 100°C. Endogenous peroxidase activity was blocked in a blocking solution with 3% H₂O₂ in PBS for 10 min at room temperature, and then the sections were incubated with PBS containing 1% bovine serum albumin (Amresco, Solon, OH, USA) for 10 min at room temperature to block non-specific binding. Following washing in PBS, the tissue sections were incubated at room temperature for 1 h with antibodies against CD44 (cat no. ab6124; dilution, 1:2,000; Abcam), followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (cat no. A4416; 1:1,000; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 1 h at room temperature. Then, the slides were visualized with 0.1% 3,3-diaminobenzidine (Sigma-Aldrich; Merck KGaA) for 2 min, and counterstained with one drop of 1% hematoxylin for 10 min at room temperature. According to the percentage of cells with complete membrane staining in the tumor tissue section, the section was classified as follows: Negative, <15%; weak positive, 15–30%; moderate positive, 30–75%; and strong positive, 75–95%. In the present study, CD44 expression was either defined as negative, or positive (either weak, moderate, or strong positive).

Human CD44 open reading frame mammalian expression plasmids were purchased from YouBio (Chongqing, China, http://www.youbio.cn/). H1299 cells were seeded onto 6-well plates at 80% confluence, and then transfected with 4.0 µg of CD44 plasmids or pcDNA3.0 empty plasmids (Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. After 24 h, cells were cultured and propagated in media containing 500 µg/ml G418 (Sigma-Aldrich; Merck KGaA) at 37°C with an atmosphere containing 5% CO₂ in a humidified incubator until a stable cell line was established. Small interfering RNAs (siRNAs) targeting CD44 were designed using BLOCK-iT^™ RNAi Designer (Thermo Fisher Scientific, Inc.) within open reading frame, with the default criteria on the webpage (http://rnaidesigner.thermofisher.com/rnaiexpress), and then synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). siRNAs (50 nmol/l) were transfected into A549 cells using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's protocol. The CD44 siRNA target sequences were as follows: siRNA-1, 5′-CUCCCAGUAUGACACAUAUTT-3′; siRNA-2, 5′-GGACCAAUUACCAUAACUATT-3′; siRNA-3, 5′-GCAGUCAACAGUCGAAGAATT-3′; and NC, 5′-UUCUCCGAACGUGUCACGUTT-3′.

RNA was extracted using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. 1 µg total RNA was reverse transcribed using TransScript^® All-in-One First-Strand cDNA Synthesis SuperMix for PCR (TransGene, Beijing, China, http://www.transgen.com.cn), according to the manufacturer's protocol. qPCR reactions were performed using a Light-Cycler 480 Real-Time PCR System (Roche Diagnostics GmbH) and the LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics GmbH). GAPDH was used as the internal control. The primers sequences are as follows: CD44, forward 5′-ACACGAAGGAAAGCAGGACC-3′, and reverse 5′-TTTGCTCCACCTTCTTGACTC-3′; GAPDH, forward 5′-TGAAGGTCGGAGTCAACGG-3′, and reverse 5′-CTGGAAGATGGTGATGGGATT-3′. The cycling conditions were as follows: 5 min at 95°C, followed by 42 cycles of 10 sec at 95°C, 30 sec at 60°C, and 20 sec at 72°C. The relative gene expression levels were calculated by the 2^−ΔΔCq method (where ΔCq=Cq_target- Cq_control) (21).

Cells were lysed in RIPA buffer (Roche Diagnostics GmbH) containing a complete protease inhibitor cocktail for 15 min on ice. The bicinchoninic acid assay method (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) was used to determinate protein quantity and equivalent amounts of total proteins (30 µg) were separated by 10% SDS-PAGE and transferred topolyvinylidene fluoride membranes (Millipore, Billerica, MA, America). Membranes were blocked with 5% nonfat dried milk dissolved in TBST containing 0.1% Tween 20 for 1 h at room temperature and then incubated with following specified antibodies for 1 h at room temperature: Anti-CD44 (cat no. ab6124; dilution, 1:5,000; Abcam) and mouse anti-GAPDH (cat no. 97166; dilution, 1:5,000; Cell Signaling Technology, Inc., Danvers, MA, USA), followed by HRP-conjugated goat anti-mouse IgG (cat no. A4416; dilution, 1:5,000; Sigma-Aldrich; Merck KGaA) for 1 h at room temperature. Signals were visualized using an enhanced ECL Chemiluminescence reagent (cat no. WBKLS0500; Merck KGaA) and detected using AI600 version 1.2.0 on Amersham Imager 600 (GE Healthcare, Chicago, IL, USA).

Briefly, a total of 10,000 live cells were incubated with anti-CD44 (cat no. ab6124; dilution, 1:100; Abcam) at 37°C for 30 min. Following washing with PBS, cells were then incubated with FITC-labeled goat anti-mouse IgG (cat no. F0257; dilution, 1:100; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min. Following washing three times with PBS, the fluorescence intensity was detected with the BD Accuri^™ C6 Flow Cytometer with BD Accuri C6 software version 1.0.264.21 (BD Bioscience, San Diego, CA, USA).

Cells were seeded into 96-well plates (1,500 cells/well). To assess cell proliferation, cells were subjected to Cell Counting Kit-8 (CCK-8) assays (Dojindo, Kumamoto, Japan) at 0, 12, 24, 36, 48, 60 and 72 h. Each time, supernatant was replaced by RPMI-1640 medium containing 10% CCK-8 reagent. After 2 h incubation at 37°C, the absorbance at 450 nm was measured to determine the number of viable cells, according to the manufacturer's protocol. The experiment was repeated three times independently. The cell doubling time (DT) was calculated by the equation: TD=ΔTx[lg2/(lgNt-lgN0)], where ΔT is the time interval; N0 is the initial cell number and Nt is the end point cell number.

A total of ~500 cells were seeded into 60-mm dishes, and the culture medium of RPMI-1640 medium supplemented with 10% FBS was changed every 3 days. Visible colonies were fixed after 8 days in 4% paraformaldehyde for 15 min at room temperature, stained with 0.1% crystal violet solution for 30 min at room temperature, and then washed with PBS. The number of colonies number was counted by naked eye from three independent experiments.

All statistical analysis was performed using SPSS 22.0 software (IBM Corp, Armonk, NY, US). Associations between CD44 expression and clinicopathological factors were analyzed using Pearson's χ² test and Fisher's exact tests. The mRNA levels, colony number and size were presented as the mean ± standard deviation from three independent experiments. The difference between two groups was assessed using independent samples t-test. Dunnett's Multiple Comparison tests were used to compare differences between treatment groups and the control group following analysis of variance (ANOVA). Two-way ANOVA tests were used to analyze the difference of cell viability curves for two groups. P<0.05 (two-tailed) was considered to indicate a statistically significant difference.

As presented in Fig. 1, CD44 was predominantly expressed in the cell membrane of NSCLC cells. CD44 expression was observed in all pathological types of NSCLC tissue, including squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma and carcinoid. The percentage of CD44-stained tumor cells in the samples ranged from 0–95%. To facilitate analysis, CD44 expression was classified into two groups; negative and positive. When >15% of tumor cells stained positively for CD44 in a sample, it was classified as positive CD44 expression; otherwise it was defined as negative. In total, 64 (68.1%) of 94 NSCLC cases were identified as positive for CD44 expression.

To predict the clinical significance of CD44 expression, we analyzed the associations between CD44 expression and clinicopathological features in NSCLC. There was no significant difference between CD44 expression and age, sex, smoking status, pathology type, N classification or TNM stage (22). However, the level of CD44 expression was positively associated with an advanced T classification (P<0.05; Table I). A total of 58.9% of cases (33 of 56) stained positive for CD44 in the T1 group, while 80.0% of cases (28 of 35) stained positive for CD44 in the T2 group, and all cases (3 of 3) stained positive for CD44 in the T3 group. This result suggested that the higher the T stage was, the stronger the CD44 staining in the tumor tissue would be, indicating that CD44 potentially promotes tumor growth.

To further investigate the relationship between CD44 expression and cell proliferation, CD44 expression (Fig. 2A) and cell doubling time (Fig. 2B) were determined in NSCLC cell lines. Among these cells, A549 cells and CLC82 cells had relative high CD44 expression, while PC9, H520 and H1299 had the relative low CD44 expression. Thus, H1299 were selected to perform the CD44 over-expression experiments and A549 cells to do the CD44 interference experiments. The data from the present study suggested that A549 cells had the highest CD44 expression and the shortest doubling time of the 6 NSCLC cell lines (Fig. 2A and B). The line graph in Fig. 2C also suggested a positive association between CD44 expression and cell doubling time, indicating that higher CD44 expression tended to lead to faster cell proliferation.

To investigate the forced expression of CD44 on cell malignancy, we forced expression of CD44 in H1299 cells with a low endogenous CD44 expression (Fig. 2A). This was conducted using plasmid transfection and G418 screening, a pool of H1299 cells that stably overexpressed CD44, named H1299-CD44, were established, and the negative control cells transfected with empty vectors were named H1299-control. CD44 mRNA and protein expression of the two cell lines by RT-qPCR (Fig. 3A) and western blotting (Fig. 3B). As presented in Fig. 3A, the expression of CD44 increased ~17.4 fold in H1299-CD44 cells compared with H1299-control cells. In addition, the protein expression of CD44 also visibly increased in H1299-CD44 cells compared with H1299-control cells (Fig. 3B; lane 2), indicating a successful CD44 overexpression in H1299-CD44 cells. Consistent with this result, CCK-8 assays suggested that H1299-CD44 cells proliferated significantly faster than the negative control cells (Fig. 3C; P<0.001). The difference between H1299-CD44 cells and H1299-control cells became significant 12 h after seeding the cells. As time increased, the difference became increasingly significant, and the OD₄₅₀ value of the H1299-CD44 cells was almost 2 times that of the H1299-control cells. These results confirmed our hypothesis that CD44 promoted cell proliferation in vitro.

In order to further validate the hypothesis that CD44 promotes cell proliferation, a clonogenic assay was performed. A total of 500 cells were seeded on 60-mm plates and cultured for 8 days, with a medium changed each 3 days. As presented in Figs. 3D and E, the number of colonies of H1299-CD44 cells was significantly increased compared with H1299-control cells. In addition, the colonies formed by the H1299-CD44 cells were larger than those formed by the H1299-control cells (Fig. 3F). These results suggested that overexpression of CD44 improved the colony forming ability of H1299 cells.

To further confirm this result, CD44 expression was knocked down by siRNAs in A549 cells with relatively high levels of CD44 expression (Fig. 2A). As presented in Fig. 4A, CD44 expression was decreased by 94.7, 97.7 and 93.2%, respectively for siRNA-1, siRNA-2 and siRNA-3, compared with cells transfected with siRNA-NC. Although the positive CD44 population of the A549 cells did not alter following siRNA transfection (data not shown), the average fluorescein intensity of CD44 also decreased by 64.6, 68.5 and 60.7% respectively for siRNA-1, siRNA-2 and siRNA-3, compared with cells transfected with siRNA-NC (Fig. 4B), indicating that CD44 expression was significantly inhibited by the siRNAs. As presented in Fig. 4C, A549 cells transfected with CD44 siRNA-1, siRNA-2 and siRNA-3 proliferated more slowly than the negative control cells (P<0.05). A clonogenic assay was then performed. The number of cell colonies formed by the siRNA-transfected cells was significantly decreased compared with the negative control cells (Fig. 4D and E; P<0.01). These results suggested that knockdown of CD44 expression suppressed A549 cell proliferation in vitro.