The bronchial-tracheal-derived human ASMC cell line was purchased from the American Type Culture Collection (Manassas, VA, USA) and incubated in Dulbecco's Modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) in a humidified atmosphere containing 5% CO₂ at 37°C. ASM cells at passage 9 to 20 were used.

For siRNA transfection, 1×10⁵ cells were seeded in 6-cm dishes for 24 h in DMEM medium (Gibco; Thermo Fisher Scientific, Inc.). The cells (1×10⁵ cells/well) were subsequently transfected with 1 µl (10 nM) FSTL1 siRNA (5′-UGCAAAUACUUACGGACUUUU-3′) or non-targeting siRNA (5′-UCUCCGAACGUGUCACGUTT-3′) (Shanghai Sangong Pharmaceutical Co., Ltd., Shanghai, China) for 18 h using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Protein silencing was confirmed by western blot analysis. ASM cells were subsequently stimulated with 10 ng/ml PDGF-BB (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 24 h.

Cell proliferation was evaluated using an MTT assay. Cells were suspended at 1×10⁴ cells/well and 200 µl of this suspension was plated into each well of a 96-well plate. The ASMC cells were cultured with DMEM and cells were growth-arrested prior to every experiment. They were pre-treated with FSTL1 siRNA for 24 h, followed by PDGF-BB stimulation. Following this, the medium was removed, and 20 µl 5 mg/ml MTT in DMEMwas added. The cells were further incubated in 5% CO₂ at 37°C for 4 h. Formazan was solubilized with 100 µl dimethyl sulfoxide for 10 min. The absorbance of the solution was measured at a wavelength of 570 nm using a microplate reader (Spectra Max 190; Molecular Devices, LLC, Sunnyvale, CA, USA).

The growth-arrested cells were pre-treated with FSTL1 siRNA for 24 h, followed by PDGF-BB stimulation. The cells were harvested and fixed with ice-cold 70% (v/v) ethanol for 24 h. Following centrifugation at 2,000 × g for 10 min at 37°C, the cell pellet was washed with PBS and resuspended in PBS containing 50 µg/ml propidium iodide, 0.1% (v/v) Triton X-100 and 1 µg/ml DNase-free RNase. The cells were subsequently incubated for 1 h in the dark at room temperature. A flow cytometer (FC 500; Beckman Coulter, Inc., Brea, CA, USA) was used to elucidate cell cycle progression, and this was analyzed using Modfit software (version 3.2; Verity Software House, Topsham, ME, USA).

ASM cell migration was measured using a Transwell migration assay. ASM cells at a concentration of 5×10⁴ cells/well, transfected with FSTL1 siRNA, were resuspended gently in DMEM/F-12 medium containing 2% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and added to into the upper chamber. Following this, 600 µl DMEM/F-12 medium containing 10% FBS with or without PDGF-BB was added into the lower chamber. Following incubation at 37°C for 24 h, the cells on the upper membrane surface were removed by washing with PBS, and those that had migrated to the bottom side of the filter were fixed in methanol and stained with Harris hematoxylin solution for 5 min. The mean number of migrated cells from five randomly selected optical fields under a light microscope (magnification, ×100; Olympus Corporation, Tokyo, Japan) was determined.

Total RNA was extracted from ASM cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. For mRNA detection, cDNA was generated using Moloney Murine Leukemia Virus Reverse Transcriptase (Clontech Laboratories, Inc., Mountainview, CA, USA). The expression levels of gene mRNA transcripts were analyzed using specific primers, SYBR^®-Green I reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and the Bio-Rad iQ5 qPCR system (Takara Biotechnology Co., Ltd., Dalian, China), according to the manufacturer's protocol. The specific primers for FSTL1 were as follows: Sense, 5′-CGAGGTGGAGTTGACGAGAAAC-3′ and antisense, 5′-AGGACTGGATCATCATGACGTTCT-3′. GAPDH were 5′-GGCAAATTCAACGGCACAGTC-3′ (sense), 5′-GCTGACAATCTTGAGTGAGTT-3′ (antisense). The PCR procedure was as follows: An initial denaturation step at 94°C for 4 min, followed by 40 cycles of denaturation at 94°C for 20 sec, annealing at 55°C for 30 sec and extension at 72°C for 20 sec, and a melting curve from 65 to 95°C. GAPDH served as a control for normalizing the gene expression levels and the results were analyzed using the 2⁻ΔΔ^Cq method (14). The sequences for GAPDH are as follows: 5′-GGCAAATTCAACGGCACAGTC-3′ (sense) and 5′-GCTGACAATCTTGAGTGAGTT-3′ (antisense).

Cells were lysed using RIPA Cell lysis buffer (Takara Biotechnology, Co., Ltd., Dalian, China) and protein concentrations were determined using the Bicinchoninic Acid assay kit (Pierce; Thermo Fisher Scientific, Inc.). Equal amounts of protein (30 µg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Whatman; GE Healthcare Life Sciences, Chalfont, UK). The membranes were blocked in 5% nonfat milk in TBS with Tween-20 (TBST; 5 mM Tris-HCl at pH 7.4, 136 mM NaCl, 0.1% Tween-20) for 1 h at room temperature prior to hybridization with the following primary antibodies overnight at 4°C. Detection of rabbit anti-FSTL1 (dilution, 1:1,500; Abcam, Cambridge, UK; cat. no. 111969), mouse anti-phospho-extracellular signal-regulated kinase (ERK; dilution, 1:2,000; Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. sc-81492), rabbit anti-ERK (dilution, 1:2,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-292838), mouse anti-AKT (dilution, 1:2,000; Santa Cruz Biotechnology, Inc.; sc-5298), rabbit anti-phospho-AKT (dilution, 1:2,000; Santa Cruz Biotechnology, Inc.; cat. no. sc-135650) was performed. The membranes were subsequently washed three times for 5 min with TBST and incubated with bovine anti-rabbit horseradish peroxidase-conjugated secondary antibodies (dilution, 1:3,000; cat. no. sc-2385) for 1 h at room temperature. The resulting protein bands were visualized using an Enhanced Chemiluminescence reagent according to the manufacturer's protocol. The relative expression levels of each protein compared with GAPDH were analyzed. BandScan software (version 5.0; Glyko, Novato, CA, USA) was used for the quantification of all proteins following western blot analysis.

All experiments were performed in at least triplicate and results are expressed as the mean ± standard deviation. Statistical comparisons were performed using one-way analysis of variance followed by the Student's t-test. P<0.05 was considered to indicate a statistically significant difference.

The present study initially measured the expression levels of FSTL1 in PDGF-BB-treated ASM cells. As presented in Fig. 1A, after 6 h PDGF-BB markedly increased the mRNA expression levels of FSTL1, reaching a peak at 24 h. Additionally, the expression levels of the FSTL1 protein were increased by PDGF-BB (Fig. 1B) in a time-dependent manner.

Following this, ASM cells were transfected with FSTL1 siRNA, and the efficiency of transfection was evaluated by western blot and RT-qPCR analysis. Western blot data revealed that transfection of FSTL1 siRNA markedly decreased the expression levels of FSTL1 protein (P=0.016; Fig. 2A). Consistent with the results of western blot, western blot analysis demonstrated that FSTL1 siRNA inhibited the mRNA expression levels of FSTL1 (P=0.029; Fig. 2B).

The role of FSTL1 in PDGF-BB-stimulated proliferation of ASM cells was subsequently investigated. As presented in Fig. 3A, compared with the control group, PDGF-BB treatment markedly increased cell proliferation (P=0.034; P=0.026). However, knockdown of FSTL1 significantly inhibited PDGF-BB-induced ASM cell proliferation (P=0.043; P=0.036).

To further examine the effect of FSTL1 on PDGF-BB-induced ASM cell proliferation, cell cycle progression was evaluated by flow cytometry. As presented in Fig. 3B, PDGF-BB induced the entry of ASM cells to the synthesis phase (S-phase) to undergo proliferation, with 62.5±1.6% cells in G0/G1 phase (compared with 73.1±2.5% in the control; P=0.041) and 31.2±1.9% in S-phase (compared with 19.4±1.3% in the control; P=0.031), whereas the knockdown of FSTL1 significantly arrested the cell cycle at the G2/M phase (18.6±1.7 vs. 5.8±1.1% in the PDGF-BB group; P=0.028).

Following this, the effect of FSTL1 on ASM cell migration induced by PDGF-BB was examined using a Transwell migration assay. As presented in Fig. 4, PDGF-BB resulted in a 2.59-fold increase in the number of cells that migrated through the membrane compared with the untreated control (P=0.023), whereas knockdown of FSTL1 markedly inhibited PDGF-BB-induced ASM cell migration (P=0.036).

To investigate the underlying mechanisms by which FSTL1 affected PDGF-BB-induced ASM cell proliferation and migration, the effect of FSTL1 on the activation of ERK and AKT in PDGF-BB-stimulated ASM cells was examined. As presented in Fig. 5, the protein expressions levels of p-ERK1/2 and p-AKT increased following PDGF-BB stimulation, whereas the knockdown of FSTL1 markedly inhibited PDGF-induced expression levels of p-ERK1/2 and p-AKT.