Forkhead box F1 (FOXF1) has been reported to be associated with lung development. However, the role of FOXF1 in asthma is still not fully understood. In the present study, the biological role and the potential mechanism of FOXF1 was explored in transforming growth factor β1 (TGF-β1)-induced bronchial epithelial cell injury. Reverse transcription-quantitative PCR and western blotting were performed to detect the expression levels of FOXF1 and cadherin (CDH) 11 in TGF-β1-induced bronchial epithelial cells. Proliferation, apoptosis and inflammation were assessed using Cell Counting Kit-8 assay, flow cytometry, western blotting and ELISA. Fibrosis and epithelial-mesenchymal transition (EMT) were evaluated using immunofluorescence and western blotting. The expression levels of the proteins involved in the Wnt/β-catenin pathway were detected by western blotting. The results indicated that FOXF1 expression was downregulated, while CDH11 expression was upregulated in TGF-β1-treated BEAS-2B cells. FOXF1 overexpression promoted proliferation, inhibited induction of apoptosis and suppressed the inflammatory response of BEAS-2B cells exposed to TGF-β1. In addition, FOXF1 overexpression restrained TGF-β1-induced bronchial epithelial fibrosis and EMT and inhibited the activation of the Wnt/β-catenin pathway. CDH11 overexpression reversed the effects of FOXF1 overexpression on proliferation, apoptosis, fibrosis, EMT and inflammation by regulating the Wnt/β-catenin pathway. Collectively, the results of the present study suggested that FOXF1 regulated TGF-β1-induced BEAS-2B cell injury by inhibiting CDH11-mediated Wnt/β-catenin signaling. This may provide a novel therapeutic strategy for the treatment of asthma.
Asthma, also known as bronchial asthma, is a common chronic airway inflammatory disease. It is characterized by reversible airflow obstruction, airway inflammation, persistent airway hyperresponsiveness and airway remodeling and the release of inflammatory mediators that can cause tissue damage and airway dysfunction (
The forkhead box (FOX) transcription factor family is composed of a group of evolutionarily conserved transcriptional regulators that play an important role in embryonic growth and development and maintaining cellular homeostasis (
The human bronchial epithelial cell line BEAS-2B was purchased from the American Type Culture Collection. The cells were cultured in DMEM containing 10% FBS, 1% penicillin/streptomycin in an incubator at 37˚C in the presence of 5% CO2 for 24 h. Subsequently, BEAS-2B cells were stimulated with 10 ng/ml TGF-β1 for 24 h at 37˚C. LiCl (β-catenin activator; 20 mM; Sigma-Aldrich; Merck KGaA) was used to treat the cells for 3 h at 37˚C. Untreated cells were regarded as the control group.
FOXF1-specific pcDNA overexpression vector (pcDNA-FOXF1; 20 µg), cadherin (CDH) 11-specific pcDNA overexpression vector (pcDNA-CDH11; 20 µg), and the corresponding negative control (pcDNA3.1; 20 µg) were synthesized by Suzhou GenePharma Co., Ltd. These recombinant plasmids were transfected into BEAS-2B cells using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) for 48 h at 37˚C according to the manufacturer's protocols. Cells were collected after 48 h transfection for the following experiments.
BEAS-2B cell viability was evaluated using the CCK-8 assay. The transfected cells were seeded into 96-well plates at a density of 5x104 cells/ml and cultured in DMEM with 10% FBS at 37˚C. A total of 10 µl CCK-8 solution (Sangon Biotech Co., Ltd.) was added to each well following 24, 48 and 72 h of culture and incubated for 2 h. The absorbance was measured at 450 nm with a microplate reader (Bio-Rad Laboratories, Inc.).
Apoptosis was detected by the FITC Annexin V/propidium iodide (PI) Apoptosis Detection Kit I (Guangzhou RiboBio Co., Ltd.). Briefly, the cells were collected by centrifugation (1,000 x g for 5 min) at room temperature, washed with precooled PBS at 4˚C and re-suspended in binding buffer (Guangzhou RiboBio Co., Ltd.). The cells were incubated with 5 µl Annexin V-FITC (20 µg/ml) at room temperature for 15 min and with 10 µl PI (10 mg/ml) in a dark room for 5 min at room temperature. Apoptotic cells were subsequently analyzed using a BD FACS Calibur flow cytometer (BD Biosciences) and FlowJo software (v10.4; FlowJo LLC) was used for apoptosis analysis.
The cell supernatants of BEAS-2B cells were collected. The concentration levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β in each group were measured using Human TNF-alpha Quantikine ELISA kit (cat. no. DTA00D), Human IL-6 Quantikine ELISA kit (cat. no. D6050) and Human IL-1 beta/IL-1F2 Quantikine ELISA kit (cat. no. DLB50) (R&D Systems, Inc.), respectively. The optical density of each well was assayed at 450 nm using a microplate spectrophotometer.
Total RNA was extracted from BEAS-2B cells using the TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocols. The quality and concentration of RNA were detected using NanoDrop 2000 (Thermo Fisher Scientific) at 260 and 280 nm. Reverse transcription of first-strand cDNAs was performed using PrimeScript RT Master Mix (Perfect Real Time; Takara Bio, Inc.) according to the manufacturer's instructions. cDNA amplification was performed by RT-qPCR using the SYBR Premix Ex Taq™ II kit (Takara Bio, Inc.). The following thermocycling conditions were used for qPCR: Pre-denaturation at 95˚C for 1 min, followed by 40 cycles of denaturation at 95˚C for 15 sec, annealing at 60˚C for 40 sec and extension at 72˚C for 15 sec. The primer sequences for PCR are: FOXF1, forward 5'-GCCATCCAGAGTTCACCCAC-3', and reverse 5'-GAAGCCGAGCCCGTTCAT-3'; CDH11, forward 5'-GGGTTGCCCAAGCTTAATGG-3', and reverse 5'-TTTGATGTCTTTGCGGGGGA-3'; GAPDH, forward 5'-GGGAAACTGTGGCGTGAT-3', and reverse 5'-GAGTGGGTGTCGCTGTTGA-3'. The results were normalized to the expression levels of GAPDH and were measured using the 2-ΔΔCq method (
The collected BEAS-2B cells were fixed in 4% polyoxymethylene for 1 h at room temperature and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Following blocking with 2% BSA for 1 h at room temperature, cells were incubated with an anti-alpha smooth muscle actin primary antibody (α-SMA; 1:500; cat. no. ab32575; Abcam) at 4˚C overnight. Subsequently, the secondary antibody (1:400; cat. no. ab150077; Abcam) was added and incubated for 1 h at room temperature. The cells were subsequently counterstained with DAPI (Beyotime Institute of Biotechnology) for 10 min at 37˚C and examined using a confocal microscope.
The total protein was extracted from the cells using RIPA buffer (Hunan Auragene Biotechnology Co., Ltd.). The BCA Protein Assay kit (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was used to detect the protein concentration according to the manufacturer's protocols. An equal amount of protein (60 µg/lane) was loaded on 8% SDS-polyacrylamide gels and subsequently transferred to a pure nitrocellulose blotting membrane (Pall Life Sciences). Following blocking with 5% non-fat milk in 0.1% Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature, the membranes were incubated with primary antibodies at 4˚C overnight for the following proteins: FOXF1 (cat. no. ab168383), Bcl-2 (cat. no. ab32124), Bax (cat. no. ab32503), poly(ADP ribose) polymerase (PARP; cat. no. ab191217), cleaved PARP (cat. no. ab32064), α-SMA (cat. no. ab108531), fibronectin (cat. no. ab2413), collagen IV (cat. no. ab6586), E-cadherin (cat. no. ab40772), N-cadherin (cat. no. ab76011), vimentin (cat. no. ab92547), CDH11 (cat. no. ab151302) (all 1:1,000), β-catenin (1:5,000; cat. no. ab32572), c-Myc (1:1,000; cat. no. ab32072), c-jun (1:1,000; cat. no. ab40766), and β-actin (1:1,000; cat. no. ab8227) (all Abcam). The membranes were washed and incubated with horseradish peroxidase-labeled secondary antibody (Cell Signaling Technology, Inc.) for 1 h at room temperature. Finally, bands were visualized using an enhanced chemiluminescence detection system (Merck KGaA) and immunoreactivity was detected using Image J software (version 1.49; National Institutes of Health).
Statistical analysis was conducted using SPSS 22.0 (IBM Corp.) and GraphPad Prism 6 (GraphPad Software, Inc.). The unpaired Student's t-test was used for the comparison between two groups. The differences among multiple groups were analyzed using one-way ANOVA with a post hoc Bonferroni multiple comparison test. The data are presented as mean ± standard deviation of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.
To explore the role of FOXF1 in bronchial epithelial cells, the expression levels of FOXF1 were initially investigated in untreated and TGF-β1-treated BEAS-2B cells. RT-qPCR and western blotting indicated a significant decrease in both mRNA and protein expression levels of FOXF1 in TGF-β1-treated BEAS-2B cells compared with the corresponding levels observed in the control cells (
To investigate the roles of FOXF1 in proliferation, apoptosis and inflammation in BEAS-2B cells, FOXF1 was overexpressed in untreated and TGF-β1-treated BEAS-2B cells. The transfection efficiency was evaluated using RT-qPCR and western blotting, which revealed that FOXF1 expression was significant increased following transfection of pcDNA-FOXF1 compared with transfection control (
The biological role of FOXF1 was investigated with regard to the induction of BEAS-2B cell fibrosis and EMT. Treatment of the cells with TGF-β1 markedly enhanced the relative fluorescence intensity of α-SMA in BEAS-2B cells, which was subsequently reduced following FOXF1 overexpression (
The mechanism underlying the regulatory role of FOXF1 in TGF-β1-treated BEAS-2B cells was investigated. As presented in
The CCK-8 assay revealed that CDH11 overexpression and LiCl treatment significantly reduced the optical density values of TGF-β1-treated BEAS-2B cells transfected with the FOXF1 overexpression plasmid compared with the TGF-β1 + pcDNA-FOXF1 group (
Asthma is a chronic airway inflammatory disease, with children being mainly susceptible to the disease (
FOXF1 plays an important role in regulating lung development and lung injury (
CDH11 is a member of the cadherin family of proteins, with its gene located on chromosome 16q22.1(
CDH11 has been revealed to induce cancer cell apoptosis, suppress cell motility and invasion and inhibit cancer progression via the Wnt/β-catenin pathway (
In summary, the results of the present study suggested that FOXF1 overexpression increased BEAS-2B cell proliferation and repressed apoptosis and inflammation. In addition, overexpression of FOXF1 was demonstrated to reduce fibrosis and EMT in TGF-β1-treated BEAS-2B cells. These protective effects may rely on the regulation of the CDH11-mediated Wnt/β-catenin pathway, which may provide a novel fundamental insight into the pathogenesis of asthma and may be useful in developing therapeutic strategies for the treatment of pediatric asthma. Limitations of the present study included the use of only one cell line and the use of an
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All data generated and/or analyzed during this study are included in this published article.
QC and QT designed the study, drafted and revised the manuscript. XL, LL and LW analyzed the data and searched the literature. All authors performed the experiments. All authors read and approved the final manuscript. QC and QT confirm the authenticity of all the raw data.
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The authors declare that they have no competing interests.
TGF-β1 reduces the FOXF1 expression in BEAS-2B cells. (A) mRNA and (B) protein expression levels of FOXF1 in BEAS-2B cells were detected using reverse transcription-quantitative PCR and western blotting, respectively. ***P<0.001. FOXF1, forkhead box F1; TGF-β1, transforming growth factor β1.
Upregulation of FOXF1 reduces TGF-β1-induced damage and release of inflammatory factors in BEAS-2B cells. (A) mRNA and (B) protein expression level of FOXF1 in TGF-β1-treated BEAS-2B cells were detected using reverse transcription-quantitative PCR and western blotting, respectively. (C) Cell Counting Kit-8 assay was used to assess proliferation. Flow cytometry was carried out to (D) identify and (E) quantify apoptosis in TGF-β1-treated BEAS-2B cells transfected with pcDNA-FOXF1. (F) Western blotting was performed to detect the protein expression level of Bcl-2, Bax, PARP and cleaved PARP. (G) Levels of IL-6, TNF-α and IL-1β were detected using ELISA. **P<0.01 and ***P<0.001. FOXF1, forkhead box F1; TGF-β1, transforming growth factor β1; PI, propidium iodide; FITC-A, FITC-Annexin-V; PARP, poly(ADP ribose) polymerase; IL, interleukin; TNF-α, tumor necrosis factor-α.
Upregulation of FOXF1 suppresses TGF-β1-induced BEAS-2B cell fibrosis and epithelial-mesenchymal transition. (A) Immunofluorescence was used to detect the expression of α-SMA (magnification, 200x). (B) Western blotting was performed to detect the protein expression level of α-SMA, fibronectin and collagen IV. (C) The levels of E-cadherin, N-cadherin and vimentin were assessed using western blotting. **P<0.01, ***P<0.001. FOXF1, forkhead box F1; TGF-β1, transforming growth factor β1; α-SMA, smooth muscle α-actin.
FOXF1 regulates the Wnt/β-catenin signaling by inhibition of CDH11. (A) mRNA and (B) protein expression level of CDH11 in TGF-β1-induced BEAS-2B cells transfected with pcDNA-FOXF1 were detected using RT-qPCR and western blotting, respectively. (C) mRNA and (D) protein expression level of CDH11 in TGF-β1-induced BEAS-2B cells transfected with pcDNA-CDH11 were detected using RT-qPCR and western blotting, respectively. (E) Levels of β-catenin, c-Myc and c-JUN in TGF-β1-induced BEAS-2B cells transfected with pcDNA-FOXF1 with or without pcDNA-CDH11. *P<0.05, **P<0.01, ***P<0.001. FOXF1, forkhead box F1; TGF-β1, transforming growth factor β1; CDH11, cadherin 11; RT-qPCR, reverse transcription-quantitative PCR.
FOXF1 reduces TGF-β1-induced BEAS-2B cell injury by inhibiting CDH11-mediated Wnt/β-catenin signaling. (A) Cell Counting Kit-8 assay was used to detect cell proliferation. Flow cytometry was performed to (B) identify and (C) quantify apoptosis. (D) Western blotting was performed to detect the protein expression level of Bcl-2, Bax, PARP and cleaved PARP. (E) Immunofluorescence was used to detect the expression of α-SMA (magnification, 200x). **P<0.01, ***P<0.001. FOXF1, forkhead box F1; TGF-β1, transforming growth factor β1; OD, optical density; PI, propidium iodide; FITC-A, FITC-Annexin-V; PARP, poly(ADP ribose) polymerase; α-SMA, smooth muscle α-actin.
FOXF1 reduces TGF-β1-induced BEAS-2B cell fibrosis, epithelial-mesenchymal transition and inflammation by inhibiting CDH11-mediated Wnt/β-catenin signaling. (A) Western blotting was performed to detect the protein levels of α-SMA, fibronectin and collagen IV. (B) Protein expression levels of E-cadherin, N-cadherin and vimentin were assessed using western blotting. (C) Levels of IL-6, TNF-α and IL-1β were detected using ELISA. *P<0.05, **P<0.01, ***P<0.001. FOXF1, forkhead box F1; TGF-β1, transforming growth factor β1; CDH11, cadherin 11; α-SMA, smooth muscle α actin; IL, interleukin; TNF-α, tumor necrosis factor-α.