In the present study, 18 LUAD and adjacent normal tissues were obtained surgically from April 2023 to May 2023, which included 10 men and 8 women, with an age range of 58 to 77 years. Inclusion criteria for patients were as follows: i) Individuals diagnosed with LUAD, excluding other forms of lung cancer; ii) patients confirmed through pathological examinations; and iii) individuals willing to actively participate. Exclusion criteria for patients were as follows: i) Individuals with additional health conditions, including chronic diseases; and ii) patients unable to cooperate effectively with researchers. Written consent from all participants involved in the study was acquired. All experiments involving human subjects were carried out in The First Affiliated Hospital of Anhui Medical University. Protocols involving the obtained tissues were approved (approval no. Quick-PJ 2023-04-36, Hefei, China) by The Medical Ethical Committee of The First Affiliated Hospital of Anhui Medical University.

To screen for critical genes that may be involved in LUAD progression, three paired human LUAD tissues were collected for microarray experiments. ITGB4-knocked down A549 cells were also used for microarray experiments to explore downstream signaling pathways. All microarrays were performed using Affymetrix U133 Plus 2.0 arrays (Novogene Co., Ltd.). The kits used were RNA-Quick Purification Kit (cat. no. RN001; EZBioscience) and NovoScript^®Plus All-in-one 1st Strand cDNA Synthesis SuperMix (gDNA Purge) (cat. no. E047; Novoprotein).

Data were normalized (16) and DEGs between NSCLC and adjacent normal tissues were analyzed using the limma package (http://www.bioconductor.org/). The following criteria were used to determine significant DEGs: Fold change ≥2 or ≤0.5 and P<0.05.

The ITGB4 expression pattern in LUAD in The Cancer Genome Atlas (TCGA) dataset, TCGA-LUAD, was obtained through the GEPIA2 online tool (http://gepia2.cancer-pku.cn/#index, accession number: LUAD-TCGA). Pan-cancer analysis was also conducted using the GEPIA2 online tool. In the prognostic analysis, the prognostic data were downloaded from cBioPortal for Cancer Genomics website [datasets: Lung Adenocarcinoma (TCGA, Firehose Legacy) and Lung Adenocarcinoma (MSK, 2021)]. The ITGB4 median expression level was used as the cut-off, with patients with expression levels above the median assigned to the high expression group and patients with expression levels below the median assigned to the low expression group. The prognosis between the different groups was then evaluated using the Kaplan-Meier method and the log-rank test. P<0.05 was considered to indicate a statistically significant difference. Online immunohistochemistry (IHC) data were obtained directly from the Human Protein Atlas (HPA) database (www.proteinatlas.org/). UALCAN was used to obtain the presentation data and survival information of the TCGA-LUAD dataset (17). Furthermore, ITGB4 expression was analyzed using the ONCOMINE database (18,19). Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/eg/) and Reactome pathway analyses, involving commentary, visualization and integrated discovery, were also conducted (https://reactome.org/). The analysis of protein-protein interactions (PPI) and modules was conducted using the STRING (Search Tool for the Retrieval of Interacting Genes) database. The current approach involved uploading the list of DEGs to the STRING website (www.string-db.org/) to assess protein interactions, where interactions with an experimentally validated score exceeding 0.4 were considered significant. For module and hub gene identification, Cytoscape (version 3.9.0; http://cytoscape.org/) software with molecular complex detection (MCODE) criteria (score >3 and nodes >4) was employed.

The human LUAD cell lines, A549 and PC9, were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. These cells were respectively cultured in Ham's F-12K medium and Dulbecco's Modified Eagle Medium with 10% fetal bovine serum (FBS; all from Wuhan Servicebio Technology Co., Ltd.) and 1% penicillin and streptomycin, in a humidified atmosphere at 37°C with 5% CO₂.

Total RNA was obtained using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). cDNA synthesis was then conducted using a PrimeScript™ RT reagent kit (Takara Bio, Inc.) according to the manufacturer's instructions. The thermocycling conditions for qPCR were as follows: Initial denaturation (95°C for 60 sec), then the 40 cycling steps (95°C for 20 sec, annealing for 20 sec at 60°C, and extension at 72°C for 30 sec). TB Green^® Fast qPCR Mix (Takara Bio, Inc.) was then used to quantify the ITGB4 and GAPDH (internal control) expression levels. The primers used in the qPCR were as follows: GAPDH forward, 5′-AGCCACATCGCTCAGACAC-3′ and reverse, 5′-GCCCAATACGACCAAATCC-3′; and ITGB4 forward, 5′-GCAGATCTCCGGTGTACACAAG-3′ and reverse, 5′-GCTTTTTCCCGGCATTGG-3′. mRNA expression was quantified using the 2^−ΔΔCq method (20).

IHC was conducted in accordance with standard laboratory protocols. In short, the paraffin-embedded tissue sections (5 µm) were deparaffinized using xylene and hydrated in an ethanol gradient. The sections were then incubated in 3% BSA (cat. no. A9647; Sigma-Aldrich; Merck KGaA) blocking solution, with gentle shaking at 37°C for 30 min. Then, the sections were incubated with rabbit monoclonal antibody against ITGB4 (1:300; cat. no. 14803; Cell Signaling Technology, Inc.) for 1 h, followed by the HRP-conjugated secondary antibody (1:10,000; cat. no. ab205718; Abcam) working solution at 37°C for 30 min. After further dehydration, the slices were sealed with neutral gum. Microscopic examination was performed, and images were acquired. ITGB4 staining was then scored by two independent observers (including one pathologist) to determine the expression levels. A positive reaction was scored using four graded categories, depending on the intensity of the staining and the percentage of positively stained cells. The sum of the intensity and percentage scores determined the final score.

RNAi in A549 and PC9 cell lines was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) as the transfection reagent. The sequences of the small interfering (si)RNAs (Sangon Biotech Co., Ltd.) used were as follows: siITGB4#1 sense, 5′-CCACAGAGCUGGUGCCCUATT-3′ and antisense, 5′-UAGGGCACCAGCUCUGUGGTT-3′; siITGB4#2 sense, 5′-CAGAGAAGCAGGUGGAACATT-3′ and antisense, 5′-UGUUCCACCUGCUUCUCUGTT-3′; and si-NC sense, 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense, 5′-ACGUGACACGUUCGGAGAATT-3′. The concentration of siRNA used was 20 µM. The duration of siRNA transfection was 48 h at 37°C and subsequent experiments were performed immediately after transfection. Western blot analysis and RT-qPCR were conducted to verify successful transfection of siRNA.

Cell Counting Kit-8 (CCK-8) was used to detect cell proliferation. In short, cells were carefully placed at a density of 2×10⁴ cells per well (0.1 ml) in 96-well plates and allowed to incubate overnight at 37°C. The number of adherent ITGB4-knocked down LUAD cells was calculated at 0, 24, 48 and 72 h using 10 µl of CCK-8 (cat. no. C0037; Beyotime Institute of Biotechnology) and incubated for 1 h at 37°C. The number of cells was then determined using a micro titer plate reader at 450 nm wavelength. To assess the colony formation ability, cells were diluted to 500 cells/well in a 6-well plate. After 10 days, the resulting colonies were stained using a 0.1% crystal violet staining solution (Sangon Biotech Co., Ltd.). The minimum number of cells forming a colony was 50 cells and ImageJ software (version: 1.42q; National Institutes of Health) was used to quantify colonies. An Annexin V-FITC Apoptosis Detection kit (cat. no. CA1020; Beijing Solarbio Science & Technology Co., Ltd.) combined with flow cytometry (model, FC500; Beckman Coulter, Inc.; analysis software, FlowJo 10; FlowJo LLC) and Hoechst staining (cat. no. C0003; Beyotime Institute of Biotechnology; cells were stained for 5 min in room temperature) were performed to detect cell apoptosis.

The invasion and migration abilities of the cells were measured using Transwell inserts (8 µm) with or without Matrigel, respectively. The plates were precoated with Matrigel at 4°C. After coating, they were incubated at 37°C for 3 h. A total of 5×10⁴ cells/well were seeded into the upper chambers of the inserts and 500 µl culture medium containing 20% FBS was added to the lower chambers. After 48 h of incubation at 37°C, the non-invasive cells were carefully wiped away using a cotton-tipped gauze, whereas the invasive cells were stained using 0.1% crystal violet staining solution (Sangon Biotech Co., Ltd.) for 2 h at room temperature. Images of five randomly selected fields were captured by an inverted light microscope (Olympus Corporation) and the number of migratory or invasive cells was calculated.

Cells at a density of 1×10⁵ cells/well were transfected with the relevant siRNA. Once the cells adhered to the plate, a wound was made by scratching the cells with a micropipette tip. All cells were serum-starved (no FBS) during the wound healing assay. Images of the wound were then recorded using a light microscope at 0 or 24 h after wounding.

A total of 14 female NOD/SCID mice (6 weeks-old; weight 28–22 g) were purchased from the Animal Center of Shanghai. Mice were kept in an SPF animal room with a constant temperature of 25°C, a relative humidity of 40–70%, a 12/12-h light/dark cycle and free access to food and water. Mice were subcutaneously injected on the back with cells (10⁶ cells in 100 µl PBS) to produce xenograft tumors in Hefei Normal University. Tumor growth was monitored every 3 days before the tumor could be detected. After that, the tumor growth was monitored every day when the tumor could be detected. In the followed protocol, if the tumor weight in mice reached 10% of the body weight, or the size of the tumor in any dimension exceeded 15 mm, all the mice would be euthanized. If not, the mice would be sacrificed 54 days after cell injection. After 54 days, all the 14 mice were euthanized using CO₂ (35% vol/min) asphyxiation in chamber (630×480×500 mm). The mice were exposed to CO₂ for at least 1 additional min after breathing ceased. The methods for confirming animal death included: Cessation of heartbeat, cessation of breathing, stiffness in the animal and dilated pupils. The tumor volumes were calculated as previously reported (21). The protocol was approved (approval no. HFNU-2023-TK61-1,) by The Medical Ethical Committee of Hefei Normal University (Hefei, China), and followed the principles outlined in the Declaration of Helsinki (2013) for all human or animal experimental investigations. All animal welfare considerations were taken to minimize suffering and distress. The tumor weight in mice should not exceed 10% of the body weight, and the size of the tumor in any dimension should not exceed 15 mm.

Cells were lysed using RIPA protein extraction buffer (cat. no. P0013B) with PSMF (cat. no. ST505; Beyotime Institute of Biotechnology) and 1X SDS loading buffer. Protein concentration was determined using bicinchoninic acid (BCA) method. A total of 20 µg protein was loaded per lane. The proteins were transferred to polyvinylidene difluoride (PVDF) membrane. Blocking was conducted using QuickBlock™ blocking buffer (cat. no. P0252; Beyotime Institute of Biotechnology) for 15 min at room temperature. TBST with 1% Tween 20 was used for washing. The membrane was incubated at 4°C overnight with the following primary antibodies: anti-ITGB4 antibody (1:1,000; cat. no. 14803) and anti-GAPDH (1:1,000; cat. no. 2118; both from Cell Signaling Technology, Inc.). Subsequently, membranes were incubated at room temperature for 2 h with HRP-conjugated anti-rabbit IgG secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.). Protein bands were visualized using a ChemiDoc XRS chemiluminescence detection and imaging system (Bio-Rad Laboratories, Inc.).

Data were analyzed using SPSS v20 software (IBM Corp.). The results were presented as the mean ± standard deviation. Student's paired t-test was used for RT-qPCR, CCK-8, migration and invasion analyses. Survival curves were plotted from the date of operation using the Kaplan-Meier method and were compared using the log-rank test. P<0.05 was considered to indicate a statistically significant difference.

In microarray experiments using the collected human LUAD tissues, 167 DEGs were identified (Table SI), among which 164 DEGs were upregulated and three were downregulated (Fig. 1A and B, Table SII). Furthermore, the 167 DEGs were analyzed using UALCAN and it was found that 14 genes were associated with poor survival (P<0.01; Table I). Then, the interactions between the DEGs were studied and the top 20 identified genes were ranked by interaction level through protein-protein interaction network analysis using STRING website and Cytoscape software (Figs. 1C and S1). In addition, 1,028 genes upregulated in LUAD tissues were identified using data from TCGA dataset (Fig. 1C). After integrated bioinformatical analysis, only three genes, ITGB4, B3GNT3 and CDKN2A, were identified from the aforementioned lists (Fig. 1D). ITGB4 was selected for further study due to its importance in LUAD pathophysiology with rare mechanistical study (15).

To further understand the role of ITGB4 in cancer development, a pan-cancer analysis was first conducted. The difference in ITGB4 expression between tumor and adjacent normal tissues in different tumor types was explored using TCGA database. It was found that ITGB4 was markedly upregulated in multiple types of cancer tissues, including LUAD (Fig. 2A). The same result was also obtained following analysis using the ONCOMINE database (Fig. 2B). In the prognostic analysis, the cases were divided into high and low expression groups according to the ITGB4 expression level. The results indicated that a high ITGB4 expression level was associated with poor overall survival for adrenocortical carcinoma, kidney renal clear cell carcinoma (KIRC), low grade glioma (LGG) and LUAD (Fig. 2C). It was also identified that high ITGB4 expression was associated with poor disease-free survival for KIRC, LGG and LUAD (Fig. 2D). These data indicated the possible oncogenic role of ITGB4.

Next, the role of IGTB4 in LUAD was focused on. The copy number and mRNA expression level of ITGB4 in LUAD was analyzed and it was revealed that gain and amplification significantly promoted ITGB4 expression (Fig. 3A). Data from TCGA (Fig. 3B) and HPA (Fig. 3C) online datasets confirmed the upregulated ITGB4 mRNA and protein expression levels in LUAD. In prognostic analyses, it was found that elevated ITGB4 expression predicted an adverse clinical outcome in LUAD (Fig. 3D), which also suggested the oncogenic role of ITGB4 in LUAD. Moreover, these results were validated using the collected LUAD tissues. The results of the RT-qPCR analysis suggested that ITGB4 expression was significantly higher in LUAD tissues (Fig. 3E), which was also confirmed by IHC (Fig. 3F).

To further confirm the oncogenic role and improve understanding of its biological function, the expression of ITGB4 was knocked down in A549 and PC9 cells using specific siRNAs. The transfection results demonstrated that the two siRNAs (siITGB4#1 and #2) significantly decreased the ITGB4 expression level (Figs. 4A and S2A). Downregulated ITGB4 expression also significantly restrained A549 and PC9 cell proliferation (Fig. 4B) and colony formation (Fig. 4C). Beyond the in vitro experiments, an in vivo tumorigenesis nude mouse model was also constructed. It was found that the tumor volume of the ITGB4-knocked down cells group was significantly smaller than that of the control group (Fig. 4D). Moreover, apoptosis assays demonstrated that ITGB4 knockdown also induced cell apoptosis (Figs. 4E and S2B). Wound healing and Transwell assays indicated that ITGB4 downregulation markedly inhibited LUAD cell migration and invasion (Fig. 5A and B).

Next, the underlying mechanisms were explored. High throughput sequencing and DEG analysis was performed using ITGB4-knocked down A549 cells (Table SIII). A total of 917 DEGs were identified in ITGB4-knocked down A549 cells, including 522 upregulated and 395 downregulated genes (Fig. 5C). Pathway enrichment analysis was subsequently conducted using these DEGs. In the KEGG analysis of upregulated DEGs, cell cycle, oocyte meiosis and viral carcinogenesis pathways were identified. Meanwhile, metabolic pathways, oxidative phosphorylation and Parkinson's disease were identified for the downregulated DEGs (Fig. 5D). In the Reactome analysis demonstrated in Fig. 5E, the enriched pathways for upregulated DEGs consisted of cell cycle mitotic, cell cycle and cell cycle checkpoint pathways. The enriched pathways for downregulated DEGs consisted of metabolism, neutrophil degranulation and metabolism of steroids pathways. Notably, the cell cycle and metabolism pathways were all identified in the KEGG and Reactome pathway enrichment analyses, indicating the potential mechanism underlying the oncogenic influence of ITGB4 on LUAD.