2.1 Tissue samples

A total of 15 paired NSCLC tissues and normal lung tissues were collected after resection from Xi'an Chest Hospital between December 2020 and January 2022. A total of 9 males and 6 females were included, and the mean age was 56 years (range, 38–71 years). Eight of these patients had squamous cell carcinoma, and 7 had adenocarcinoma. The distance between normal tissue and lung cancer tissue was more than 3 cm. The frozen tissues were maintained at -80 degrees Celsius. Before surgery, the patients did not undergo any treatment. This study was approved by the Ethics Committee of Xi'an Chest Hospital (no. s2020-0029).

2.2 Cell culture

Human NSCLC cell lines were purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured at 37degrees Celsius with 5% CO2 in a humidified incubator. NCI-H1299, NCI-H1975 and 95-D cells were cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin‒streptomycin (HyClone), and A549 cells were maintained in DMEM/F12 medium (Sigma‒Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin‒streptomycin (HyClone; GE Healthcare).

2.3 Tissue immunohistochemistry

Four micron sections were dried at 60 degrees Celsius overnight. The sections were deparaffinized, rehydrated and subjected to antigen retrieval according to established methods. Endogenous peroxidase was blocked with 3% hydrogen peroxide at room temperature for 10 min, washed with PBS for 3 min three times, blocked at room temperature for 30 min with normal goat serum (Abcam), and incubated with a rabbit anti-GABPB1 primary antibody (1:400; cat. no. HPA019653; Atlas Antibodies) for 90 min at room temperature. After washing, goat anti-rabbit IgG antibody (1:1000; cat. no. Ab6721; Abcam) was subsequently added, after which the sections were incubated at room temperature for 1 h and washed with PBS. Then, 3′,3′-diaminobenzidine (Beyotime Institute of Biotechnology) was used to develop the colour for 3 min at room temperature. The slides were counterstained with haematoxylin for 5 min at room temperature, mounted after rinsing with water, and observed using light microscopy (Olympus Corporation).

2.4 Lentiviral vectors and cell transfection

For analysis of the role of GABPB1 in NSCLC, shGABPB1 was constructed with recombinant lentivirus and transfected into the A549 and H1299 cell lines. We examined the efficiency of the transfection using a fluorescence microscope. The cells transfected with shGABPB1 or shCtrl were defined as the experimental group and negative control group, respectively. Lentiviruses containing the RNA interference sequence of the target gene GABPB1 and the negative control were chemically synthesized by Shanghai Genechem (Shanghai Genechem Co., Ltd., Shanghai, China). The sequences of GABPB1 shRNA and negative control shRNA were 5ʹ-AGAACCAAATCAACACAAA-3ʹ and 5ʹ-TTCTCCGAACGTGTCACGT-3ʹ, respectively. Cell transfection was carried out according to the manufacturer’s instructions for NSCLC cell lines. NSCLC cells were cultivated in 6-well plates, and negative control lentivirus or the GABPB1-shRNA lentivirus was added according to the multiplicity of transfection. Two groups of cells were collected to determine the knockdown efficiency using a fluorescence microscope (MicroPublisher 3.3, Olympus) after 72 h of transfection. RNA was extracted using SuperfecTRI (Shanghai Pufei Biotechnology Co., Ltd.) according to the manufacturer’s protocol. An ultraviolet spectrophotometer was used to measure the concentration of the extracted RNA. A total of 5 µg of RNA was reverse transcribed using an M-MLV reverse transcriptase kit (Promega Corporation) according to the manufacturer’s protocol. The forward and reverse primers (Shanghai Genechem Co., Ltd.) used were as follows: GABPB1, 5ʹ -CCTAACAGATGAAACGGGTGT-3ʹ and 5ʹ-CCACTGGTTGGAATAGAGTGC-3ʹ; and GAPDH, 5ʹ -TGACTTCAACAGCGACACCCA-3ʹ and 5ʹ-CACCCTGTTGCTGTAGCCAAA-3ʹ. We used a SYBR Master Mixture Real-Time PCR System (TaKaRa Biotechnology Co., Ltd., Dalian, China) to perform RT‒PCR in 12 µl reactions under the following reaction conditions. GAPDH was used as the internal control. RT‒PCR was performed according to the following conditions. Predenaturation: 30 s at 95 degrees Celsius. Denaturation: 40 cycles of 5 s at 95 degrees Celsius and 30 s at 60 degrees Celsius. Dissociation: 15 s at 95 degrees Celsius, 30 s at 60 degrees Celsius, and 15 s at 95 degrees Celsius. The relative gene expression levels were measured and compared via the 2 − ΔΔCT analysis program.

2.5 Colony formation assay

shGABPB1-transfected and shCtrl-transfected cells were digested with trypsin (Sangon Biotech Co., Ltd., Shanghai, China) when they were in the logarithmic growth stage. The cells in each group were inoculated in a 6-well plate culture plate according to the growth of the cells (400–1000 cells/well), and three replicate wells were used. The cell clones were photographed under a fluorescence microscope when the inoculated cells were cultured for 14 days or the number of cells in most single clones was more than 50, 4% paraformaldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added in each pore, the cells were fixed for 30–60 min and washed by PBS, and clean and impurity-free crystal violet dye (Sangon Biotech Co., Ltd., Shanghai, China) solution was added to each pore for 20 min. Then, the samples were washed twice with phosphate-buffered saline (PBS), and colonies were counted manually.

2.6 3-(4,5-Dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide (MTT) assay

For measurement of cell viability, two groups of transfected cells were seeded into a 96-well culture plate at a density of 2000 cells/well and allowed to grow to subconfluence. MTT incubation and absorbance were performed following the manufacturer’s instructions. MTT (0.5 mg/mL; Genview PTY, Ltd., VIC, Australia) was added to each well and incubated for 4 h at 37 degrees Celsius, after which dimethyl sulfoxide (DMSO) (ShiYi Pharmaceutical Group, Shanghai, PR China) (100 µl) was added. After 10 min of shaking, cell proliferation was measured at OD490 using a microplate reader (Tecan, Durham, NC, USA). We examined the cells for five consecutive days and used the average count of three repetitions in each group as the result.

2.7 Cell proliferation assay

shGABPB1-transfected and shCtrl-transfected cells were cultured and digested with trypsin (Sangon Biotech Co., Ltd., Shanghai, China). Then, the resuspended cells were seeded in a 96-well plate at a density of 2,000 cells/100 μl/well and incubated overnight at 37 degrees Celsius in 5% CO2. Green fluorescent protein (GFP), a marker of lentivirus-transfected cells, was expressed in every cell. We counted each cell at different time points with a Celigo image cytometer (Nexcelom Bioscience, Lawrence, MA, USA) and subsequently plotted the cell proliferation curve.

2.8 Cell apoptosis assay

In this study, cell apoptosis was analysed using an Annexin V-FITC Apoptosis Detection Kit (BD Biosciences, C1063). The two groups of passaged cells were collected after transfection. When the cell confluence reached 85%, the cells were digested with 0.25% trypsin (without EDTA) and centrifuged. The cell precipitate was washed with 4 degrees Celsius prechilled D-Hanks solution (pH 7.2–7.4) and phosphate-buffered saline (PBS). The mixture was incubated with 100 μl of the cell suspension, 5 μl of Annexin V/FITC was added at room temperature for 5 min in the dark, 10 µl of 20 µg/ml propidium iodide solution (PI) and 400 µl of PBS were added, and flow detection was immediately performed. Flow cytometric analysis was performed on a Fluorescence Activated Cell Sorting (FACS) Calibur platform (Millipore Accuri™ C6 Plus; BD Biosciences).

2.9 Western blotting

The transfected cell samples were washed twice with cold PBS and lysed in lysis buffer (1 M Tris–HCl (pH 6.8), 2% mercaptoethanol, 20% glycerol, and 4% SDS) on ice for 15 min. The protein concentration was determined with a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal volumes of cellular protein were subjected to 10% SDS‒PAGE (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and electrotransferred to a PVDF membrane (EMD Millipore, Billerica, MA, USA). Then, the PVDF membrane was blocked using TBST solution buffer with 5% nonfat milk and incubated overnight at 4 °C with the following antibodies: rabbit anti-GABPB1 (1:2500; cat. no. HPA019653; Atlas antibodies); and rabbit anti-GAPDH (1:2500; cat. no. ab9485; Abcam). Then, goat anti-rabbit IgG antibody (1:1,0000; cat. no. Ab6721; Abcam) conjugated to horseradish peroxidase was used for 1.5 h. Finally, the immunoreactive bands were visualized using a Pierce™ ECL Western Blotting Substrate Kit (cat. no. K820; BioVision, Inc.) and digitized with Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

2.10 Relevant database analysis

We used the Human Protein Atlas (HPA) [14] and Gene Expression Profiling Interactive Analysis (GEPIA2) [15] databases to analyse the expression of GABPB1 at the protein and RNA levels in human tissues, particularly lung and lung cancer tissues. Using the GSCA (Gene Set Context Analysis) [16] database, we analysed four aspects of NSCLC: the correlation between GABPB1 expression and tumor stage and related pathways, its prognostic value for patients, and the impact of GABPB1 and its methylation on the tumor immune microenvironment.

2.11 Statistical analysis

The statistical analysis was performed with the SPSS 19.0 program (IBM Corporation, Armonk, NY, USA). In this study, the data collected are presented as the mean ± standard deviation (SD). Differences between two groups were determined with Student’s t test. P < 0.05 was considered to indicate statistical significance.