Family with sequence similarity 13 member A mediates TGF-β1-induced EMT in small airway epithelium of patients with chronic obstructive pulmonary disease

Abstract

Background: To explore the role of family with sequence similarity 13 member A (FAM13A) in TGF-β1-induced EMT in the small airway epithelium of patients with chronic obstructive pulmonary disease (COPD).

Methods: Small airway wall thickness and protein levels of airway remodeling markers, EMT markers, TGF-β1, and FAM13A were measured in lung tissue samples from COPD and non-COPD patients. The correlations of FAM13A expression with COPD severity and EMT marker expression were evaluated. Gain- and loss-of-function assays were performed to explore the functions of FAM13A in cell proliferation, motility, and TGF-β1-induced EMT marker alterations in human bronchial epithelial cell line BEAS-2B.

Results: Independent of smoking status, lung tissue samples from COPD patients exhibited significantly increased small airway thickness and collagen fiber deposition, along with enhanced protein levels of remodeling markers (collagen I, fibronectin, and MMP-9), mesenchymal markers (α-SMA, vimentin, and N-cadherin), TGF-β1, and FAM13A, compared with those from non-COPD patients. FAM13A expression negatively correlated with FEV₁% and PO₂ in COPD patients. In small airway epithelium, FAM13A expression negatively correlated with E-cadherin protein levels and positively correlated with vimentin protein levels. In BEAS-2B cells, TGF-β1 dose-dependently upregulated FAM13A protein levels. FAM13A overexpression significantly promoted cell proliferation and motility in BEAS-2B cells, whereas FAM13A silencing showed contrasting results. Furthermore, FAM13A knockdown partially reversed TGF-β1-induced EMT marker protein alterations in BEAS-2B cells.

Conclusions: FAM13A upregulation is associated with TGF-β1-induced EMT in the small airway epithelium of COPD patients independent of smoking status, serving as a potential therapeutic target for anti-EMT therapy in COPD.

Keywords: Chronic obstructive pulmonary disease; Epithelial–mesenchymal transition; FAM13A; Small airway epithelium; TGF-β.

Fig. 1

Expression of family with sequence similarity 13 member A (FAM13A) in small airway epithelium and its correlation with COPD severity and EMT marker expression. The patients were divided into non-smokers without COPD (n = 22), smokers without COPD (n = 23), non-smokers with COPD (n = 10), and smokers with COPD (n = 14) groups. A IHC staining was carried out to detect FAM13A protein expression in the small airway epithelium of the patients. B–D Multivariate Cox proportional hazards analysis, was performed to examine the correlations of FAM13A age as the control variable, expression with pre-bronchodilator FEV₁%, PO₂, PCO₂, and expression of E-cadherin and vimentin. FEV₁%pre: pre-bronchodilator forced expiratory volume in 1 s; PO₂: partial pressure of oxygen; PCO₂: partial pressure of carbon dioxide

Fig. 2

FAM13A expression in BEAS-2B cells stimulated by TGF-β1 and transfection efficiency of lentiviral vectors overexpressing FAM13A or small hairpin RNA (shRNA) against FAM13A. A BEAS-2B cells were stimulated with different concentrations of TGF-β1 (0, 3, 5, and 10 ng/mL) for 48 h. Western blot analysis was performed to measure FAM13A protein expression. B and C BEAS-2B cells were treated with 0 or 10 ng/mL TGF-β1 for 48 h. Immunocytochemical and immunofluorescent staining was conducted to detect FAM13A protein expression. Representative images are shown. Magnification × 200. D and E BEAS-2B cells were infected with lentiviral vectors expressing FAM13A or shRNA against FAM13A. Western blot analysis and quantitative real-time PCR were performed to measure protein and mRNA levels of FAM13A at 48–72 h after transfection. shRNA-target 2 shows the highest knockdown efficiency. Data are expressed as mean ± SD. n = 3 (technical replicates). The experiment was repeated three times

Fig. 3

FAM13A promoted BEAS-2B cell proliferation. BEAS-2B cells were transfected with lentiviral vectors expressing FAM13A, shRNA against FAM13A, or corresponding negative control and incubated for 48–72 h. A Cells were stained with Ki67. Ki67-positive cells were detected by flow cytometry assay. B Cell numbers were stained with BrdU and observed under microscopy. Scale bar = 20 µm. 20 C Flow cytometry analysis was performed to examine cell apoptosis. Data are expressed as mean ± SD. n = 3 (technical replicates). The experiment was repeated three times

Fig. 4

The effects of FAM13A on cell migration, invasion, and TGF-β1-induced EMT marker alterations in BEAS-2B cells. A and B BEAS-2B cells were transfected with lentiviral vectors expressing FAM13A, shRNA against FAM13A, or corresponding negative control and incubated for 48–72 h. Wound healing and Transwell chamber assays were performed to examine the roles of FAM13A in cell migration and invasion. Representative images are shown. Magnification × 100. C Upper panel: BEAS-2B cells were transfected with lentiviral vectors overexpressing FAM13A or negative control. Western blotting was performed at 48 h after transfection to measure the protein levels of FAM13A, E-cadherin, ZO-1, vimentin, α-SMA, and N-cadherin. GAPDH was used as an internal control. *P < 0.05 vs. control, ^#P < 0.05 vs. negative control. Lower panel: BEAS-2B cells were transfected with shRNA against FAM13A or negative control. Cells were stimulated with TGF-β1 (10 ng/mL) for 48 h. Western blotting was performed at 48 h after transfection to measure the protein levels of FAM13A, E-cadherin, N-cadherin, vimentin, and α-SMA. GAPDH was used as an internal control. Data are expressed as mean ± SD. n = 3 (technical replicates). The experiment was repeated three times. NC: negative control; LV: lentiviral vectors. *P < 0.05 vs. control. ^#P < 0.05 vs. negative control. ^&P < 0.05 vs. shFAM13A. ^$P < 0.05 vs. TGF-β1 + shFAM13A