GEPIA2 (http://gepia2.cancer-pku.cn/) analyzes gene expression differences based on tumor samples and normal samples from both The Cancer Genome Atlas (TCGA; http://portal.gdc.cancer.gov) and the Genotype-Tissue Expression (https://www.gtexportal.org) databases (28). In the present study, this tool facilitated the comparison of GBP5 expression between renal tumor tissue and adjacent normal tissue.

From the GEO database (http://www.ncbi.nlm.nih.gov/geo), two datasets were acquired. GSE53757 (29) included GBP1-6 mRNA data from 72 ccRCC and 72 normal tissues. GSE36895 (30) provided GBP1-6 mRNA data from 23 ccRCC samples and 23 normal tissues. Analysis of GBP1-6 mRNA levels was conducted using these two datasets.

As an online tool, the KM plotter (https://kmplot.com/analysis) provided survival analysis capabilities for 21 tumor types by associating gene expression profiles with clinical prognosis (31). Using this resource, the KIRC dataset was selected in the pan-cancer module, and the ‘auto select best cutoff’ option was applied for patient grouping to evaluate the overall survival of GBP1-7.

GeneMANIA (http://www.genemania.org) is a publicly accessible tool for analyzing genetic and protein interactions, co-expression, pathways and gene co-localization (32). The potential interactions of GBP5 with related genes were assessed using this platform.

TIMER (https://cistrome.shinyapps.io/timer/) is based on six algorithms used to evaluate immune infiltration levels (33). The impact of GBP5 expression on immune cell infiltration in kidney renal clear cell carcinoma (KIRC) was analyzed in the present study. TIMER2.0 (http://timer.comp-genomics.org/) was also employed to investigate GBP5 expression differences between tumor tissue and adjacent tissue, alongside its association with immune cell gene markers in KIRC (34).

Based on single-cell RNA sequencing, the TISCH2 database (http://tisch.comp-genomics.org/) is designed to analyze the TME at single-cell resolution (35). The distribution and expression patterns of GBP5 in the TME were analyzed using the GSE111360 (36) and GSE121636 (37) datasets.

TCGA-KIRC RNA-sequencing data were downloaded from TCGA database. Transcript per million format expression data and the corresponding clinical information were extracted. Boxplots illustrating GBP5 expression across different clinicopathological features, including tumor-lymph node-metastasis (TNM) stages, were generated using the ‘ggplot2 v3.3.5’ R package (RStudio, Inc.). Receiver operating characteristic (ROC) curves were plotted to assess the diagnostic value of GBP5 in ccRCC. Based on the criteria of log₂ fold change ≥2 and the adjusted P-value of <0.05, GBP5-related differentially expressed genes (DEGs) were identified and visualized through a volcano plot plotted using the aforementioned ‘ggplot2’ R package. Gene Ontology (GO) biological process enrichment analysis was performed using the DAVID database (https://david.ncifcrf.gov/tools.jsp). Gene set enrichment analysis (GSEA) was conducted to explore the relevant molecular signaling pathways. Data from the Tracking Tumor Immunophenotype (TIP) database (http://biocc.hrbmu.edu.cn/TIP) were used for immune cell infiltration analysis (38). The relationship between GBP5 expression and immune cell infiltration and circulation was assessed using Spearman's rank correlation analysis.

A total of eleven pairs of clinical specimens were obtained from the University of Hong Kong-Shenzhen Hospital (Shenzhen, P.R. China) between August 2024 and November 2024. Patients were eligible if no radiotherapy or chemotherapy had been administered prior to biopsy, the pathological diagnosis confirmed ccRCC and no other malignant tumors were present. The Ethics Committee of the University of Hong Kong-Shenzhen Hospital approved the collection of renal carcinoma tumor tissues and adjacent normal tissues (approval no. 2024256), ensuring that all procedures complied with the ethical guidelines of the Declaration of Helsinki. All the donors signed an informed consent form.

Human ACHN, Caki-1, 786-O and human proximal tubular epithelial (HK-2) cell lines were procured from Procell Life Science & Technology Co., Ltd. RPMI 1640 complete medium (Procell Life Science & Technology Co., Ltd.) was used to culture the 786-O and ACHN cells. For Caki-1 cells, McCoy's 5A complete medium (Procell Life Science & Technology Co., Ltd.) was used. For HK-2 cells, Dulbecco's modified eagle medium (Gibco; Thermo Fisher Scientific, Inc.) was utilized. A 10% fetal bovine serum solution (FBS; Procell Life Science & Technology Co., Ltd.) and 1% antibiotics solution (100 µg/ml streptomycin and 100 U/ml penicillin) were added to prepare the complete medium. Cultures were placed at 37°C in a 5% CO₂ incubator, Mycoplasma contamination was checked in all cell lines and short tandem repeats analysis was used for authentication.

Lentivirus overexpression, GBP5 overexpression (GBP5 OE; 47597-2; order of vector elements: Ubi-MCS-3FLAG-SV40-puromycin), vector controls (CON254), GBP5 short hairpin RNA (shRNA; order of vector elements: hU6-MCS-CBh-gcGFP-IRES-puromycin) and negative control shRNAs (shNC) were produced by Shanghai GeneChem Co., Ltd. The target sequences of the shRNAs were as follows: shNC (CON313), 5′-TTCTCCGAACGTCACGT-3′; the GBP5-specific shRNA shGBP5#1 (103584–1), 5′-CTGGAAATAGATGGGCAACTT-3′ and the GBP5-specific shRNA shGBP5#2 (103585–1), 5′-TGCCTCATCGAGAACTTTAAT-3′. Caki-1 and 786-O cells were transfected according to manufacturer's instructions. Cells infected with lentivirus were treated with puromycin (2 µg/ml) for 7 days, followed by evaluation of target gene knockdown and overexpression efficiency using western blot analysis. The stable cell lines obtained after successful selection and maintained within 50 passages were used for subsequent experiments.

Tissue and cellular protein lysates were prepared using radioimmunoprecipitation assay buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented with protease and phosphatase inhibitors (cat. no. P1048, Beyotime Institute of Biotechnology). Protein concentrations were determined using the bicinchoninic acid method (cat. no. P0012; Beyotime Institute of Biotechnology). The proteins were resolved using 8–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins (10–50 µg) were then transferred on to nitrocellulose membranes (cat. no. 66485; Pall Life Sciences). The membranes were blocked with 5% skimmed milk for 1 h at 20–25°C, after which the membranes were incubated at 4°C for 8–12 h with primary antibodies against CD163 (1:1,000; cat. no. R381830; Chengdu Zen-BioScience Co., Ltd.), GAPDH (1:5,000; cat. no. D110016-0100; Sangon Biotech Co., Ltd.), β-actin (1:5,000; cat. no. D110022-0100; Sangon Biotech Co., Ltd.), Flag (1:5,000; cat. no. 20543-1-AP; Proteintech Group, Inc.) and GBP5 (1:2,000; cat. no. 13220-1-AP; Proteintech Group, Inc.). The membranes were incubated with secondary antibodies [horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG; 1:5,000; cat. no. 511203; Chengdu Zen-BioScience Co., Ltd.] and HRP-conjugated goat anti-mouse IgG (1:5,000; cat. no. 511103; Chengdu Zen-BioScience Co., Ltd.) at 20–25°C for 2 h. Protein signals were detected using the ChemiDoc^™ MP enhanced chemiluminescence system (Bio-Rad Laboratories, Inc.) and quantified using the ImageJ software (version 1.40 g; National Institutes of Health).

786-O and Caki-1 cells were seeded in 12-well plates and cultured until they reached full confluence. A 1,000 µl pipette tip was used to create a linear scratch in the cell monolayer. The wells were rinsed twice with PBS to remove detached cells and the adherent cells were maintained in a medium containing 1% FBS. Images of the wound area were captured at 0, 10, 12, 24 and 36 h using time-lapse imaging under a light microscope (magnification, ×4).

Transwell assay was performed to assess the migratory potential of ccRCC cells. Cell migration was evaluated using uncoated Transwell chambers (cat. no. 14341, 8 µm pore size; LABSELECT^®; Lanjieke Technology Co., Ltd.) and cell invasion was assessed using Matrigel-coated inserts (Corning, Inc.) pre-incubated at 37°C for 30 min. A total of 3×10⁴ 786-O and Caki-1 cells were seeded into the upper chambers of 24-well plates containing serum-free medium. Culture medium supplemented with 15% FBS was added to the lower chambers, making up 500 µl as a chemoattractant. After incubation at 37°C for 24 h for the invasion assay and 12 h for the migration assay, the cells that had moved to the lower surface of the membrane were fixed with 4% paraformaldehyde at 20–25°C for 30 min, followed by crystal violet staining at 20–25°C for 10 min. Following staining, the cells were imaged using a light microscope (magnification, ×10) and cell counts were performed in three or more randomly selected fields of view.

GraphPad Prism (version 7.0; GraphPad; Dotmatics) was used for statistical analyses. Each experiment was performed in triplicate. Continuous variables are presented as mean ± standard deviation. Both unpaired and paired two-tailed Student's t-tests were used for comparisons between groups. For comparisons involving multiple groups, Tukey's post hoc test was applied after one-way ANOVA. Spearman's or Pearson's correlation coefficients were determined, depending on suitability, to perform correlation analyses. P<0.05 was considered to indicate a statistically significant difference. Additionally, the following criteria indicated statistical significance: *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Research suggests that the GBP gene family members regulate the biological behavior of various cancers, indicating their role in cancer initiation and progression (12). Therefore, the GEPIA2.0 tool was initially applied to assess the expression of the GBP family members in ccRCC. Results revealed that GBP1-7 exhibited a consistent expression pattern, characterized by upregulation in tumor tissues (Fig. 2A-G). Notably, the upregulation of GBP1, GBP2 (39), GBP4 and GBP5 was statistically significant. This observation was consistent with the results of the analysis of ccRCC datasets from the GEO database, further reinforcing their validity (Fig. 2H and I). Furthermore, the Kaplan-Meier survival analysis results indicated that elevated levels of GBP2 (39) and GBP5 were associated with a poor prognosis, whereas low expression of GBP3, GBP4 and GBP7 was associated with unfavorable outcomes (Fig. 2J-P). Collectively, these findings support the hypothesis that the GBP gene family is critically involved in ccRCC, with individual members exhibiting distinct clinical relevance. Research specifically targeting GBP5 in ccRCC has been limited. Therefore, the present study focused on elucidating the function of GBP5 in ccRCC.

To comprehensively assess the expression profile of GBP5 across multiple tumor types, the TIMER database was employed. Analysis showed a marked increase in GBP5 expression across a wide range of malignancies, including KIRC and kidney renal papillary cell carcinoma (Fig. 3A). To investigate the clinical relevance of GBP5 in ccRCC, an in-depth analysis was performed using transcriptomic and clinical data from TCGA. Elevated GBP5 expression was significantly associated with notable clinicopathological factors, including tumor size (T stage), lymph node involvement (N stage), distant metastasis (M stage) and both clinical and pathological stage classifications (Fig. 3B-E). Notably, GBP5 expression increased progressively with advancing tumor stage. In addition, univariate Cox regression analysis indicated that elevated GBP5 expression was associated with unfavorable clinical characteristics in patients with ccRCC (Fig. 3F). To evaluate the diagnostic potential of GBP5 in ccRCC, ROC curve analysis was performed, revealing a good diagnostic efficacy with an area under the curve (AUC) value of 0.934 (Fig. 3G), suggesting high sensitivity and specificity. These results imply that GBP5 is a promising diagnostic biomarker involved in the progression of ccRCC.

To further investigate the potential biological function of GBP5 in ccRCC, DEG analysis was performed and the results were visualized using a volcano plot (Fig. 4A). Notably upregulated and downregulated genes were identified using the criteria: Log₂ fold change >2 and adjusted P-value <0.05. Enrichment analysis was performed on these DEGs, revealing significant associations with various GO terms. Specifically, GO biological process analysis revealed significant enrichment in ‘humoral immune response’, ‘positive regulation of cell activation’, ‘positive regulation of leukocyte activation’ and ‘immune response-regulation signaling pathways’ (Fig. 4B). The enriched GO cellular component terms were the ‘immunoglobulin complex’, ‘external side of plasma membrane’, ‘plasma membrane signaling receptor complex’ and ‘T cell receptor complex’-related components (Fig. 4C). Regarding GO molecular functions, enrichment was observed in functions such as ‘antigen binding’, ‘immunoglobulin receptor binding’, ‘passive transmembrane transporter activity’ and ‘channel activity’-associated functions (Fig. 4D).

Subsequently, GeneMANIA was used to build a protein-protein interaction network for GBP5 in order to identify interacting proteins potentially involved in tumorigenesis. Results indicated that GBP5 exhibited strong co-expression with proteins such as GBP1, GBP2, NLR family pyrin domain containing 3 (NLRP3) and GBP4 (Fig. 4E), implying a potential interaction between GBP5 and these genes, which could promote tumor progression.

To further elucidate the biological pathways regulated by GBP5 across the high and low expression groups, GSEA was performed. As shown in Fig. 4F, several biological pathways showed notable enrichment in the high GBP5 expression group, including IFN-γ response, IL-6/JAK/STAT3 signaling, inflammatory response, G₂/M damage checkpoint and TNF-α signaling through NF-κB. By contrast, pathways such as oxidative phosphorylation, fatty acid metabolism, adipogenesis, estrogen response late and the p53 pathway were enriched in the low GBP5 expression group (Fig. 4G). According to these findings, GBP5 may influence ccRCC progression through mechanisms related to tumor immunity, cell cycle regulation and inflammation.

To investigate whether GBP5 affects immune cell infiltration in ccRCC, the TIMER online tool was used to explore the relationship between GBP5 expression and the abundance of various immune cell types. In ccRCC, a strong positive correlation was observed between GBP5 expression and the levels of immune cell infiltration, including B cells, CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils and dendritic cells (Fig. 5A). These findings suggest that GBP5 serves a role in immune cell infiltration in ccRCC. TIMER2.0 was then utilized to conduct a deeper analysis of the association between GBP5 expression and marker genes of various immune cells (Table I). The data revealed strong positive correlations between GBP5 and marker genes of T cells, CD8⁺ T cells, M2 macrophages and exhausted T cells, further supporting the involvement of GBP5 in immune regulation in ccRCC.

To validate the relationship between GBP5 expression and immune cell infiltration in ccRCC, two single-cell RNA datasets of ccRCC were analyzed from the TISCH2 database. GBP5 expression was assessed in different TME-associated cell subsets. The results demonstrated that in both KIRC GSE111360 and GSE121636 datasets, GBP5 was highly expressed in CD4⁺ T cells, proliferating T cells, regulatory T cells, CD8⁺ T cells and natural killer (NK) cells (Fig. 5B and C).

Further analysis of the immunological characteristics of GBP5 in ccRCC was conducted. Results indicated a strong positive correlation between GBP5 and various immune cells, including macrophages, NK cells and CD8⁺ T cells (Fig. 5D). To validate the findings from public databases, western blotting of tumor and adjacent normal tissues from 11 patients with ccRCC was performed. In ccRCC tissues, the levels of GBP5 and CD163 were markedly higher compared with those in adjacent normal tissues. The relative expression levels of GBP5 and CD163 exhibited a positive correlation (Fig. 5E). These findings underscore the association between GBP5 and M2 macrophages and suggest a potential interaction between them.

Several in vitro experiments were performed to investigate the role of GBP5 in ccRCC cells. Western blotting was performed to assess GBP5 expression in Caki-1, 786-O, ACHN and HK-2 cells. Findings demonstrated that GBP5 protein levels were markedly higher in renal cancer cell lines compared with HK-2 cells (Fig. 6A). For subsequent experiments, 786-O and Caki-1 cell lines were selected.

The efficiency of lentivirus-mediated gene silencing was evaluated using western blotting. As shown in Fig. 6B, two lentiviral constructs carrying different shRNA sequences targeting GBP5 were tested in Caki-1 and 786-O cells. Both shRNA constructs effectively knocked down GBP5 expression, with shGBP5#1 showing improved knockdown efficiency. Thus, the shGBP5#1 lentivirus was selected to establish stable GBP5 knockdown cell lines. Similarly, GBP5 OE cell lines were generated using a lentiviral system. Following the exogenous introduction of GBP5 into 786-O and Caki-1 cells, western blotting analysis confirmed a significant upregulation of GBP5 expression (Fig. 6C).

To explore the biological function of GBP5 in cancer progression, a wound healing assay was performed. Notably, the suppression of GBP5 expression significantly decreased the migratory capacity of the cells (Fig. 7A). By contrast, GBP5 overexpression enhanced the wound-closure rate (Fig. 7B). These findings were consistently observed in Caki-1 and 786-O cell lines, indicating that the GBP5-mediated regulation of cell migration is not cell-type specific.

The Transwell migration assays demonstrated that GBP5 knockdown inhibited cell migration, whereas GBP5 overexpression significantly promoted it (Fig. 7C and D). In addition, the Matrigel-coated Transwell invasion assays demonstrated that GBP5 depletion markedly suppressed the invasive capabilities of Caki-1 and 786-O cells, whereas the overexpression of GBP5 exerted the opposite effect, significantly enhancing cell invasion (Fig. 7C and D).