The human CRC cell lines SW480 (cat. no. CCL-228™) and HCT116 (cat. no. CCL-247™) were purchased from the American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin. Cultures were maintained in a humidified incubator at 37°C with 5% CO₂. All cells used in experiments were in the logarithmic growth phase. Cell line authentication was performed prior to the study, and routine testing confirmed the absence of mycoplasma contamination.

For transient knockdown, siRNAs targeting ITGB4 (siITGB4) and a negative control siRNA (siNC) were synthesized by Shanghai GenePharma Co., Ltd. For EZR overexpression, a human EZR open reading frame was cloned into the pcDNA3.1 vector (pcDNA3.1-EZR), with the empty vector serving as a control (pcDNA3.1-vector); these were also obtained from Shanghai GenePharma Co., Ltd. For stable knockdown, shRNAs targeting ITGB4 (shITGB4) and a non-targeting control (shNC) were cloned into a lentiviral vector by Shanghai GenePharma Co., Ltd. The sequences for all siRNAs and shRNAs used are listed in Table SI. Transfections were performed using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol when cell confluence reached 80-90%. For experiments performed in 6-well plates, 1.2 μg of plasmid or 50 nM of siRNA was transfected per well. The transfection was performed at 37°C for 6-8 h. Following transfection, subsequent experiments were performed after 24 h (for RNA extraction) or 48 h (for protein extraction).

To establish stable ITGB4-knockdown cell lines, HCT116 cells, cultured to 70% confluence, were transfected with shITGB4 or shNC vectors. A total of 48 h post-transfection, the cells were subjected to selection with 600 μg/ml G418 (Neomycin). The selection medium was refreshed every 2-3 days. After ~1 week, when significant cell death was observed, the G418 concentration was reduced to 300 μg/ml for maintenance. After 10-14 days of selection, resistant cell colonies were pooled, expanded, and cultured without G418. The efficiency of stable knockdown was confirmed by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis.

Total RNA was extracted from cells using the RNA-Easy Kit (Vazyme Biotech Co., Ltd.). cDNA was synthesized from 1 μg of total RNA using the PrimeScript RT Reagent Kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. RT-qPCR was performed on a 4800 Real-Time PCR System using SYBR Green Master Mix (Vazyme Biotech Co., Ltd.). The thermal cycling conditions were: 95°C for 5 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. Relative gene expression was calculated using the 2^−ΔΔCq method (19), with β-actin serving as the internal control. Primer sequences are listed in Table SII.

Cells were lysed in RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with a protease inhibitor cocktail (PMSF; Beijing Solarbio Science & Technology Co., Ltd.). Protein concentrations were determined using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). Equal amounts of protein (30 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck KGaA). The membranes were blocked with 5% non-fat milk in TBST (0.1% Tween-20) for 1 h at room temperature and then incubated overnight at 4°C with primary antibodies. The primary antibodies used were: anti-ITGB4 (1:1,000; cat. no. ab261778; Abcam), anti-EZR (1:1,000; cat. no. 26056-1-AP; Proteintech Group, Inc.), anti-β-catenin (1:1,000; cat. no. 51067-2-AP; Proteintech Group, Inc.), anti-c-Myc (1:1,000; cat. no. 10828-1-AP; Proteintech Group, Inc.), anti-cyclin D1 (1:500; cat. no. 60186-1-Ig; Proteintech Group, Inc.) and anti-β-actin (1:1,500; cat. no. TA-09; ZSBG-Bio; http://www.zsbio.com). After washing, membranes were incubated with corresponding HRP-conjugated secondary antibodies (1:2,500; cat. no. A23920; Abbkine Scientific Co., Ltd.). Immunoreactive bands were visualized using an Odyssey scanning system (LI-COR Biosciences). Band intensities were quantified using ImageJ software (version 1.53k; National Institutes of Health).

For the Cell Counting Kit-8 (CCK-8) assay, cells were seeded into 96-well plates at a density of 1×10³ cells/well. At 24, 48, 72 and 96 h, 10 μl of CCK-8 reagent (Dojindo Laboratories, Inc.) was added to each well, followed by a 2 h incubation at 37°C. The absorbance at 450 nm was measured using a microplate reader (Promega Corporation). For the colony formation assay, cells were seeded into 6-well plates at 500 cells/well and cultured for 10-14 days, with the medium changed every 3 days. Colonies were fixed with 4% paraformaldehyde (PFA) at room temperature for 30 min, stained with 0.1% crystal violet at room temperature for 15 min, and colonies containing >50 cells were counted.

For the wound healing assay, cells were cultured to 100% confluence in 6-well plates. A scratch was made using a 200-μl pipette tip. After washing with PBS, cells were cultured in serum-free medium. Images were captured at 0 and 48 h. The migration rate was calculated based on the change in wound width. For Transwell assays, 1×10⁵ cells in 200 μl of serum-free medium were seeded into the upper chamber of a Transwell insert (8.0 μm pore size; Corning, Inc.). The lower chamber contained 700 μl of DMEM with 10% FBS. For the invasion assay, the upper chamber was pre-coated with Matrigel (BD Biosciences) at 37°C for 2 h. After 48 h of incubation, non-migratory/invasive cells were removed from the upper surface. Cells on the lower surface were fixed with 4% PFA at room temperature for 30 min, stained with 0.1% crystal violet at room temperature for 20 min, and counted in five random fields under a light microscope.

Cell apoptosis was assessed using the Annexin V-FITC/PI Apoptosis Detection Kit (Neobioscience Technology Co., Ltd.). After 48 h of transfection, cells were harvested, washed with PBS, and resuspended in binding buffer. Cells were then stained with Annexin V-FITC and Propidium Iodide (PI) for 15 min in the dark. The percentage of apoptotic cells was determined by flow cytometry (BD Biosciences) and analyzed using FlowJo software (version 10; BD Biosciences).

A total of 12 5-week-old male BALB/c nude mice, weighing 18-22 g, were housed in a specific pathogen-free (SPF) facility under a 12/12-h light/dark cycle with free access to food and water. They were randomly divided into two groups (n= 6 per group). HCT116 cells (2×10⁶) stably expressing shNC or shITGB4 were suspended in 100 μl of serum-free DMEM and injected subcutaneously into the right flank of each mouse. Tumor growth was monitored every 3 days by measuring the length (L) and width (W) with calipers. Tumor volume was calculated using the formula: V=(L × W²)/2. After 21 days, all animals were euthanized by cervical dislocation after anesthesia with intraperitoneal injection of 40-50 mg/kg sodium pentobarbital, and tumors were excised, weighed, and processed for histological analysis. Tumors were fixed at 4°C overnight in 4% PFA for subsequent H&E and immunohistochemistry (IHC) staining. The study protocols were approved by the Experimental Animal Care and Use Committee and Ethics Committee of The First Hospital of Hebei Medical University (approval no. 20220395; Shijiazhuang, China).

Total RNA was extracted from SW480 cells transfected with siNC or siITGB4 (three biological replicates per group) using TRIzol™ reagent (cat. no. 5596026CN; Thermo Fisher Scientific, Inc.). RNA quality and integrity were verified using the Agilent 2100 Bioanalyzer. The type of sequencing was paired-end, with a nucleotide length of 150 bp, with TruSeq RNA Exome Kit (cat. no. RS-301-2001; Illumina, Inc.) used for sequencing. Library construction and sequencing were performed by Novogene Technology Co., Ltd. on an Illumina NovaSeq 6000 platform. The loading concentration of the final library was 1.5 ng/μl and was measured using a Qubit 2.0 Fluorometer. After quality control, clean reads were aligned to the human reference genome (GRCh38). Differential expression analysis was performed using DESeq2 (version 1.20.0). Genes with an adjusted P<0.05 and a log2|Fold Change| >1.0 were considered differentially expressed genes (DEGs).

Paraffin-embedded tumor sections (4 μm) were deparaffinized with xylene, rehydrated using ethanol series, and subjected to antigen retrieval. Sections were then incubated with an anti-Ki67 antibody (1:100; cat. no. 27309-1-AP; Proteintech Group, Inc.) overnight at 4°C. Staining was performed using a two-step detection kit (ZSBG-Bio) and visualized with DAB. Images were captured using a light microscope, and the staining intensity was quantified as the average optical density using ImageJ software.

For Co-IP, SW480 cell lysates were incubated with anti-ITGB4 antibody (10 μg; Proteintech Group, Inc.), anti-EZR antibody (10 μg; Proteintech Group, Inc.), or control IgG (10 μg; Proteintech Group, Inc.) using the Pierce™ Classic Magnetic IP/Co-IP Kit (cat. no. 88804; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Immunoprecipitated proteins were analyzed by western blotting. For Co-IF, SW480 cells cultured on glass coverslips were fixed with 4% PFA at room temperature for 30 min, permeabilized with 0.2% Triton X-100, and blocked with 2% BSA at room temperature for 30 min. Cells were then incubated with primary antibodies against ITGB4 (1:50; cat. no. sc-13543; Santa Cruz Biotechnology, Inc.) and EZR (1:50; cat. no. 26056-1-AP; Proteintech Group, Inc.) overnight at 4°C. After washing, cells were incubated with Cy3-conjugated anti-rabbit and FITC-conjugated anti-mouse secondary antibodies (cat. nos. A0516 and A0568; Beyotime Institute of Biotechnology. Nuclei were counterstained with DAPI (1 mg/ml; cat. no. C1002; Beyotime Institute of Biotechnology). Images were acquired using a Zeiss laser scanning confocal microscope (Carl Zeiss AG).

ITGB4 expression data in CRC and adjacent normal tissues were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2; http://gepia2.cancer-pku.cn/) databases. The TCGA and GEPIA2 datasets were prioritized due to their large sample sizes, standardized high-throughput sequencing platforms, and comprehensive clinical annotations, which provide robust statistical power for differential expression and survival analyses compared with smaller, individual datasets. Inclusion criteria involved datasets containing paired tumor and adjacent normal tissues with complete follow-up information. Although clinicopathological variables such as tumor stage and subtype are available in these datasets, the current analysis focused on overall differential expression and survival correlations to identify potential targets, without stratification by specific parameters. The prognostic value of gene expression was assessed using the Kaplan-Meier plotter (https://kmplot.com/analysis/) followed by the log-rank test. Subcellular localization data were retrieved from GeneCards (https://www.genecards.org/). STRING database (https://string-db.org/) was used for PPI interaction network.

All experiments were performed with at least three independent replicates. Data are presented as the mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 8.0 (Dotmatics) and SPSS 21.0 (IBM Corp.). Differences between two groups were analyzed using an unpaired two-tailed Student's t-test. Comparisons among multiple groups were performed using one-way or two-way ANOVA followed by Bonferroni post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

To corroborate previous findings, ITGB4 expression was first analyzed using public databases. Analysis of TCGA and GEPIA2 datasets confirmed that ITGB4 mRNA expression was significantly higher in CRC tissues compared with adjacent normal tissues (Fig. 1A-C). Furthermore, Kaplan-Meier survival analysis revealed that patients with high ITGB4 expression had significantly poorer OS than those with low expression (Fig. 1D). These results are consistent with the authors' prior study (6) and collectively underscore that ITGB4 is an oncogenic factor in CRC, and its overexpression is associated with unfavorable patient outcomes, highlighting its potential as a prognostic biomarker.

To investigate the biological function of ITGB4 in CRC, its expression in SW480 and HCT116 cells was first silenced using siRNAs. Four different siRNA sequences were tested, and the siITGB4-681 sequence, which demonstrated the most potent knockdown efficiency at both mRNA and protein levels, was selected for subsequent experiments (Fig. 2A and B). Transient transfection with siITGB4-681 effectively reduced ITGB4 mRNA and protein expression in both cell lines (Fig. 2C-F). Functional assays revealed that ITGB4 knockdown significantly inhibited cell proliferation, as determined by CCK-8 assays (Fig. 2G and H), and suppressed clonogenic ability, as shown by colony formation assays (Fig. 2I and J). Moreover, flow cytometric analysis indicated that ITGB4 silencing significantly increased the proportion of apoptotic cells (Fig. 2K and L). Transwell assays demonstrated a significant reduction in both the migratory and invasive capacities of CRC cells upon ITGB4 knockdown (Fig. 2M and N). Collectively, these in vitro results suggest that ITGB4 plays a critical role in promoting CRC cell proliferation, survival, migration and invasion.

To extend the in vitro findings, the role of ITGB4 in tumor growth was assessed using a xenograft model. An HCT116 cell line with stable ITGB4 knockdown (shITGB4) was established, confirming sustained suppression of ITGB4 expression (Fig. 3A and B). These cells, along with control cells (shNC), were subcutaneously injected into nude mice. Tumor growth was monitored over 21 days. The tumors formed by shITGB4 cells grew significantly slower and were substantially smaller and lighter at the end of the experiment compared with tumors from the shNC group (Fig. 3C-F). The reduced ITGB4 expression in the xenograft tumors was verified by RT-qPCR and western blotting (Fig. 3G and H). H&E staining confirmed the CRC tissue morphology in the tumors (Fig. 3I). Furthermore, IHC staining for the proliferation marker Ki-67 showed significantly lower positivity in the shITGB4 group, indicating reduced cell proliferation (Fig. 3J and K). These in vivo data provide strong evidence that ITGB4 is essential for promoting CRC tumorigenesis.

To uncover the molecular mechanisms downstream of ITGB4, RNA-seq was performed on SW480 cells following ITGB4 knockdown. This analysis identified 572 DEGs, of which 296 were downregulated and 276 were upregulated (Fig. 4A and B). To pinpoint key downstream effectors, a stringent, multi-step filtering strategy was devised. First, focus was addressed on the 296 downregulated genes from our RNA-seq data. This list was then intersected with two externally derived gene sets: (i) Genes known to be co-expressed with ITGB4 in CRC patient cohorts (from the GEPIA database) and (ii) genes that are significantly upregulated in CRC tissues compared with normal tissues (from the GEPIA2 database). This intersection yielded a refined list of 35 candidate genes. To further narrow the selection, genes whose protein products share a subcellular localization with ITGB4 (that is, plasma membrane or cytoplasm, based on GeneCards data), were prioritized. This criterion was selected because ITGB4 is a transmembrane receptor; therefore, its immediate downstream signal transducers are likely to be physically proximal, residing at the membrane or in the sub-membrane cortical cytoplasm. This systematic process identified five high-confidence candidate target genes: ITGA6, CXADR, EZR, GPRC5C and EPHA2 (Fig. 4C). The regulatory relationship was validated by confirming that knockdown of ITGB4 led to a significant decrease in the mRNA levels of all five candidate genes in both SW480 and HCT116 cells (Fig. 4D and E). Among the candidates, EZR, CXADR and ITGA6 showed predominantly plasma membrane localization, similar to ITGB4, whereas GPRC5C showed mixed localization. This spatial proximity is a prerequisite for direct protein-protein interaction (PPI). Among these candidates, EZR was selected for further investigation based on its strong positive correlation with ITGB4 expression in CRC patient samples (Fig. 5C), its consistent upregulation in CRC tissues (Fig. 5B), its shared subcellular localization (Fig. 5A) and its strong association with poor patient prognosis (Fig. 5D), which mirrored the prognostic significance of ITGB4.

Given the strong correlational evidence, a potential physical interaction between ITGB4 and EZR was next investigated. Co-IP assays in SW480 cells demonstrated that endogenous ITGB4 could be immunoprecipitated with an anti-EZR antibody, and conversely, EZR was pulled down with an anti-ITGB4 antibody, confirming that the two proteins exist in the same complex (Fig. 6A). Co-IF staining further revealed that ITGB4 and EZR extensively co-localize at the plasma membrane of CRC cells (Fig. 6B). This interaction is also supported by PPI network analysis from the STRING database (Fig. 6C). It was then confirmed that ITGB4 regulates EZR expression. As shown in Fig. 6F and G, knockdown of ITGB4 in SW480 cells resulted in a significant reduction of both EZR mRNA and protein levels. To assess the functional hierarchy, rescue experiments were performed. After establishing an efficient EZR overexpression system (Fig. 6D and E), cells were co-transfected with siITGB4 and the EZR overexpression plasmid. Interestingly, overexpression of EZR partially restored the suppressed levels of ITGB4 mRNA and protein induced by siITGB4 (Fig. 6H and I). Given that EZR functions as a downstream effector that activates Wnt/β-catenin signaling, this concomitant increase in ITGB4 suggests the potential existence of a positive feedback loop. It was hypothesized that hyperactive Wnt/β-catenin signaling may transcriptionally upregulate ITGB4 to further sustain the malignant progression. Collectively, these data establish that ITGB4 interacts with EZR and positively regulates its expression at both transcriptional and post-transcriptional levels.

To determine the signaling pathway through which the ITGB4/EZR axis functions, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was performed on the DEGs from the current RNA-seq data. The analysis revealed a significant enrichment in the Wnt/β-catenin signaling pathway (Fig. 7A). To validate this, key proteins of the pathway were examined by western blotting. ITGB4 knockdown in HCT116 cells led to a marked decrease in the levels of active β-catenin and its downstream transcriptional targets, c-Myc and Cyclin D1. Crucially, this downregulation was reversed by the ectopic overexpression of EZR (Fig. 7B). This indicates that ITGB4 activates the Wnt/β-catenin pathway in an EZR-dependent manner. Finally, functional rescue experiments were conducted to confirm that EZR mediates the oncogenic effects of ITGB4. As revealed in Fig. 7C-K, while ITGB4 knockdown suppressed CRC cell proliferation, migration and invasion, concomitant overexpression of EZR significantly rescued these phenotypes. Taken together, these results demonstrate that ITGB4 promotes CRC progression by regulating EZR, which in turn leads to the activation of the Wnt/β-catenin signaling cascade.