Human type II alveolar epithelial cells (BEAS-2B; BCRC 60074) and lung cancer cell lines (H1975, HCC827 and A549) were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). The cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO₂. All cell lines used were verified by STR through Applied Biosystems (Thermo Fisher Scientific, Inc.).

For transfection, 20 µM small interfering (si)RNA-negative control (NC; 5′-CACUGAUUUCAAAUGGUGCUAUU-3′), overexpression (Oe) plasmid-NC, si-TXNDC9 (5′-TTTGGTAGTCTGAAGCAGC-3′) and Oe-YWHAG were obtained from Shanghai GenePharma Co., Ltd. Cells were incubated with 5% CO₂ at 37°C and were used in subsequent experiments after 48 h of transfection. Cell transfection was performed using Lipofectamine 2000^® Transfection Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Total protein extraction from A549 cells was performed using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) and the protein concentrations were quantified using a BCA kit (Beyotime Institute of Biotechnology). Proteins (40 µg/lane) were separated by 12% SDS-PAGE and then transferred onto PVDF membranes. After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated overnight at 4°C with the following primary antibodies (all purchased from Abcam): Anti-TXNDC9 (1:5,000; cat. no. ab185959), anti-YWHAG (1:1,000; cat. no. ab237732), anti-matrix metalloproteinase (MMP)2 (1:1,000; cat. no. ab92536), anti-MMP9 (1:1,000; cat. no. ab76003), anti-BCL-2 associated X (Bax; 1:1,000; cat. no. ab32503), anti-B-cell lymphoma-2 (Bcl-2; 1:1,000; cat. no. ab32124) and anti-GAPDH (1:2,500; cat. no. ab9485). Following primary incubation, membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated IgG secondary antibody (1:5,000; cat. no. ab6721; Abcam) at room temperature for 2 h. Protein bands were visualized using enhanced chemiluminescence reagent (cat no. P0018AS; Beyotime Institute of Biotechnology) and protein expression levels were detected and semi-quantified using ImageJ software (version 1.46; National Institutes of Health) with GAPDH as the loading control.

Total RNA from was isolated from the BEAS-2B and A549 cells using TRIzol^® reagent and reverse transcribed into cDNA using a SuperScript^™ Double-Stranded cDNA Synthesis kit (both Invitrogen; Thermo Fisher Scientific, Inc.). qPCR for gene quantification was performed using SYBR Premix Ex Taq (Takara Bio, Inc.) and an ABI 7500 Fast Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The following thermocycling conditions were used for qPCR: 95°C for 10 min; followed by 40 cycles of 95°C for 10 sec and 60°C for 60 sec. The following primers (purchased from GenScript) were used for qPCR: TXNDC9 forward, 5′-GTGAAAATGTGGTTTGCCATT-3′ and reverse, 5′-TGCTTTTTCCACATTCAGCTT-3′; YWHAG forward, 5′-GGAGGGTCATCAGTAGCATTG-3′ and reverse, 5′-AGTTATCCAGCAGGCTCAGC-3′ and GAPDH forward, 5′-AGCCACATCGCTCAGACAC-3′ and reverse, 5′-GCCCAATACGACCAAATCC-3′. GAPDH served as the endogenous control and the calculation of relative gene expression was determined using 2^−ΔΔCq method (16).

A549 cells (1×10³ cells/well) were inoculated into 96-well plates and incubated at 37°C for 24, 48 and 72 h. Each well was then supplemented with 10 µl CCK-8 reagent (Beyotime Institute of Biotechnology) and incubated for a further 2 h. The absorbance of each well was detected at a wavelength of 50 nm, using a microplate reader (Bio-Rad Laboratories, Inc.).

A549 cells were resuspended in DMEM supplemented with 10% FBS for colony formation assay; 5×10² cells/well were seeded in 6-well plates and incubated at 37°C with 5% CO₂ for 14 days. Subsequently, cells were fixated using 4% paraformaldehyde for 15 min at 37°C and stained with 0.5% crystal violet solution for 30 min at room temperature. Finally, colonies (>50 cells) were counted manually using an inverted fluorescent microscope (Nikon Corporation; magnification, ×100).

A549 cells (1×10⁵ cells/well) were inoculated in 6-well plates and incubated at 37°C until the cell confluency reached 90–100%. A linear scratch in the cell monolayer was then made using a pipette tip. The cells were washed three times with PBS to remove cellular debris and incubated at 37°C and 5% CO₂. Images were captured by a light microscope at 0 and 24 h, and the area of migrated cells in the linear scratch).

The effect of TXNDC9 silencing on A549 cell apoptosis was detected using TUNEL detection solution (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In brief, A549 cells (1×10⁶ cells/well) were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized in 0.25% Triton X-100 for 20 min at room temperature. Subsequently, after cells were rinsed with PBS, TdT solution and dUTP solution were added and incubated at 37°C for 1 h in the dark. Cells were treated with 10 µg/ml DAPI for nucleus staining for 5 min at 37°C and mounted in an anti-fade reagent (Beijing Solarbio Science & Technology Co., Ltd.). In total, three fields of view were selected at random and an inverted fluorescence microscope (magnification, ×100; Olympus Corporation) was used to observe the excitation and emission wavelengths at 450–500 and 515–565 nm, respectively.

STRING (string-db.org) and GEPIA (gepia.cancer-pku.cn) databases revealed that TXNDC9 was positively associated with YWHAG and bound to YWHAG. To verify this, co-IP assays were performed. Total proteins from the A549 cells were isolated using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) and quantified using BCA kit (Beyotime Institute of Biotechnology). For immunoprecipitation, 500 µg protein was incubated with 2 µg appropriate antibodies including TXNDC9 (1:50; cat. no. ab185959; Abcam), YWHAG (1:200; cat. no. ab237732; Abcam) and IgG (1:2,000; cat. no. A0208; Beyotime Institute of Biotechnology) overnight at 4°C. Subsequently, 40 µl Protein G/A agarose beads (Invitrogen; Thermo Fisher Scientific, Inc.) were added to cell lysate and incubated for 2 h. After beads were washed with PBS three times, precipitated proteins were re-suspended in 2X SDS-PAGE loading buffer, boiled for 5 min and eluted from the beads. Finally, western blot analysis was used to measure the products from IP as aforementioned.

ENCORI database (https://starbase.sysu.edu.cn/panCancer.php) was used to detect TNXDC9 expression in LUAD and to analyze the association between TNXDC9 and the overall survival rate of LUAD patients. GEPIA database (http://gepia.cancer-pku.cn/) was used to explore the correlation between TNXDC9 and YWHAG in LUAD.

Data are presented as the mean ± SD. All experiments were performed in triplicate. Statistical analysis was performed using SPSS 20.0 software (IBM Corp.). One-way ANOVA was used to perform statistical analysis followed by Tukey's multiple comparisons post hoc test. The survival of LUAD patients was subjected to Kaplan-Meier analysis. Correlation between TNXDC9 and YWHAG was evaluated using Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The results from ENCORI suggested that TXNDC9 was markedly upregulated in LUAD cells (Fig. 1A). Additionally, poor survival of patients with LUAD was observed in the high TXNDC9 expression group compared with the low TXNDC9 expression group (Fig. 1B). The relative mRNA and protein expression levels of TXNDC9 in normal lung epithelial (BEAS-2B0 and lung cancer cells (A549, H1975 and HCC827 cells) were detected using RT-qPCR and western blot analysis. Compared with BEAS-2B normal epithelial cells, relative TXNDC9 mRNA and protein expression in lung cancer cells was significantly upregulated, particularly in A549 cells (Fig. 1C and D, respectively). Therefore, A549 cells were selected for use in subsequent experiments.

To investigate the effects of TXNDC9 on LUAD cells, A549 cells were transfected with si-TXNDC9-1/2. mRNA and protein expression levels of TXNDC9 were significantly decreased in the TXNDC9-silenced A549 cells compared with the si-NC group (Fig. 2A and B). si-TXNDC9-2 was chosen for the subsequent experiments as it displayed an improved interference efficiency compared with si-TXNDC9-1. In addition, CCK-8 and colony formation assay results demonstrated that the viability and the proliferation, respectively, of A549 cells were significantly diminished by TXNDC9 silencing compared with the controls (Fig. 2C and D), revealing that TXNDC9 knockdown exerted inhibitory effects on the proliferation of LUAD cells.

Wound healing and Transwell assays were used to determine the relative migration rate and invasive ability. TXNDC9 silencing significantly suppressed the migration rate of A549 cells compared with the si-NC group (Fig. 3A and B); TXNDC9 silencing similarly inhibited A549 cell invasiveness (Fig. 3C and D). Additionally, the effects of TXNDC9 knockdown on apoptosis of A549 cells was detected using TUNEL assay. TXNDC9 knockdown significantly promoted apoptosis of A549 cells (Fig. 3E and F). Moreover, the protein expression levels of matrix metalloproteinase (MMP)2, MMP9 and Bcl-2 were significantly decreased by TXNDC9 knockdown, whereas relative Bax protein expression was significantly increased (Fig. 3G).

The mRNA and protein expression levels of YWHAG were higher in A549 LUAD cells compared with BEAS-2B normal lung epithelial cells (Fig. 4A and B). According to STRING and GEIPA databases, YWHAG and TXNDC9 were co-expressed and positively correlated with each other (Fig. 4C and D). Considering the positive association between YWHAG and TXNDC9, co-IP assay was performed to verify the binding between YWHAG and TXNDC9. TXNDC9 was observed with anti-YWHAG and YWHAG was observed with anti-TXNDC9, revealing that TXNDC9 bound to YWHAG (Fig. 4E). Compared with the si-NC group, the expression of YWHAG was decreased in TXNDC9-silenced A549 cells (Fig. 4F).

A549 cells were transfected with Oe-YWHAG, and the relative mRNA and protein expression of YWHAG were significantly upregulated compared with the Oe-NC-transfected group (Fig. 5A and B). Decreased cell viability and proliferation induced by TXNDC9 knockdown were partially reversed by YWHAG overexpression (Fig. 5C-E), suggesting that overexpression of YWHAG decreased the inhibitory effects of TXNDC9 silencing on A549 cell viability and proliferation. Furthermore, compared with the si-TXNDC9 + Oe-NC group, the decrease in migratory (Fig. 5F and G) and invasive rates (Fig. 5H and I) of the A549 cells were partially reversed following transfection with YWHAG overexpression vector.

TUNEL assay was used to detect the effects of YWHAG overexpression on the apoptosis of TXNDC9-silenced A549 cells. The results demonstrated that the increased apoptosis observed following TXNDC9 silencing was decreased by overexpression of YWHAG compared with the si-TXNDC9 + Oe-NC group (Fig. 6A and B), which indicated that YWHAG overexpression diminished the promotive effects of TXNDC9 on cell apoptosis. Furthermore, the protein levels of apoptosis-related factors including Bax and Bcl-2 and metastasis-related factors including MMP2 and MMP9 were tested. It was found that the downregulated expression levels of MMP2, MMP9 and Bcl-2 were upregulated by YWHAG overexpression, whereas upregulated Bax expression was downregulated (Fig. 6C).