Paired cancerous and adjacent normal tissue (ANT) samples from 36 patients with bladder urothelial carcinoma were obtained from a commercially available paraffin-embedded tissue microarray provided by Shanghai Weiao Biotechnology Co., Ltd. The inclusion criteria were as follows: (i) histopathological confirmation of bladder urothelial carcinoma; (ii) availability of matched tumor and ANT samples; (iii) no history of neoadjuvant chemotherapy or radiotherapy to minimize the risk of treatment-related molecular alterations; and (iv) complete clinical data, including age, sex, tumor stage, pathological grade, and survival information. Patients with other malignancies or incomplete clinical records were excluded. Based on HOXB7 expression levels, the patients were classified into a high-expression group (HOXB7-high) and a low-expression group (HOXB7-low). Kaplan–Meier survival analysis was performed to compare the overall survival between the two groups, and differences were assessed using the log-rank test. A p-value < 0.05 was considered statistically significant. The 95% confidence intervals were shown by shaded areas in the survival curves. All statistical analyses and visualizations were carried out using R software (version 4.1.3) with the “survival” and “survminer” packages.

The paraffin sections were baked at 59˚C for 60 min, deparaffinized in xylene (three times, 10 min each), and dehydrated in anhydrous ethanol (three times, 10 min each). After immersion in distilled water and rinsing with running water for 10–15 min, slides were incubated in 3% H₂O₂+NaN₃ for 10 min to block endogenous peroxidase activity, followed by a 10-min rinse. Antigen retrieval was performed using a microwave (medium-high power) in 1x sodium citrate antigen retrieval solution, with an initial preheating step (1–2 min), followed by 10 min of heating. The slides were then allowed to cool to room temperature and reheated for 12 min under the same conditions. After retrieval, slides were washed twice in PBS (15 min each). Tissue sections were incubated with 5% BSA blocking solution at room temperature for 35 min before primary antibody incubation. Primary antibody incubation was performed overnight at 4°C without prior washing. After four PBS washes (15 min each), slides were incubated with the secondary antibody at 37°C for 35 min, followed by four additional PBS washes (15 min each). Manual hematoxylin counterstaining was performed for 2 min, followed by washing under running water for 10 min. After counterstaining, slides were sequentially washed in PBS (three times, 5 min each). Finally, slides were sequentially dehydrated in ethanol and xylene, mounted with a coverslip, and observed under the microscope (IX73P2F, Olympus Corporation).

Gene Expression Profile Interactive Analysis (GEPIA; http://gepia.cancer-pku.cn/) was used to compare the mRNA expression levels of HOXB7 in patients with BC from TCGA/Gene Tissue Expression (GTEx) using a |Log₂FC| cutoff of 1 and a P-value cutoff of 0.01. The FPKM-normalized mRNA expression data for 414 BC tissues and 19 adjacent non-tumor tissues were obtained from the TCGA-BLCA cohort. For differential expression analysis, the expression values were log2-transformed as log2(FPKM+1) and analyzed using the “limma” package and Wilcox-test in R. The differentially expressed genes (DEGs) were identified with a threshold of |log2FC|≥1 and adjusted P-value <0.05. The UALCAN database (http://ualcan.path.uab.edu/) was used to assess HOXB7 expression levels in tumor tissue compared with normal samples across different tumor subgroups.

Bioinformatics analysis of the DEGs and signaling pathway. The R packages “limma” and “ggpubr” were used to identify the DEGs associated with HOXB7 expression between BC and normal samples from the TCGA dataset. The screening criteria included a |log₂FC| ≥1 and an adjusted P-value of <0.05. The R package “pheatmap” was used to visualize the DEGs. To explore the biological pathways associated with HOXB7 expression, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed based on the DEGs using the ‘clusterProfiler’, ‘org.Hs.eg.db’, ‘enrichplot’, and ‘ggplot2’ packages, with a statistical threshold of P < 0.05. Gene annotation was based on the ‘org.Hs.eg.db’ database. DEGs between the high- and low-HOXB7 expression groups were identified based on a threshold of |log₂FC| ≥1 and adjusted P-value <0.05. Enrichment analysis was performed using the enrichKEGG function. P < 0.05 was used as the initial cutoff for identifying potentially enriched pathways. The Benjamini-Hochberg (BH) method was used, and pathways with Q-values [false discovery rate (FDR)-adjusted P-values) <0.05 were considered significantly enriched. Q-values reflect the estimated FDR and help ensure the robustness and biological relevance of the identified pathways.

The human BC cell lines 5637 (CL-0002) and T24 (CL-0227) and the urothelial cell line SV-HUC-1 (CL-0222) were obtained from Procell Life Science & Technology Co., Ltd (Wuhan, China). The human BC cell lines TCCSUP (cat. no. CBP60752) and J82 (cat. no. CBP60311) were purchased from Nanjing Cobioer Biosciences Co., Ltd. The T24, 5637, and J82 cells were cultured in McCoy’s 5A medium (Biological Industries), RPMI 1640 medium, and MEM, respectively, each supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.). The TCCSUP cells were cultured in MEM supplemented with 10% FBS, 1% non-essential amino acids, and 1 mM sodium pyruvate. The SV-HUC-1 cells were cultured in Ham’s F-12K medium with 10% FBS. All cells were maintained in a humidified incubator at 37°C with 5% CO₂ and passaged every 2–3 days based on their growth. All experiments were conducted using cells at passages 3–8 to ensure consistency and minimize genetic drift. For loss-of-function and gain-of-function experiments, 1 μM Ro67-7476 (phospho-ERK1/2 activator, MedChemExpress) was added to HOXB7-knockdown 5637 cells, while 10 μM PD98059 (p-ERK1/2 inhibitor, MedChemExpress) was applied to HOXB7-overexpressing T24 cells. All treatments were incubated under identical conditions for 24 h. After incubation, the medium was replaced with fresh media without drugs.

The siRNA sequences targeting HOXB7 mRNA and si-NC as negative control were synthesized by Shanghai GenePharma Co., Ltd. The sequences were as follows: si-HOXB7#1 forward, 5’-GAGAGUAACUUCCGGAUCUTT-3’ and reverse, 5’−AGAUCCGGAAGUUACUCUCTT−3’; si-HOXB7#2 forward, 5’-CGGAAAGACAGAUCAAGAUTT-3’ and reverse, 5’−AUCUUGAUCUGUCUUUCCGTT− 3’; si-HOXB7#3 forward, 5’-CGAGAGTAACTTCCGGATCTA-3’ and si-NC forward, 5’-UUCUCCGAACGUGUCACGUTT-3’ and reverse, 5’-ACGUGACACGUUCGGAGAATT-3’.

si-HOXB7 and si-NC were transfected into 5637 cells using Lipofectamine 3000 (cat. no. L3000-015, Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. After a 4-h transfection period, the cells were cultured in normal medium for 48 h. Subsequently, the cells were harvested for further experiments. The siRNA achieving the highest knockdown efficiency was selected for subsequent functional assays in the 5637 cells.

HOXB7 overexpression was achieved using the CV702 plasmid (Shanghai GenePharma, Co., Ltd.). T24 cells were cultured in a 6-well plate and transfected with 5 μg HOXB7 overexpression plasmid or empty vector using Lipofectamine 3000 when confluence reached 70-90%. After 48 h of transfection, reverse transcription-quantitative (RT-q)-PCR and western blot analyses were performed to evaluate transfection efficiency. Given the differential expression of HOXB7 between 5637 and T24 cells, HOXB7 expression was knocked down in 5637 cells (which exhibit high baseline HOXB7 expression) and overexpressed HOXB7 in T24 cells (which exhibit low baseline HOXB7 expression) to investigate its functional role. A similar approach was previously used to study the functional effects in BC cells (14).

Lentiviral vectors encoding a short hairpin RNA (shRNA) for the knockdown of HOXB7 and a scramble shRNA control were provided by Shanghai GenePharma Co., Ltd. The transfection process followed the established protocols. To establish a stably transfected cell line, the transfected cells were cultured in media supplemented with 5 µg/ml puromycin. RT-qPCR was used to assess the mRNA expression levels 72 hours after infection.

Total RNA was extracted using TRIzol^®, according to the manufacturer’s protocol. RNA (2µg) was reverse-transcribed into cDNAs using a Hifair^® III First Strand cDNA Synthesis Kit (cat. no. 1114ES10; Shanghai Yeasen Biotechnology Co., Ltd.). For reverse transcription, the temperature protocol was 25°C for 5 min, 55 °C for 15 min, and 85°C for 5 min. qPCR was conducted using primers synthesized by Sangon Biotech Co., Ltd. The primer sequences for qPCR were as follows:

GAPDH forward, 5’-CAGGAGGCATTGCTGATGAT-3’ and reverse, 5’-GAAGGCTGGGGCTCATTT-3’; and HOXB7 forward, 5’-CGAGAGTAACTTCCGGATCTAC-3’ and reverse, 5’-GGTCTTGTTCTCCTTTTTCCAC-3’.

The qPCR thermocycling conditions were 40 cycles of 25°C for 5 sec, 55°C for 15 sec, and 85°C for 5 sec. Each sample was measured in triplicate to ensure reliability. Gene expression levels in the cells were normalized relative to GAPDH expression using the 2^−ΔΔCq method.

A total of 4x10³ transfected cells were seeded into each well of a 96-well plate. Cell viability was measured on days 0, 1, 2 and 3 after transfection. For each time point, 10 μl CCK8 solution was added to each well, and cells were incubated for a further 2-h. Then, the absorbance was measured at 450 nm using a microplate reader (Synergy H1, Omega Bio-Tek, Inc.).

The Cell-Light™ EdU Apollo643 In Vitro Imaging Kit (cat. no. C10310-2, Guangzhou RiboBio Co., Ltd.) was used to evaluate cell proliferation according to the manufacturer’s instructions. Briefly, BC cells (3x10³) were seeded into each well of a 96-well plate and allowed to adhere for 24 h. Subsequently cells were cultured with1.0 mg/ml EdU solution for 2 h to label the DNA of proliferating cells. The cells were fixed with 50 μl of 4% paraformaldehyde for 30 min. After fixation, the cells were treated with 0.5% Triton X-00 (cat. no. T8200; Beijing, Solarbio Science & Technology Co., Ltd.) for 10 min. Next, 100 μl Apollo staining solution was added to each well, and the plates were incubated in the dark for 30 minutes at room temperature. DNA staining was performed using Hoechst33342 to visualize the cell nuclei. Cell proliferation was assessed by observing the fluorescence of the stained cells under fluorescence microscopy (IX71, Olympus Corporation).

A linear wound was created in a cell monolayer using a 1-ml sterile plastic pipette tip when the transfected cells had reached ~95% confluence in a 6-well plate. The cells were cultured in a media containing 2% FBS. Images of the wound area were captured using a microscope (BX53, Olympus Corporation) at 0, 24, and 48 h after wounding to monitor the rate of cell migration into the wound space.

Transfected cells (500 cells/well) were seeded into a 6-well plate and complete media was added to each well to a final volume of 2 ml. The cells were cultured in a cell incubator, and the medium was refreshed every 3 days to maintain optimal growth conditions. After 2 weeks, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Visible colonies were counted and imaged to assess the efficiency of the transfection and the proliferative capacity of the cells.

For the migration assay, 2x10⁴ treated and control BC cells were added to the upper chamber of a 24-well Transwell (8.0-mm, NEST Biotechnology, Wuxi, China) in 200 μl serum-free culture medium. The lower chamber was filled with 600 μl complete media.

For the invasion assay, Matrigel (cat. no. 356234, Corning Inc) was used to precoat the upper chamber for 24 h. Subsequently, 4x10⁴ treated and control BC cells were seeded into the upper chamber in 200 μl serum-free culture medium. The lower chamber was filled with 600 μl of 10% FBS complete culture medium.

After 24 or 48 h of incubation, the cells on the upper layers of upper chamber membranes were gently removed using a cotton swab. The cells that had migrated or invaded the lower surface of the membrane were fixed with 4% paraformaldehyde at room temperature for 30 min, followed by staining with 0.1% crystal violet for 30 min at room temperature. The number of cells was quantified in four randomly selected fields of view using a light microscope at x100 magnification.

Cells were harvested and resuspended in 500 µl binding buffer. Subsequently, the cells were stained using an Annexin V-FITC/PI Apoptosis kit according to the manufacturer’s protocol (cat. no. KGA108, Nanjing KeyGen Biotech Co., Ltd.). Staining was performed in the dark for 15 min. After staining, the percentage of apoptotic cells was determined using a BD FACS Canto II flow cytometer (BD Biosciences.).

Cells were lysed in RIPA lysis buffer with protease and phosphatase inhibitors (cat. no. P1045, Beyotime Institute of Biotechnology). Protein concentrations were measured using a BCA protein assay kit (cat. no. P0010, Beyotime Institute of Biotechnology). Equal quantities of protein were mixed with SDS buffer, denatured, and prepared for gel electrophoresis. Sample were resolved on 6-15% SDS gels and resolved using SDS-PAGE (cat. on. P0012A, Beyotime Institute of Biotechnology) and transferred to a PVDF membrane (cat. on. 0021, MilliporeSigma). Membrane were blocked using 5% skimmed milk and incubated overnight at 4˚C with the following primary antibodies: HOXB7 (cat. on. 12613-1-AP; 1:1000; ProteinTech Group, Inc.), Vimentin (cat. on. 10366-1-AP; 1:2000; ProteinTech Group, Inc.), E-cadherin (cat. on. 20874-1-AP; 1:1000; ProteinTech Group, Inc.), N-Cadherin (cat. on. 22018-1-AP; 1:1000; ProteinTech Group, Inc.), H-Ras (cat. on. 18295-1-AP; 1:1000; ProteinTech Group, Inc.), Raf-1(cat. on. 347271; 1:1000; ZenBio), P38 (cat. on. A14401; 1:1000; ABclonal), p-P38(cat. on. AP0526; 1:2000; ABclonal), JNK1/2 (cat. on. A18287; 1:500; ABclonal), p-JNK1/2 (cat. on. AP0473; 1:1000; ABclonal), ERK (cat. on. A16686; 1:1000; ABclonal), p-ERK (cat. on. AP0472; 1:1000; ABclonal), Bax (cat. on. 50599-2-lg; 1:2000; ProteinTech Group, Inc.), Bcl2 (cat. on. 26593-1-AP; 1:1000; ProteinTech Group, Inc.), MEK (cat. on. 11049-1-AP; 1:5000; ProteinTech Group, Inc.). p-MEK (cat. on. 310050; 1:1000; Zenbio) and β-actin (cat. on. 81115-1-RR; 1:500; ProteinTech Group, Inc.). After incubation, the membranes were washed with TBST and incubated with appropriate secondary antibodies (cat. on. PMK-014-097M/PMK-014-097S, HRP-conjugated goat anti-rabbit IgG, H+L, Bioprimacy, 1:5000). Following three washes with TBST, the target protein bands were detected using a high-sensitivity ECL chemiluminescence kit (cat. on. P2200, NCM Biotech).

A total of ten 6-week-old NSG mice were purchased from Shanghai Model Organisms Center, Inc. (Shanghai, China) and maintained under specific pathogen-free (SPF) conditions. The mice were randomly divided into two groups (n=5 per group). Each mouse in the scramble control group was subcutaneously injected with 1x10⁷ lentivirus-infected scramble 5637 cells, while each mouse in the shHOXB7 group was injected with 1x10⁷ lentivirus-infected shHOXB7–5637 cells. All injections were performed in the right axilla using 100 μl PBS as the suspension medium. Tumor growth was monitored by measuring the tumor length and width every 3 days, and tumor weights were recorded at the end of the study. Tumor volume was calculated using the formula: volume = length x width²/2 at the end of the 7-week experimental period. The maximum tumor diameter permitted in this study was 15 mm, in accordance with ethical guidelines. The mice were euthanized by cervical dislocation, and the tumors were harvested for weight measurement, immunohistochemical staining, and western blot analysis. The animal experiment was approved by the Animal Research Ethics Committee of Guizhou Medical University (approval no. 2201615) and performed in accordance with the Guidelines for Animal Experiments of Laboratory Animals.

Data are presented as the mean ± standard deviation (SD) of at least three independent experiments. Differences between the two groups were determined using an unpaired Student’s t-test. The correlations between the clinical characteristics in the BC TMA were analyzed using a chi-square (χ²) test. ANOVA was used to compare differences between three or more groups. A Levene’s test was used to assess the homogeneity of variances, followed by either a Tukey’s post-hoc test (for equal variances) or the Games-Howell test (for unequal variances), depending on the uniformity of variance. All statistical analyses were performed using GraphPad version 8.0 (GraphPad Software Inc.). A two-sided P < 0.05 was considered to indicate a statistically significant difference.

To evaluate the expression pattern of HOXB7 in BC, the GEPIA database was first used to analyze data from TCGA and GTEx, revealing that HOXB7 expression was significantly higher in BC tumor tissues compared with normal bladder tissues ( Figure 1A ). Consistently, UALCAN analysis based on TCGA datasets confirmed the elevated expression of HOXB7 in primary BC tissues relative to normal samples ( Figure 1B ). Further stratification by tumor stage and lymph node metastasis status demonstrated that HOXB7 expression was significantly correlated with tumor progression, showing increased levels in advanced stages and in patients with lymph node involvement ( Figures 1C, D ). To validate these findings at the protein level, immunohistochemical (IHC) staining was performed on 36 paired BC and adjacent normal tissue (ANT) specimens. The results demonstrated markedly higher HOXB7 expression in BC tissues compared with matched normal counterparts ( Figure 1E ). Quantification of IHC staining confirmed this difference to be statistically significant (p < 0.0001). Survival analysis based on clinical follow-up data from these 36 patients revealed that high HOXB7 expression was significantly associated with poorer overall survival (p < 0.001; Figure 1F ). Moreover, upregulated expression of HOXB7 was significantly associated with pathological grade and tumor stage ( Table 1 ). Additionally, we assessed HOXB7 expression in four human BC cell lines (5637, T24, J82, and TCCSUP) and in the immortalized normal urothelial cell line SV-HUC-1. Both western blot and RT-qPCR analyses revealed elevated HOXB7 expression in BC cell lines, with the highest expression observed in 5637 cells and the lowest in T24 cells ( Figures 1G, H ). Based on these results, 5637 and T24 cell lines were selected for subsequent functional assays.

To modulate HOXB7 expression in BC cells, siRNA-mediated knockdown was performed in 5637 cells, while HOXB7 was ectopically overexpressed in T24 cells using a pcDNA3.1-HOXB7 plasmid. Western blotting and RT-qPCR confirmed efficient knockdown and overexpression of HOXB7, respectively ( Figures 2A, B ). The effect of HOXB7 on cell proliferation was assessed using CCK-8 and EdU assays. Silencing HOXB7 significantly suppressed the proliferation of 5637 cells, while HOXB7 overexpression markedly enhanced the proliferation of T24 cells ( Figures 2C, D ). Consistent results were obtained in colony formation assays, where HOXB7 knockdown reduced the number of colonies formed in 5637 cells, and HOXB7 overexpression increased clonogenic growth in T24 cells ( Figure 2E ). To investigate whether the observed proliferative changes were associated with apoptosis, flow cytometry analysis was performed. HOXB7 knockdown significantly increased the apoptosis rate in 5637 cells, whereas HOXB7 overexpression suppressed apoptosis in T24 cells ( Figure 2F ). Further analysis of apoptosis-related protein expression revealed that HOXB7 silencing downregulated anti-apoptotic Bcl-2 and upregulated pro-apoptotic Bax in 5637 cells. Conversely, in T24 cells, HOXB7 overexpression led to increased Bcl-2 and decreased Bax levels ( Figure 2G ).

EMT is a key process involved in cancer metastasis. To evaluate the role of HOXB7 in BC cell migration, wound healing assays were performed. Knockdown of HOXB7 significantly reduced the migratory ability of 5637 cells, while overexpression of HOXB7 enhanced migration in T24 cells ( Figures 3A, C ). Consistently, Transwell assays demonstrated that HOXB7 silencing significantly inhibited both migration and invasion of 5637 cells, whereas HOXB7 overexpression in T24 cells promoted these abilities ( Figures 3B, D ). To further explore whether HOXB7 affects EMT in BC cells, the expression levels of EMT-related markers were examined by western blot. In 5637 cells, HOXB7 knockdown led to increased expression of the E-cadherin and decreased levels of N-cadherin and Vimentin. Conversely, HOXB7 overexpression in T24 cells suppressed E-cadherin while upregulating N-cadherin and Vimentin ( Figures 3E, F ). These results indicate that HOXB7 facilitates the malignant progression of BC cells, at least in part, by promoting EMT.

To elucidate the regulatory mechanisms of HOXB7 in the occurrence and development of BC, patients with BC were stratified into high- and low- HOXB7 expression groups using data from the TCGA. The analysis revealed a significant difference in gene expression patterns between the two groups ( Figure 4A ). KEGG pathway analysis indicated that the DEGs influenced by HOXB7 were predominantly involved in the MAPK signaling pathway ( Figure 4B ). To further explore this relationship, key targets within the MAPK signaling pathway were identified and their correlation with HOXB7 expression in BC was validated using the GEPIA database. The analysis revealed a positive correlation between HOXB7 and key components of the MAPK pathway, including MAPK3, MAPK1, MAPK14, MAPK8, and MAPK9 ( Figure 4C ). Functionally, these components are known as ERK1, ERK2, P38, JNK1, and JNK2, respectively ( Figure 4C ). The results from the western blot analysis further confirmed that HOXB7 knockdown resulted in a significant reduction in the phosphorylation levels of ERK1/2, while total ERK levels remained unchanged in 5637 cells. No significant alterations were observed in the phosphorylation or total expression of P38 and JNK1/2. Conversely, in T24 cells, HOXB7 overexpression markedly increased the phosphorylation levels of ERK1/2 without affecting total ERK levels, whereas P38 and JNK1/2 levels, both phosphorylated and total, showed no significant changes ( Figure 4D ). These findings confirm that HOXB7 specifically regulates the activation of ERK1/2 within the MAPK signaling pathway in both 5637 and T24 cells, further emphasizing its pivotal role in modulating this pathway’s activity. In addition, cell function assays indicated that HOXB7 promoted the EMT phenotype of BC cells. Previous research established the importance of the Ras-mediated Raf/MEK/ERK pathway in EMT regulation. These findings support the hypothesis that HOXB7 may regulate the occurrence and development of BC by modulating the MAPK/MEK/ERK signaling pathway. Nevertheless, the crucial role of HOXB7 and the MAPK/MEK/ERK pathways in BC development has not been extensively reported and requires additional experimental validation. Thus, the changes in the H-Ras/Raf-1/MEK/ERK pathway following HOXB7 downregulation and overexpression were investigated, including H-Ras, Raf-1, MEK, p-MEK, ERK, and p-ERK. In 5637 cells, HOXB7 downregulation reduced the phosphorylation levels of MEK and ERK and decreased the expression of H-Ras and Raf-1. In contrast, HOXB7 overexpression significantly increased the expression of H-Ras, Raf-1, p-MEK, and p-ERK in T24 cells ( Figure 4E ).

To investigate whether the Ras/ERK signaling pathway mediates the functional effects of HOXB7 in BC cells, the ERK1/2-specific activator Ro67–7476 and the MEK inhibitor PD98059 were used for rescue experiments in 5637 and T24 cells. CCK-8 assays demonstrated that the proliferation-inhibitory effect of HOXB7 knockdown in 5637 cells was partially reversed by Ro67–7476 treatment (1 μM, 24 h), while the proliferation-enhancing effect of HOXB7 overexpression in T24 cells was suppressed by PD98059 (10 μM, 24 h) ( Figure 5A ). Similarly, Transwell assays showed that Ro67–7476 significantly restored the invasive capacity of HOXB7-silenced 5637 cells, whereas PD98059 attenuated the invasion-promoting effect of HOXB7 overexpression in T24 cells ( Figure 5B ). Wound healing assays further confirmed that the impaired migration of 5637 cells upon HOXB7 silencing was rescued by Ro67-7476, while PD98059 suppressed the enhanced migration caused by HOXB7 overexpression in T24 cells ( Figure 5C ). Flow cytometry analysis revealed that HOXB7 knockdown significantly increased apoptosis in 5637 cells, which was partially reversed by Ro67-7476. In T24 cells, HOXB7 overexpression reduced apoptosis, whereas PD98059 treatment increased the apoptotic rate, particularly in HOXB7-overexpressing cells ( Figure 5D ). Western blot analysis showed that HOXB7 knockdown decreased MEK and ERK phosphorylation in 5637 cells, and this effect was rescued by Ro67-7476. Conversely, HOXB7 overexpression enhanced phosphorylation of MEK and ERK in T24 cells, which was effectively suppressed by PD98059 ( Figure 5E ). These findings indicate that the Ras/MEK/ERK signaling pathway plays a critical role in mediating the tumor-promoting effects of HOXB7 in BC cells.

To investigate the in vivo role of HOXB7 in tumor development, a subcutaneous xenograft model was established using 5637 BC cells stably transfected with either shHOXB7 or scramble control. RT-qPCR confirmed efficient knockdown of HOXB7 expression ( Figure 6A ). Tumors derived from shHOXB7-transfected cells were significantly smaller in both volume and weight compared with those in the control group ( Figures 6B–D ). Immunohistochemical staining revealed a marked reduction in HOXB7 and Ki-67 expression in the shHOXB7 group, indicating decreased proliferative activity ( Figure 6E ). Western blot analysis of tumor tissues showed that HOXB7 knockdown led to downregulation and decreased phosphorylation of several key components in the H-Ras/MEK/ERK signaling pathway, including H-Ras, RAF1, p-MEK, and p-ERK ( Figure 6F ). A schematic diagram illustrating the proposed mechanism is shown in Figure 6G .