The H460 human NSCLC line was obtained from Chemical Biology Research Center (Wenzhou Medical University, Wenzhou, China). The cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), and 1% antibiotic-antimycotic (Gibco) at 37°C and 10% CO₂.

A 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (Beyotime Institute of Biotechnology, Haimen, China) was used to determine the proliferation of H460 cells. The cells (4,000 cells/well) were seeded and cultured for 24 h in 96-well plates, and serum-free medium was added for 4 h, following which cells were stimulated with (0, 5, 10, 50, 100 and 200 ng/ml rhFGF18 (Bioreactor, Wenzhou, China) (19), 5 µmol ERK inhibitor (FR180204, Sigma-Aldrich), 5 µmol p38 inhibitor (SB203580, Sigma-Aldrich) and further incubated for 0, 24, 48 and 72 h. Subsequently, 20 µl of 5 mg/ml MTT (BioSharp, Hefei, China) was added to each well, the plate was incubated for 4 h and the absorbance at 490 nm (SpectraMax M2, Molecular Devices, Sunnyvale, CA, USA) was subsequently detected. In each group, three wells were measured for cell proliferation; the data are shown as the mean ± standard deviation (SD).

Cell cycle distribution was analyzed by propidium iodide (PI; BD Bioscience, San Jose, CA, USA) staining and flow cytometry. The H460 cells (20,000 cells/well) were seeded in 6-well plates and exposed to (0, 5, 10 and 50 ng/ml) FGF18 for 48 h. The cells were then harvested, fixed with 70% ice-cold ethanol, and stored at −20°C until analysis. After fixation, the cells were washed twice with cold phosphate-buffered saline (PBS) and centrifuged, following which the supernatants were removed. The pellet was resuspended and stained with PBS containing 50 mg/ml PI and 100 mg/ml RNaseA for 20 min in the dark. The DNA content was analyzed by flow cytometry using a FACSCalibur instrument and CellQuest software (BD Bioscience). The cell cycle data were analyzed using FlowJo 7.6 software, version (FlowJo, LLC, Ashland, OR, USA) and the data are shown as the mean ± SD.

H460 cells were seeded into 6-well plates and were cultured at 37°C until they reached 100% confluence. The monolayers were scratched with a pipette tip and cultured under normal conditions or (0, 5, 10 and 50 ng/ml) FGF18 after the cell fragments were removed by washing with PBS. Images (Olympus IX51, magnification: ×40, Olympus Corporation, Tokyo, Japan) were captured at 0, 24, 48 and 72 h, and the data are shown as the migration distance between the two edges. The migration distance was assessed using ToupView software. The data are shown as the mean ± SD.

Chemically synthesized FGF18 siRNA (Guangzhou Ribobio Co., Ltd., Guangzhou, China) were used for transfection. To make the transfection mixture, riboFECT™ CP buffer and siRNA were first prepared in 1.5 ml micro-centrifuge tubes. Subsequently, they were mixed with riboFECT CP reagent and allowed to incubate at room temperature for 15 min. Cells in 6-well plates were washed with PBS twice and the transfection mixture was added to the cells. The plates were gently rocked following which the medium was added to the cells. Subsequently, the H460 cells were transfected with FGF18 siRNA for 48 h.

Total RNA was isolated from the cells using a TRIzol plus kit (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's protocol. RNA was reverse transcribed using PrimeScript™ RT Master Mix (Takara Bio, Inc.) according to the manufacturer's instructions. The PCR amplifications were performed for 40 cycles of 94°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec, using a Applied CFX96™ Real-Time PCR (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with 1.0 µl of cDNA and SYBR Green Real-time PCR Master Mix (Takara Bio, Inc.). Data were collected and analyzed by Bio-Rad CFX Manager software. The expression level of each sample was internally normalized against that of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The relative quantitative value was calculated using the 2^−∆∆Ct method (20). Each experiment was performed in triplicate. The primers used in real-time PCR were as follow: Matrix metalloproteinase 26 (MMP26), forward 5′-GGCCAGGTGGTATCTTAGGC-3′ and reverse 5′-AGCTGACCAGTGTTCATTCTTG-3′; FGF18 forward sequence 5′-GGACATGTGCAGGCTGGGCTA-3′ and reverse 5′-GTAGAATTCCGTCTCCTTGCCCTT-3′; and GAPDH forward 5′-ACAACAGCCTCAAGATCATCAG-3′ and reverse 5′-GGTCCACCACTGACACGTTG-3′. The primers were designed and chemically synthesized in Genewiz, Inc. (South Plainfield, NJ, USA).

Cell samples were digested in lysis buffer (Beyotime Institute of Biotechnology), and the protein concentrations were measured using the bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology). Total protein (40 µg) was resolved by 12% odium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto 0.22 µm polyvinylidene fluoride membranes and probed with the following primary antibodies, overnight at −4°C: Anti-human FGF18 and anti-MMP26 (Abcam, Cambridge, MA, USA), anti-phosphorylated (p)-ERK1/2, anti-ERK1/2, anti-p-p38, anti-p38 and anti-β-actin (Cell Signaling Technology, Inc., Danvers, MA, USA). Following three washes, the membranes were incubated with horseradish peroxidase conjugated anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h at room temperature. The detection of specific proteins was carried out using an enhanced chemiluminscence western blotting kit (Santa Cruz Biotechnology, Inc.).

For each experiment, three independent replicates were performed. All data are expressed as the mean ± SD. Statistical evaluation was conducted using Student's test. The intergroup differences were compared using one-way analysis of variance followed by Dunnett's test. P<0.05 was considered to indicate a statistically significant difference. *P<0.05, **P<0.0l, and ***P<0.001 vs. control.

A cell proliferation assay was used to investigate the effect of FGF18 on H460 cells. Cells were treated with 0, 5, 10, 50, 100 and 200 ng/ml of FGF18 for 0, 24, 48 or 72 h. As shown in Fig. 1, stimulation with FGF18 significantly increased H460 cell proliferation in a time and concentration-dependent manner. At concentrations of 5, 10 and 50 ng/ml, FGF18 had a remarkable effect on H460 cell proliferation. When time reached 72 h, however, FGF18 was unable to affect the growth as a consequence of the too long culture. The results suggest that FGF18 has a role in promoting the proliferation of lung cancer cells.

Different concentrations of FGF18 promoted proliferation of H460 cells, indicating that FGF18 may alter cell cycle-related events in H460 cells. Therefore, the effects of FGF18 on cell cycle kinetics were investigated to understand how FGF18 may regulate the cell cycle. As shown in Fig. 2A-E, stimulation with FGF18 increased the proportion of cells in the G0/G1 phase and reduced the proportion of cells in the S or G2/M phases compared with untreated H460 cells. These results suggest that the FGF18-mediated increase in H460 cell proliferation occurred via increasing the proportion of cells in G0/G1 phase and S phase.

A wound healing assay was used to investigate the effects of FGF18 on cell migration. Following treatment with 5, 10 and 50 ng/ml FGF18 for 24, 48 or 72 h, the migration distance was increased in H460 cells compared with the control untreated cells (Fig. 3A and B). Additionally, the effect of FGF18 on the expression of the migration-related factor MMP26, was investigated in H460 cells. As shown in the results of the western blot and RT-qPCR assays in Fig. 3C and E, FGF18 increased the mRNA and protein expression levels of MMP26 in a dose-dependent manner, particularly at 10 and 50 ng/ml concentrations. These results suggest that FGF18 promotes the migration of H460 cells and affects the migration-related factor MMP26.

The above findings indicate that FGF18 significantly promotes the proliferation and migration of H460 cells. Moreover, the expression of MMP26 was promoted by FGF18 in H460 cells. However, the underlying mechanisms responsible for the effect of FGF18 are unclear. Hence, the effect of FGF18 on the signal transduction of MAPKs was further assessed by western blot analysis. H460 cells were treated with 5, 10 and 50 ng/ml FGF18 for 48 h, and the total protein lysates collected and subjected to western blotting with p-ERK1/2, ERK1/2, p38 MAPK and p-p38 MAPK antibodies. As shown in Fig. 4A and B, FGF18 increased the levels of p-ERK1/2 and p-p38. To address which of these pathways are regulating these effects, we used the specific inhibitors, FR180204 (ERK inhibitor) and SB203580 (p38 inhibitor). Results showed that the effect of FGF18 stimulation on proliferation and migration was inhibited by application of 5 µmol/l FR180204 and 5 µmol/l SB203580 (Fig. 4C-E). However, MMP26 protein did not change by the inhibitors (Fig. 4F and G). These data suggest that the effects of FGF18 in proliferation and migration of H460 cells is potentially mediated via modulations of the ERK1/2 pathway and p38 MAPK pathways.

The RT-qPCR results indicated that the mRNA expression of FGF18 in H460 cells transfected with siFGF18 was reduced compared with control group (Fig. 5A). Additionally, FGF18 protein expression was examined using western blot analyses (Fig. 5B and C), with the results consistent with the mRNA data. These results demonstrate the successful knockdown of FGF18 using siRNA.

The proliferation and migration of cancer cells are key steps in the progression of cancer. Following cell transfection, the effect of FGF18 siRNA on cell proliferation was evaluated in H460. The OD values were significantly reduced in the siFGF18 group compared with the control group (Fig. 6A), indicating that FGF18 siRNA inhibited cell proliferation in H460 cells. However, as shown in Fig. 6B and C, the siFGF18 group did not exhibit alterations in the cell cycle of H460 cells. In addition, the migration distance of siFGF18-transfected H460 cells was reduced compared with the control groups (Fig. 6D and E). Expression of MMP26 gene and protein was additionally significantly reduced following siFGF18, with a 57% gene inhibition rate and 62% protein inhibition rate, respectively (Fig. 6F-H). The levels of p-ERK and p-p38 protein were significantly lower in the siFGF18 group compared with the cells in the control group (Fig. 6I and J). Taken together, these results suggest that FGF18 siRNA is able to repress cell proliferation and migration, and this effect is potentially mediated through the ERK and pP38 signaling pathways in H460 cells.