Human lung cancer tissue samples and matched adjacent noncancerous lung tissue samples were obtained from 52 patients who underwent resection at the First Affiliated Hospital and Cancer Hospital Affiliated to Zhengzhou University (Zhengzhou, China) between March 2012 and September 2013. All patients had received a definite diagnosis by a clinical pathologist prior to surgery. No patients had received chemotherapy or radiotherapy prior to surgery. Each patient gave signed informed consent for the use of their tissues for research purposes. The study protocol was approved by the Institutional Ethics Committee of Zhengzhou University. The clinicopathological characteristics of the 52 patients are summarized in Table I. All lung cancer tissue specimens and adjacent noncancerous lung tissues were immediately frozen in liquid nitrogen and stored at −80°C until required.

The lung cancer cell lines, A549 and H1299 (American Type Culture Collection, Manassas, VA, USA), were cultured in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) medium supplemented with 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml streptomycin and penicillin (Ameresco, Inc., Framingham, MA, USA) at 37°C in a humidified incubator with an atmosphere containing 5% CO₂. The human bronchial epithelial (HBE) cell line was obtained from Xiangfu Bio (Shanghai, China), and originated from the American Type Culture Collection. HBE cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% FBS; Gibco; Thermo Fisher Scientific, Inc.), 2 µM glutamine (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China), 100 IU/ml penicillin, and 100 µg/ml streptomycin (Ameresco, Inc.) at 37°C in a humidified incubator with an atmosphere containing 5% CO₂. The siRNA sequences used for silencing of human AP-4 (NM_003223) were designed by GenScript siRNA Target Finder online software 2015 (GenScript, Piscataway, NJ, USA) and synthesized by GenScript. The nucleotide sequences were as follows: Forward, 5′-CGGGAUUCCAGUCCCUCAATT-3′ and reverse, 5′-UUGAGGGACUGGAAUCCCGCG-3′. The negative control siRNA (scrambled siRNAs) was supplied by GenScript and the sequences were as follows: Forward, 5′-CAGUCGUGCGCACUCACUATT-3′ and reverse, 5′-UAGUGAGUGCGCACGACUGCG-3′. The siRNAs were transfected into the cultured cells using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Total RNA was extracted from lung cancer tissues and matched adjacent noncancerous lung tissues using RNAiso Reagent (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. RNA (500 ng) was reverse-transcribed to complementary DNA using a PrimeScript RT Reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol. The RT reaction mixture was prepared on ice, and contained 2 µl 5X PrimeScript Buffer, 0.5 µl 1X PrimeScript RT Enzyme Mix I, 0.5 µl Oligo dT Primer (50 µM) and 0.5 µl Random 6 mers (100 µM; Takara Bio, Inc.). After dispensing aliquots of the mix into microtubes (Corning Incorporated, Corning, NY, USA), 500 ng RNA sample and RNase Free dH₂O was added to total 10 µl. The reaction mixture was subsequently incubated at 37°C for 5 min, followed by 85°C for 5 sec and 4°C for a minimum of 10 min. No reverse transcriptase was used as a negative control. Expression of AP-4 messenger RNA (mRNA) was evaluated by RT-qPCR on a 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) with Premix Ex Taq (Probe qPCR) Master Mix and a ROX reference DyeII (50x) (Takara Bio, Inc.). The level of expression was calculated based on the PCR cycle number, and the relative gene expression level was determined using the 2^−ΔΔCq method as previously described (17). The ratios of AP-4, p21 and cyclin D1 to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to normalize the relative levels of AP-4 expression. The primers and probes used were as follows: AP-4 forward primer, 5′-GCCGATCGCTATGGAGTATT-3′, AP-4 reverse primer, 5′-TTAGTGGAATGTTGGCAAGG-3′ and AP-4 probe, 5′-6-carboxyfluorescein (FAM)-TGCCCACTCAGAAGGTGCCC-tetramethylrhodamine (TAMRA) 3′; GAPDH forward primer, 5′-CATCAATGGAAATCCCATCA-3′, GAPDH reverse primer, 5′-TTCTCCATGGTGGTGAAGAC-3′ and GAPDH probe, 5′-FAM-TACTCAGCGCCAGCATCGCC-TAMRA 3′.

Cells were lysed with a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, China). Protein (100 µg) was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked for 1 h in Tris buffered saline with Tween-20 (TBST; Beyotime Institute of Biotechnology) containing 5% non-fat dried milk (Nestlé, Beijing, China) at room temperature, and subsequently incubated with polyclonal rabbit primary antibodies against AP-4 (dilution, 1:1,000; #sc-292568; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), p21 (dilution, 1:1,000; #sc-756; Santa Cruz Biotechnology, Inc.), cyclin D1 (dilution, 1:1,000; #sc-718; Santa Cruz Biotechnology, Inc.) and GAPDH (dilution, 1:1,000; #sc-25778; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following washing three times with TBST, the membranes were incubated with horseradish-peroxidase-conjugated goat secondary antibody (dilution, 1:3,000; #sc-2004; Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Protein bands were detected using enhanced chemiluminescence reagents (GE Healthcare Life Science, Chalfont, UK), and quantified using Adobe Photoshop CS4 software (Adobe Systems, Inc., San Jose, CA, USA).

Cell proliferation following transfection with siRNA was assessed by Cell Counting Kit-8 (CCK-8) (Beyotime Institute of Biotechnology). A549 and H1299 cells were seeded into 96-well plates (Corning Incorporated) at 3×10³ cells/well. The cells were cultured at 37°C for 0, 24, 48 and 72 h. CCK-8 reagent (10 µl) was added to each well. Following incubation for 4 h at 37°C, the absorbance was measured at 450 nm with a multi-detection microplate reader (Synergy HT; Bio-Tek Instruments, Inc., Winooski, VT, USA).

Cells were collected in tubes 48 h subsequent to transfection with siRNA, washed once using cold phosphate-buffered saline (PBS; Beijing Dingguo Changsheng Biotechnology Co., Ltd.), suspended in binding buffer (eBioscience, Inc., San Diego, CA, USA) and stained with Annexin V-fluorescein isothiocyanate (eBioscience, Inc.) in the dark at room temperature for 15 min. In total, 10 µl propidium iodide (PI; 25 µg/ml; eBioscience, Inc.) was added to each tube and cells were incubated at room temperature for 10 min. Within 1 h of staining, the cells underwent flow cytometry, which was performed a FACSCantoII flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and cells were analyzed using BD FACSDiva Software 6.0 (BD Biosciences). Viable cells were Annexin V⁻/PI⁻, early apoptotic cells were Annexin⁺/PI⁻ and late apoptotic/dead cells were Annexin⁺/PI⁺. Nonviable cells that had undergone necrosis were Annexin⁻/PI⁺.

Cells were harvested 48 h subsequent to transfection with siRNA, fixed in 70% ice-cold ethanol (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) overnight, washed with 1X PBS and stained by PI (50 µg/ml) with RNase (50 µg/ml) in the dark at room temperature for 30 min. Cell cycle stage analysis was performed by flow cytometry (FACSCantoII) using ModFit software version 3.0 (Verity Software House, Topsham, ME, USA).

All data are presented as the mean ± standard deviation. Means were compared two groups by Student's t-test, while means of three or more groups were compared with one-way analysis of variance using SPSS for Windows version 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Expression of AP-4 in 52 human lung cancer tissue samples and matched adjacent noncancerous lung tissue samples was assessed using RT-qPCR. The level of AP-4 mRNA in lung cancer tissues was three times higher compared with that in the noncancerous tissue samples (P=0.00005; Fig. 1A). The association between AP-4 mRNA expression and clinicopathological characteristics of the lung cancer patients was additionally analyzed (Table I). The levels of AP-4 mRNA were significantly associated with tumor size (P=0.002), lymph node status (P<0.001) and TNM stage (P=0.0006), but not with sex (P=0.343), age (P=0.615), differentiation degree (P=0.828) or histology (P=0.240) in tumor tissue samples. In addition, western blotting indicated that the levels of AP-4 protein in A549 and H1299 human lung cancer cells were significantly increased compared with those in human bronchial epithelial (HBE) cells (P=0.006; Fig. 1B).

In order to investigate the involvement of AP-4 in the proliferation of human lung cancer cells, A549 and H1299 cells were transfected with scrambled siRNA or AP-4-specific siRNA, and cell proliferation was analyzed using CCK-8. The proliferation of A549 and H1299 cells was decreased following transfection with AP-4-specific transfection for 24 h (P=0.0276, P=0.0322, respectively). Following 72 h of transfection, the proliferation of the two cell lines was significantly decreased (A549, P=0.0017; H1299, P=0.0035) (Fig. 2). These results demonstrated that silencing of AP-4 expression was able to inhibit the proliferation of human lung cancer cells.

AP-4 silencing inhibited the proliferation of lung cancer cells; therefore, the present study aimed to establish whether this effect was caused by cell cycle arrest. Following transfection with scrambled siRNA or AP-4-specific siRNA for 48 h, the cell cycles of A549 and H1299 cells were analyzed using flow cytometry. There was an increased proportion of AP-4 siRNA-transfected cells in the G0/G1 phase (A549, P=0.0083; H1299, P=0.012) and a reduced proportion of these cells in the S phase compared with the G0/G1 and S phase proportions observed in untransfected and scrambled siRNA-transfected cells (Fig. 3A and B). Therefore, silencing of AP-4 may be capable of inducing cell cycle arrest at the G0/G1 phase.

The present study measured the effects of AP-4 silencing on the apoptosis of A549 and H1299 cells. As demonstrated in Fig. 4, the percentages of apoptotic cells in non-transfected or scramble siRNA-transfected A549 and H1299 cells were 4.5% and 2.8%, respectively. Following transfection with AP-4-specific siRNA, the percentages of apoptotic A549 and H1299 cells significantly increased to 15% (P=0.005) and 10% (P=0.006), respectively. These findings indicated that silencing of AP-4 may promote the apoptosis of lung cancer cells.

In order to investigate the mechanisms underlying the effect of AP-4 silencing on the cell cycle of human lung cancer cells, the expression of certain cell-cycle-associated regulators, cyclin-dependent kinase inhibitor p21 and cyclin D1, were examined using western blotting in siRNA-transfected A549 and H1299 cells. Transfection with AP-4-specific siRNA in A459 and H1299 cells reduced the expression of AP-4 (P=0.0009 and P=0.0002, respectively) and cyclin D1 protein (P=0.0023 and P=0.0007, respectively), but increased the levels of p21 expression (P=0.0062 and P=0.0039, respectively) (Fig. 5A and B). This suggested that AP-4 silencing may be capable of suppressing the proliferation of human lung cancer cells and inducing cell cycle arrest at the G0/G1 phase by modulating the expression of certain cell cycle regulators.