Primers were designed based on the TRB3 sequence published by NCBI GenBank (https://www.ncbi.nlm.nih.gov/nuccore?db=nucleotide; ref. no. MGI:3562334). The target gene was amplified using TRB3 cDNA as a template, of which the product was constructed into a vector for the TRB3 recombinant plasmid (Ad-TRB3). The TRB3-specific short hairpin (sh)RNA was designed as follows: mmu-TRB3-small interfering RNA1 (5′-GAAGAAACCGTTGGAGTTTG-3′) was constructed for the recombinant plasmid Ad-TRB3-shRNA. The plasmids were cultured and purified, and adenovirus packaging was performed. The infectious titer of the recombinant adenovirus was evaluated using a TCID50 assay, which was 1.1×10¹¹ (PFU/ml).

The MLE-12 murine alveolar type II epithelial cells, from Fuxiang Biotechnology Co., Ltd. (Shanghai, China) were cultured in a 5% CO₂ atmosphere at 37°C with saturated humidity.

The cells were seeded (the density was 1.2×10⁶ cells/ml) in a six-well plate and divided into five groups randomly: i) control group (Group C); ii) TGF-β1 group (Group T); iii) TGF-β1 + Ad-GFP group (Group T + G); iv) TGF-β1 + Ad-TRB3 group (Group T + TRB3); and v) TGF-β1 + Ad-TRB3-shRNA group (Group T + TRB3-shRNA). The cells were infected with the corresponding adenovirus vectors when at 60% confluency, MLE-12 cells in logarithmic growth phase were infected with multiple types of virus. Each virus was infected at three multiplicities of infection: 50, 100 and 200 for Ad-GFP; 200, 400 and 800 for Ad-TRB3; 100, 200 and 400 for Ad-TRB3-shRNA. The transduction time was 48 h. Subsequently, the cells were harvested, and total protein and RNA were extracted from the cells in each group.

Total RNA was extracted from the MLE-12 cells using TRIzol (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reverse transcription was performed using the PrimeScript RT reagent kit (Takara Bio, Inc., Otsu, Japan) and incubated at 37°C for 15 min and at 85°C for 5 sec. The target genes were then amplified using the SYBR Premix Ex Taq kit (Takara Bio, Inc.). Thermocycling conditions were as follows: Initial denaturation at 94°C for 4 min, followed by 40 cycles of 94°C for 30 sec, 58°C for 30 sec and 72°C for 30 sec. The results were analyzed using the software of the ABI 7500 system (version 2.0.6; Applied Biosystems; Thermo Fisher Scientific, Inc.). The primers for target genes are listed in Table I. β-actin was used as an internal control, and values for each target gene were normalized using the expression level of β-actin (10).

Total cell protein was extracted using cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), according to the manufacturer's protocol. A total of 5 µg protein was loaded in each lane. SDS-PAGE (10%) was performed for each group, and the proteins were transferred from the gel onto PVDF membranes. Following blocking for 2 h using 1X Bovine lacto transfer technique optimizer (Thermo Fisher Scientific, Inc.), the membranes were incubated with anti-TRB3 (cat. no. sc-390242; Santa Cruz Biotechnology Inc., Dallas, TX, USA), anti-phosphorylated (p-)Smad3 (cat. no. 9523S; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-E-cadherin (cat. no. wl01482; Wanleibio Co., Ltd., Shanghai, China), anti-Vimentin (cat. no. wl00742; Wanleibio Co., Ltd.), anti-Fibronectin (cat. no. sc-69681; Santa Cruz Biotechnology, Inc.) and anti-GAPDH (cat. no. sc-47724; Santa Cruz Biotechnology, Inc.) antibodies at 4°C, overnight. The concentration of each primary antibody was 1:1,000. Subsequently, the membranes were incubated with secondary antibody labeled with horseradish peroxidase (1:10,000; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) at room temperature for 1.5 h. Following incubation with the secondary antibody, all membranes were washed, incubated in the Western Lightningä Chemiluminescence reagent (PerkinElmer, Inc., Waltham, MA USA), and photographic films were exposed to the membrane in a darkroom. Finally, images of the membranes were captured using the gel imaging system from LabWorksä 4.5 (UVP, LLC, Phoenix, AZ, USA), and the band brightness of each group was calculated.

The cells were seeded onto coverslips and fixed using paraformaldehyde. Following this, rabbit TRB3 antibody (1:1,000; sc-390242; Santa Cruz Biotechnology, Inc.) was used as the primary antibody and was incubated at 4°C for 24 h. Donkey anti-rabbit immunoglobulin G labeled with FITC (1:1,000; cat. no. sab4600003; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used as the secondary antibody and was incubated at 37° for 1.5 h. The nucleus was stained with DAPI (Sigma; Merck KGaA). Finally, the coverslip was placed onto the object slide, examined under a confocal laser scanning microscope (CLSM, FV1000, Olympus Corporation, Tokyo, Japan), and images were captured.

The MLE-12 cells were plated into a six-well plate and infected according to the grouping. The cells were harvested and resuspended 48 h after infection and stained following the protocols of the ApoScreen Annexin V Apoptosis kit (SouthernBiotech, Birmingham, AL, USA) and Propidium Iodide kit (Roche Diagnostics, Basel, Switzerland). Following staining for 1 h, the cells were examined and analyzed using CellQuest software (version 6.0; Becton, Dickinson and Company, Franklin Lakes, NJ, USA).

The MLE-12 cells were cultured in 96-well plates and infected according to the grouping. After 48 h, 10 µl CCK-8 solution was pipetted into each well, and the incubation was continued for 2 h. The absorbance was measured at 450 nm using a microplate reader. The OD value was in proportion to the living cell number.

Using data from the present study, a database was established using SPSS19.0 (IBM Corp., Armonk, NY, USA). All data were consistent with normal distribution and are presented as the mean ± standard deviation. Differences between two means were compared using a t-test, while that among multiple groups using analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The mRNA and protein expression levels of TRB3 were low in normal MLE-12 cells but were significantly higher in the TGF-β1-stimulated cells, particularly in Group T + TRB3. When TRB3-shRNA was added, the mRNA and protein levels of TRB3 were the lowest (P<0.05; Table II). Furthermore, the IF assay suggested that the expression of TRB3 in the nucleus was increased in the TGF-β1-stimulated MLE-12 cells (Fig. 1).

The protein expression level of p-Smad3 in the MLE-12 cells was assessed when TRB3 was upregulated or downregulated, respectively (P<0.05). The results demonstrated that, when TRB3 was overexpressed, the protein expression of p-Smad3 increased (P<0.05). By contrast, the downregulation of TRB3 led to decreased protein expression of p-Smad3 (P<0.05; Table III).

Further investigation found that, compared with the TGF-β1 stimulation group, the overexpression of TRB3 enhanced the mRNA expression levels of α-smooth muscle actin (α-SMA) and collagen type I (CollaI), two fibroblast markers, in MLE-12 cells (P<0.05; Table IV). In addition, the results of the western blot analysis indicated that the expression of E-cadherin (epithelial marker) decreased, whereas the expression levels of Vimentin and Fibronectin (mesenchymal markers) increased (P<0.05; Table V; Fig. 2). When the expression of TRB3 was downregulated by RNA interference, the mRNA expression levels of α-SMA and CollaI were also downregulated (P<0.05; Table IV). Furthermore, the expression of E-cadherin increased, and the expression levels of Vimentin and Fibronectin decreased (P<0.05) (Table V; Fig. 2). Compared with Group C, the morphology of cells in Group T changed from polygonal or rectangular to spindle-shaped (Fig. 3A and B).

The results of the flow cytometry and CCK assays demonstrated that, following TGF-β1 stimulation, MLE-12 cell apoptosis increased (P<0.05) and proliferation decreased (P<0.05). The overexpression of TRB3 promoted the cell apoptosis induced by TGF-β1, leading to the significant inhibition of cell proliferation. Furthermore, the downregulated expression of TRB3 reduced the cell apoptosis induced by TGF-β1 (P<0.05), thereby attenuating the inhibitory effect of TGF-β1 on cell proliferation (Fig. 4, Table VI).