The A549 human NSCLC cell line was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science (Shanghai, China). The cells were cultured at 37°C in Dulbecco's modified Eagle's medium (DMEM; Shanghai Huiying Biotechnology Co., Ltd., Shanghai, China) supplemented with 10% fetal bovine serum (FBS; Shanghai Huiying Biotechnology Co., Ltd.), 100 U/ml penicillin and 100 µg/ml streptomycin (Shanghai Huiying Biotechnology Co., Ltd.). Subsequently, 70–80% of the cells were transferred to 96-well plates and cultured in fresh medium without antibiotics. X-treme™ GENE siRNA Transfection Reagent (Roche Diagnostics, Basel, Switzerland) was used to transfect the cells with siEphA7 (Shanghai GenePharma Co., Ltd., Shanghai, China; sequence, 5′-GUG GGA AGU UAU GUC UUA UTT AUA AGA CAU AAC UUC CCA CTT-3′) or negative control siRNA (Shanghai GenePharma Co., Ltd.; sequence, 5′-UUC UCC GAA CGU GUC ACG UTT ACG UGA CAC GUC CGG AGA ATT-3′). The transfection concentration determined for use by transfection efficiency testing was 50 nM siEphA7, transfection was conducted for 24 h at 37°C. A549 cells were used as the untransfected control group.

The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Beyotime Institute of Biotechnology, Haimen, China) was used to assess the viability of cells cultured in 96-well plates by measuring mitochondrial dehydrogenase activity. The MTT assay is based on the notion that viable cells, but not dead cells, can reduce MTT. Following siRNA transfection, the cells were incubated with 10 µl MTT (0.5 mg/ml) at 37°C for 4 h. The purple formazan crystals were then dissolved using 100µl dimethyl sulfoxide. The absorbance was subsequently measured at 490 nm using an 340 AT Easy Microplate Reader (SLT Lab Instruments GmbH, Salzburg, Austria).

Cellular mRNA expression levels of EphA7 were determined using RT-qPCR. Total RNA (1 µg) was extracted from each sample of cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and was used to generate cDNA using M-MLV Reverse Transcriptase (Promega Corporation, Madison, WI, USA). The cycling conditions were as follows: Initial denaturation at 25°C for 10 min; 37°C for 120 min; 85°C for 5min; the temperature was dropped to 4°C and the product was reached at 20°C. qPCR was conducted using SYBR Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), and the PCR cycling conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec, followed by a final extension step at 72°C for 10 min. The PCR primers used were as follows: Sense, 5′-CTA ATG TTG GAT TGT TGG CAA AAG-3′ and antisense, 5′-TTG ATC CAG AAG AGG GCT TATTG-3′ for EphA7 and sense, 5′-AAG AAG GTG GTG AAGCAGGC-3′ and antisense, 5′-TCC ACC ACC CAG TTG CTGTA-3′ for GAPDH. A 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used for cDNA amplification, with routine product checking using dissociation curve software (QuantStudio™ Real-Time PCR software, version 2.0.6; Applied Biosystems; Thermo Fisher Scientific, Inc.). Transcript quantities were compared using a relative quantification method. The amount of detected mRNAs was normalized to the amount of the endogenous control GAPDH. The relative value to the control sample was determined using the 2^−ΔΔCq method (17).

The cells were fixed in 4% paraformaldehyde (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) and pretreated with ethanol, proteinase K (Tiangen Biotech Co., Ltd., Beijing, China), Triton X-100 (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China), and pepsin (Shanghai Haoran Bio Technologies Co., Ltd., Shanghai, China). Following washing in PBS three times, the samples were treated with the TUNEL reaction mixture and TUNEL Dilution buffer (In Situ Cell Death Detection kit; Roche Diagnostics, Basel, Switzerland) at a ratio of 1:9, and incubated in a humidified chamber for 1 h at 37°C. After washing in phosphate-buffered saline (PBS), anti-digoxigenin peroxidase conjugate (Roche Diagnostics) was added, and incubation continued in a humidified chamber for 30 min at room temperature. The samples were washed with PBS and the nuclei were stained with DAPI (Roche Molecular Diagnostics, Pleasanton, CA, USA) for 5 min at room temperature. The number of total cells and TUNEL-positive cells were automatically counted using Image-Pro Plus (Media Cybernetics, Inc., Rockville, MD, USA), in order to calculate the apoptotic rate, which was defined as the ratio of apoptotic cells to total cells.

The migration and invasion assays were carried out using Transwell plates (EMD Millipore, Billerica, MA, USA). The filter surfaces (8 µm pore size) of the Transwell plates were uniformly coated with 25 mg Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) overnight at 4°C prior to experimentation. The lower chamber was filled with culture medium supplemented with 10% FBS. The A549 cells were carefully transferred onto the matrigel-coated (BD Biosciences, Franklin Lakes, NJ, USA) upper surface of the chamber at a seeding density of 1–4×10⁵ cells per flask (Corning Incorporated, Corning, NY, USA). Following a 24 h incubation at 37°C, the filter was gently removed, and the upper surface was wiped to remove the attached cells. The cells that had invaded through the Matrigel and attached to the lower surface of the filter were fixed with 4% paraformaldehyde and stained with Giemsa (GefanBio Co. Ltd., Shanghai, China). Three replicates were conducted for each condition. A total of 15 random fields in each replicate were selected and the number of cells was counted using an Olympus CKX41 inverted microscope (Olympus Corporation, Tokyo, Japan). The results are presented as the ratio of invasive cells relative to the invasive cells in the control conditions (cells seeded in serum-free media, which invaded towards DMEM containing 10% FBS). Cells that had invaded to the lower sides of the Transwell were fixed with 4% (w/v) paraformaldehyde and stained using 0.05% crystal violet (Amresco, LLC, Solon, OH, USA) for cell counting as described above. Cells on the upper side of the membrane were scraped off and cells that had migrated to the lower side of the membrane were fixed in 4% paraformaldehyde, stained with 0.1% crystal violet for 30 min at room temperature, and washed 3 times with PBS.

Total protein was extracted from the cells for protein immunoblotting using radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology) and protease inhibitors (Shenergy Biocolor, Shanghai, China) and the protein concentration was quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology). Briefly, protein samples (80 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes (Beyotime Institute of Biotechnology). The blots were blocked with 5% non-fat milk for 2 h at room temperature, and then incubated with the following primary antibodies: Rabbit polyclonal anti-EphA7 IgG (1:1,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-918), rabbit polyclonal anti-B-cell lymphoma 2 (Bcl-2; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA; cat. no. 2876), rabbit polyclonal anti-Bcl-2-associated X protein (Bax; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 2774), rabbit polyclonal anti-caspase-3 (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9662), rabbit polyclonal anti-phosphatase and tensin homolog (PTEN; 1:1,000; Cell Signaling Technology, Inc.; cat. no. 9552), rabbit polyclonal anti-AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9272) and anti-phosphorylated (p)-AKT (1:1,000; Cell Signaling Technology, Inc.; cat. no. 9271), and anti-GAPDH (1:1,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-48166) in PBS at 4°C overnight. Subsequently, the membranes were washed with PBS containing 0.1% Tween and incubated with Alexa Fluor^® 700-conjugated goat anti-mouse IgG (1:8,000; Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. A-21036) and IRDye^® 800CW goat anti-rabbit IgG (1:8,000; LI-COR Biosciences, Lincoln, NE, USA) for 1 h at room temperature. The bands were visualized using an imaging system (LI-COR Biosciences), and semi-quantified using Odyssey v1.2 software (LI-COR Biosciences) by measuring intensity (area x optical density). GAPDH was used as an internal control. The results were expressed as fold changes by normalizing the data to the control values.

All data are expressed as the mean ± standard error of mean. Statistical analyses, including an unpaired two-tailed Student's t-test and one-way analysis of variance followed by the Bonferroni's multiple comparison post hoc test were carried out using GraphPad Prism v6.0 (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

To confirm the silencing effect of siEphA7 on EphA7 expression, the EphA7 mRNA expression levels were detected using RT-qPCR. As shown in Fig. 1A, as compared with the control group, there were no significant alterations in the non-specific siRNA-transfected control group, however the EphA7 mRNA expression levels were significantly decreased following transfection with siEphA7 for 24 h. Furthermore, western blotting demonstrated that siEphA7 also downregu-lated the protein expression levels of EphA7 (Fig. 1B). These results indicate that EphA7 expression was significantly silenced post-transfection with siEphA7.

An MTT viability assay was conducted to determine the effects of siEphA7 on cell viability. As shown in Fig. 2A, transfection with siEphA7 for 24 h resulted in a ~50% reduction in proliferation rate, as compared with the control group. Furthermore, the results of the MTT assay were confirmed by TUNEL assay. Transfection with siEphA7 markedly induced apoptosis in A549 cells (Fig. 2B). These results suggest that EphA7 has a key role in the regulation of lung cancer cell growth.

An important factor in tumor metastasis and progression is the ability of tumor cells to invade beyond the limitations of the primary tumor environment. Therefore, a Transwell assay was conducted, in order to investigate the ability of cells to invade through a biological matrix. As shown in Fig. 3A and B, transfection with siEphA7 markedly inhibited the ability of A549 cells to invade through the Matrigel-coated filter; the rate of invasion of A549 cells was decreased by >50%, as compared with the control group.

To explore whether the decreased invasiveness mediated by siEphA7 was associated with cell motility, the effects of siEphA7 on cell migration were detected. As shown in Fig. 3A and C, the migratory capabilities of the A549 cells transfected with siEphA7 were reduced by ~62.9%, as compared with the control group. These results indicate that knockdown of EphA7 significantly inhibits cell invasion and migration in vitro.

Alterations in the expression levels of apoptosis-inducing signaling molecules were evaluated in A549 cells post-transfection with siEphA7 for 24 h. The Bcl-2 family proteins, which include pro-apoptotic Bax and anti-apoptotic Bcl-2, are known regulators of apoptotic pathways (18). Therefore, in the present study, Bcl-2 and Bax protein expression levels were detected, in order to investigate whether these proteins were associated with siEphA7-induced apoptosis. As compared with the control group, transfection with siEphA7 upregulated Bax protein expression levels (Fig. 4A), and significantly inhibited Bcl-2 protein expression levels (Fig. 4B). The inhibition rate was ~50%. These results indicate that siEphA7 induced apoptosis via regulation of the Bcl-2 family protein-mediated mitochondrial pathway.

Since caspase-3 is well established as a major caspase, and its activation ultimately leads to cell death (19), it is often used to detect apoptosis. The present study evaluated whether siEphA7-induced apoptosis was associated with alterations in caspase-3 protein expression. Caspase-3 protein expression levels were significantly upregulated (~1.5-fold) in A549 cells post-transfection with siEphA7 (Fig. 4C). These results indicate that siEphA7 induced apoptosis via upregulation of caspase-3 protein expression.

PTEN, which is an important tumor suppressor that is mutated in various malignancies, is a central negative regulatory factor of the AKT pathway (20). In order to investigate the effects of EphA7 knockdown on the activity of the PTEN/AKT signaling pathway in A549 cells, the protein expression levels of PTEN, AKT and P-AKT were measured using western blotting. The protein expression levels of PTEN were significantly higher in A549 cells transfected with siEphA7, as compared with the control group (Fig. 5A). In addition, the protein expression levels of total AKT were unchanged between the groups, whereas the expression levels of P-AKT were reduced (Fig. 5B and C). These results suggest that the inhibition of EphA7 may inhibit the AKT signaling pathway via upregulation of PTEN in A549 cells.