The role of microRNAs-23b and 27b in migration, invasion and proliferation of T24 bladder cancer cells

Abstract

Background

Bladder cancer (BC) is the sixth most common cancer among men worldwide and represents a significant cause of morbidity and mortality. High-grade BC is associated with an increased risk of progression to muscle-invasive and metastatic disease, negatively impacting patient prognosis. Despite advances in molecular characterization, therapeutic strategies remain limited, and the identification of novel molecular targets is essential. MicroRNAs (miRNAs) are small non-coding RNAs involved in post-transcriptional gene regulation and play critical roles in tumor development and progression. Among them, miRNA-23b and miRNA-27b have been implicated in several malignancies; however, their functional role in high-grade BC remains incompletely understood. This study aimed to evaluate the expression levels of miRNAs-23b and 27b in a high-grade BC cell line and to investigate their effects on cell migration, invasion, and proliferation, exploring their potential therapeutic relevance.

Methods

The high-grade BC T24 cell line was used. Cells were divided into four groups: Control (no transfection), negative control (Scramble), miRNA-23b mimic, and miRNA-27b mimic. Relative miRNA expression levels were determined by quantitative polymerase chain reaction (qPCR). Functional assays included wound healing (migration), Matrigel invasion assay, and colony formation assay (proliferation). Statistical analyses were performed to compare groups, and p-values < 0.05 were considered statistically significant.

Results

Transfection resulted in significant overexpression of miRNA-23b and miRNA-27b compared to both Scramble (p = 0.0344 and p = 0.0386, respectively) and Control groups (p = 0.0343 and p = 0.0390, respectively). Both miRNA-23b and miRNA-27b significantly reduced cell migration compared to Scramble (p = 0.0286). Additionally, miRNA-23b significantly decreased invasion compared to Scramble and Control (p < 0.0001), with similar findings observed for miRNA-27b (p < 0.0001). No statistically significant differences were observed in colony formation among groups.

Conclusions

Overexpression of miRNA-23b and miRNA-27b significantly reduced migration and invasion in a high-grade BC cell line, without affecting proliferation. These findings suggest that both miRNAs may act as tumor suppressors in high-grade BC and represent promising candidates for future therapeutic development in bladder cancer.

Background

Bladder cancer (BC) is the sixth most common cancer in men worldwide, with an estimated 82,290 new cases in the United States in 2023, including 62,420 in men and 19,870 in women [1]. In Brazil, according to the National Cancer Institute (INCA), approximately 11,370 new cases per year are expected between 2023 and 2025, with a higher incidence in men (7,870 cases) compared to women (3,500 cases) [2]. Globally, BC represents a significant public health concern due to its high recurrence rates, need for long-term surveillance, and substantial economic burden on healthcare systems. This type of cancer affects the urothelial lining of the bladder and is histologically classified into urothelial carcinoma, squamous cell carcinoma, and adenocarcinoma, each associated with distinct etiological mechanisms [3].

The grading of bladder urothelial carcinoma is primarily determined by the degree of cellular differentiation and architectural tumor patterns. The World Health Organization (WHO) classification system, established in 2004 and updated in 2016, categorizes tumors into low-grade and high-grade based on cytologic and architectural atypia. In parallel, tumor staging follows the TNM classification system of the American Joint Committee on Cancer (AJCC), which stratifies tumors into non-muscle-invasive bladder cancer (NMIBC), including pTa, pT1, and carcinoma in situ (pTis), and muscle-invasive bladder cancer (MIBC), defined as stage pT2 or higher, characterized by invasion of the muscularis propria. These classifications are essential for predicting recurrence and progression risk and for guiding therapeutic strategies, including intravesical therapy, radical cystectomy, and systemic chemotherapy [4].

BC occurs predominantly in individuals over 65 years of age, with a median age at diagnosis of 73 years, and is approximately four times more common in men than in women [4]. Although most cases are diagnosed at a localized stage, with a five-year relative survival rate of 76.9%, a substantial proportion of high-grade tumors demonstrate aggressive biological behavior, with increased risk of invasion, metastasis, and disease-specific mortality. Therefore, identifying molecular mechanisms associated with tumor progression remains critical to improving prognostic stratification and expanding therapeutic options [3].

Recent advances in molecular oncology have enabled the identification of distinct BC subtypes based on genomic alterations, transcriptomic profiles, and tumor microenvironment characteristics. Large-scale initiatives such as The Cancer Genome Atlas (TCGA) have contributed to a deeper understanding of the molecular landscape of BC, aiming to support the development of more personalized therapeutic strategies [5, 6]. However, despite these advances, the incorporation of molecular biomarkers into routine clinical practice remains limited, and further functional validation of candidate regulatory molecules is necessary.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level and are involved in key cellular processes such as proliferation, apoptosis, differentiation, migration, and invasion. Dysregulation of specific miRNAs has been consistently associated with carcinogenesis and tumor progression in BC. Among them, miRNA-23b and miRNA-27b, which belong to the same genomic cluster, have emerged as potential modulators of oncogenic signaling pathways [7]. Evidence suggests that miRNA-23b may inhibit aggressive tumor cell properties by targeting oncogenic pathways, supporting its tumor suppressor role. Conversely, some studies indicate that it may also regulate tumor suppressor proteins, suggesting context-dependent oncogenic activity. Differential expression of miRNA-23b between high-grade MIBC and low-grade NMIBC has been reported, reinforcing its relevance in tumor progression [8, 9].

Similarly, miRNA-27b has been described as deregulated in multiple malignancies, including BC, where reduced expression may be associated with enhanced tumor aggressiveness. Both miRNAs have been implicated in the regulation of pathways directly related to epithelial–mesenchymal transition, migration, and invasion. Despite growing evidence regarding their expression patterns, the functional impact of restoring miRNA-23b and miRNA-27b expression in high-grade BC cells remains insufficiently characterized. This gap highlights the need for experimental studies that evaluate their direct effects on tumor cell behavior.

In this context, the present study aimed to evaluate the expression levels of miRNA-23b and miRNA-27b in a high-grade BC cell line and to investigate their effects on cell migration, invasion, and proliferation, thereby clarifying their potential role as therapeutic targets in bladder cancer.

Methods

Cell line and culture conditions

The high-grade bladder cancer (BC) cell line T24 (urothelial carcinoma) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and authenticated by the Cell Authentication Service of the Medical School of the University of São Paulo (FMUSP). Cells were routinely tested for mycoplasma contamination and used within six passages after thawing. Cells were cultured in 25-cm² culture flasks containing Minimum Essential Medium (MEM; Life Technologies, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic–antimycotic solution (100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B). Cultures were maintained at 37 °C in a humidified incubator with 5% CO₂ and 95% air.

Cell transfection with miRNA-23b and miRNA-27b mimics

miRNA overexpression was achieved using synthetic miRNA mimics: miRNA-23b mimic (MC10711), miRNA-27b mimic (MC10750), and negative control (Scramble; AM17110) (Ambion, Austin, TX, USA).

The experimental groups were:

1. Control (no transfection),

2. Negative control (Scramble),

3. miRNA-23b mimic,

4. miRNA-27b mimic.

Cells were plated in 12-well plates and transfected using Lipofectamine™ RNAiMAX (Invitrogen, Carlsbad, CA, USA) in Opti-MEM^® I Reduced Serum Medium according to the manufacturer’s instructions. The final concentration of each miRNA mimic was 20 nM. Transfection complexes were incubated with cells for 48 h at 37 °C.

After incubation, cells were harvested using trypsin-EDTA, neutralized with complete medium containing 10% FBS, centrifuged at 300 g for 5 min, and processed for RNA extraction to confirm transfection efficiency.

Total miRNA extraction

Total RNA enriched for small RNAs was extracted using the miRVana^® microRNA Isolation Kit (Ambion, Austin, TX, USA) following the manufacturer’s protocol. Briefly, cell pellets were lysed with 300 µL lysis buffer and homogenized. Thirty microliters of homogenization additive were added, and samples were incubated on ice for 10 min.

An equal volume (300 µL) of acid-phenol: chloroform was added, followed by centrifugation at 10,000 g for 5 min at room temperature. The aqueous phase was transferred to a new tube, mixed with ethanol (1/3 volume), and applied to a silica-based filter column. After washing steps, miRNAs were eluted in 50 µL of nuclease-free water preheated to 95 °C.

RNA concentration and purity were determined using a NanoDrop^® spectrophotometer (Thermo Fisher Scientific), and samples with 260/280 ratios between 1.8 and 2.1 were considered acceptable. Extracted RNA was stored at − 20 °C until use.

Reverse transcription

Complementary DNA (cDNA) synthesis was performed using the TaqMan^® MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). A total of 200 ng of RNA was reverse-transcribed in a final volume of 10 µL containing stem-loop primers specific for miRNA-23b and miRNA-27b.

Thermal cycling conditions were: 30 min at 16 °C, 30 min at 42 °C,

5 min at 85 °C. Reactions were performed using a Veriti^® Thermal Cycler (Applied Biosystems).

Quantitative real-time PCR (qPCR)

Quantitative real-time PCR was performed using the ABI 7500 Fast Real-Time PCR System (Applied Biosystems) and TaqMan^® Universal PCR Master Mix. Each reaction (10 µL total volume) contained 0.5 µL of specific TaqMan probe (Table 1), 2 µL of Master Mix, 1 µL of cDNA, and nuclease-free water.

Table 1.

Probes used to quantify the expression of the miRNAs studied. List of specific TaqMan assays used to quantify miRNA-23b and miRNA-27b expression levels by quantitative real-time PCR (qPCR), including assay identification numbers and endogenous control (RNU48).

miRNAs	Tests	Brand
miRNA-23b	000400	Applied Biosystems
miRNA-27b	000409	Applied Biosystems
RNU48	001006	Applied Biosystems

Cycling conditions were: 2 min at 50 °C, 10 min at 95 °C, 45 cycles of 15 s at 95 °C and 1 min at 60 °C. All reactions were performed in duplicate. Relative expression levels were calculated using the 2⁻ΔΔCt method, with RNU48 as the endogenous control. Transfection efficiency was confirmed by comparing relative expression levels between experimental and control groups.

Migration assay (Wound healing)

Cell migration was evaluated using the wound healing assay. Cells (1 × 10⁵ cells/well) were seeded in 24-well plates and allowed to reach approximately 90–100% confluence. A linear scratch was created using a sterile 200 µL pipette tip.

Images were captured at 0 and 24 h using an inverted microscope. Wound area was quantified using ImageJ software (NIH, USA), using the freehand selection tool. For each well, three independent microscopic fields were analyzed, and the mean value was calculated. Migration was expressed as percentage of wound closure using the formula:

(A_initial − A_24h) / A_initial × 100. Experiments were performed in triplicate.

Invasion assay (Matrigel)

Cell invasion was assessed using BioCoat™ Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA) with 8-µm pore membranes coated with Matrigel. Transfected cells were serum-starved for 24 h, harvested, and resuspended in serum-free medium. A total of 1 × 10⁵ cells were added to the upper chamber. The lower chamber contained medium supplemented with 10% FBS as chemoattractant. After 48 h of incubation at 37 °C with 5% CO₂, non-invading cells were removed from the upper surface. Invading cells were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Cells were counted in nine randomly selected microscopic fields per insert at 200× magnification. All experiments were performed in triplicate.

Colony formation assay

To evaluate proliferative capacity, 5 × 10² cells per well were seeded in 12-well plates and transfected as described. Cells were maintained for 10 days, with medium replacement every 3 days. On day 10, colonies were fixed with 4% formaldehyde, stained with 3% methylene blue for 30 min, washed, and air-dried. Colonies larger than 1 mm in diameter were counted. Colony number and area were quantified using ImageJ software. Experiments were performed in triplicate.

Statistical analysis

Data are presented as mean ± standard deviation (SD). Normality of data distribution was assessed using the Shapiro–Wilk test. For normally distributed variables, comparisons between two groups were performed using Student’s t-test, and multiple group comparisons were analyzed using One-way ANOVA followed by Tukey’s multiple comparison test. For non-parametric data, the Mann–Whitney test was applied. When multiple comparisons were performed, appropriate post hoc corrections were applied to reduce type I error. All analyses were conducted using GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). A two-tailed p value < 0.05 was considered statistically significant.

Results

Efficiency of transfection with miRNA-23b and miRNA-27b mimics

qPCR analysis confirmed successful transfection. Both miRNA-23b and miRNA-27b were significantly overexpressed in the respective treated groups compared with the Scramble group (p = 0.0344 and p = 0.0386, respectively) and the Control group (p = 0.0343 and p = 0.0390, respectively) (Fig. 1A–B). These findings confirm adequate transfection efficiency and validate subsequent functional analyses.

Fig. 1.

Relative expression levels of miRNA-23b and miRNA-27b after transfection in T24 cells. A Relative expression of miRNA-23b in Control, Scramble, and miRNA-23b mimic groups. B Relative expression of miRNA-27b in Control, Scramble, and miRNA-27b mimic groups. Expression levels were determined by quantitative real-time PCR (qPCR) and normalized to RNU48. Data are presented as mean ± SD of three independent experiments. Statistical comparisons were performed between transfected groups and Control or Scramble groups. P-values are indicated in the figure

Effect of miRNA-23b and miRNA-27b on cell migration (wound healing assay)

A significant reduction in cell migration was observed in both miRNA-23b and miRNA-27b groups compared with the Scramble group (p = 0.0286 for both comparisons) (Fig. 2). The percentage of wound closure at 24 h was significantly lower in the miRNA-transfected groups, indicating impaired migratory capacity.

Fig. 2.

Effect of miRNA-23b and miRNA-27b overexpression on cell migration in T24 cells (wound healing assay). A Percentage of cell migration in Scramble and miRNA-23b mimic groups 24 h after scratch induction. B Percentage of cell migration in Scramble and miRNA-27b mimic groups 24 h after scratch induction. C Representative microphotographs of wound areas at 0 and 24 h in Scramble, miRNA-23b, and miRNA-27b groups. Data are presented as mean ± SD of three independent experiments. Statistical comparisons were performed between Scramble and miRNA-transfected groups. P-values are indicated in the figure

Effect of miRNA-23b and miRNA-27b on cell invasion (Matrigel assay)

In the Matrigel invasion assay, cells transfected with miRNA-23b demonstrated a significant reduction in invasion compared with both Scramble and Control groups (p < 0.0001 for both comparisons). Similar results were observed in the miRNA-27b group (p < 0.0001 versus Scramble and Control) (Fig. 3). These results indicate that restoration of miRNA-23b and miRNA-27b expression markedly suppresses invasive behavior in high-grade BC cells.

Fig. 3.

Effect of miRNA-23b and miRNA-27b overexpression on cell invasion in T24 cells (Matrigel assay). A Quantification of invading cells in Control, Scramble, and miRNA-23b mimic groups. B Quantification of invading cells in Control, Scramble, and miRNA-27b mimic groups. C Representative microphotographs of invaded cells stained with crystal violet in each experimental group (Control, Scramble, miRNA-23b, and miRNA-27b). Data are presented as mean ± SD of three independent experiments. Statistical comparisons were performed between transfected groups and Control or Scramble groups. P-values are indicated in the figure

Effect of miRNA-23b and miRNA-27b on cell proliferation (colony formation assay)

No statistically significant differences were observed among groups in the colony formation assay (Fig. 4A–B). Although a visual reduction in colony area was observed in the miRNA-transfected groups, statistical analysis demonstrated that these differences did not reach statistical significance. Therefore, these findings do not support a significant effect of miRNA-23b or miRNA-27b on proliferative capacity under the experimental conditions evaluated.

Fig. 4.

Effect of miRNA-23b and miRNA-27b overexpression on colony formation in T24 cells. A Quantification of average colony area in Control, Scramble, and miRNA-23b mimic groups. B Quantification of average colony area in Control, Scramble, and miRNA-27b mimic groups. C Representative images of colonies formed in each experimental group (Control, Scramble, miRNA-23b, and miRNA-27b). Data are presented as mean ± SD of three independent experiments. Statistical comparisons were performed among groups. No statistically significant differences were observed

Discussion

Recent studies have demonstrated the importance of miRNAs as therapeutic alternatives in various cancers, given their ability to regulate the expression of multiple target genes simultaneously. Through post-transcriptional modulation, miRNAs influence several biological processes, including proliferation, apoptosis, differentiation, migration, and invasion. Despite the growing body of evidence regarding the role of miRNAs in cancer biology, the functional characterization of specific miRNA clusters in high-grade bladder cancer remains insufficiently explored, particularly in experimental models [10].

miRNAs are frequently tissue-specific and may function either as tumor suppressors or oncogenes, depending on the cellular context and the molecular network in which they are embedded [11–15]. Among clustered miRNAs, miRNA-23b and miRNA-27b are located in close genomic proximity and have been described as components of a regulatory cluster with tumor-suppressive properties in several malignancies, including bladder cancer [5, 6]. Decreased expression of miRNA-23b and miRNA-27b has been reported in clinical BC specimens, supporting their putative suppressor role in tumor progression [7].

In our study, transfection with miRNA-23b and miRNA-27b mimics resulted in significant overexpression of both miRNAs in the T24 high-grade bladder cancer cell line. Functionally, restoration of these miRNAs significantly reduced cell migration and invasion capacity. Although a reduction trend in colony formation was observed, no statistically significant differences in proliferation were detected under the experimental conditions applied. These findings suggest that the primary biological effect of these miRNAs in this context may be related to modulation of invasive behavior rather than direct suppression of proliferative signaling.

The ability of miRNAs to target multiple mRNAs altered under disease conditions makes them attractive therapeutic candidates, either in the form of miRNA mimics or anti-miRNA strategies. Early therapeutic approaches using viral vectors faced important pharmacological barriers, including systemic instability and inefficient delivery. However, advances in nanotechnology and RNA delivery systems have enabled miRNA-based therapies to enter early-phase clinical trials, highlighting their translational potential [8]. In particular, nanoparticle- and liposome-based delivery platforms have emerged as promising approaches to improve miRNA stability, tissue targeting, and bioavailability, representing important steps toward the clinical application of miRNA-based therapeutics.

Mechanistically, miRNA-23b has been associated with regulation of epithelial–mesenchymal transition (EMT), an essential process in tumor invasion and metastasis. ZEB1, a key EMT transcription factor, has been identified as a direct target of miRNA-23b [16]. The suppression of EMT-related genes may partially explain the reduction in migration and invasion observed in our experiments. Supporting this hypothesis, Majid et al. demonstrated that miRNA-23b is significantly downregulated in bladder cancer tissues compared to normal urothelium, suggesting its tumor-suppressive role and potential utility as a diagnostic and prognostic biomarker [10]. Similar findings have been described in colorectal cancer, where decreased miRNA-23b expression was associated with worse prognosis [17, 18]. Furthermore, because EMT is a major driver of bladder cancer dissemination, the inhibitory effects observed after restoration of miRNA-23b and miRNA-27b expression may be related to suppression of EMT-associated pathways. Future studies evaluating EMT markers, including E-cadherin, N-cadherin, Vimentin, and ZEB1, would be valuable to further clarify the molecular mechanisms underlying these findings.

Nevertheless, the biological role of miRNA-23b appears paradoxical in different tumor types. In lung, liver, and gastric cancers, miRNA-23b has been reported to exert oncogenic effects by targeting distinct regulatory proteins [19–21]. These apparently contradictory findings reinforce the concept that miRNA function is highly context-dependent and influenced by tumor-specific transcriptional landscapes [22].

miRNA-27b is also frequently deregulated in cancers and has been described as a tumor suppressor in renal cell carcinoma, prostate cancer, gastric cancer, and oral squamous cell carcinoma [23–28]. In bladder cancer specifically, decreased miRNA-27b expression has been associated with tumor grading and aggressive disease features [24]. Our results are consistent with these findings, demonstrating that restoration of miRNA-27b significantly impaired migration and invasion in T24 cells. Similarly, Chiyomaru et al. reported that re-expression of the miRNA-23b/27b cluster inhibited proliferation, migration, and invasion in bladder cancer models [7].

Conversely, studies in cervical and breast cancer have demonstrated oncogenic roles for miRNA-27b, further reinforcing its tumor-specific behavior [29–31]. Such variability likely reflects differences in target gene repertoires and microenvironmental signaling across tumor types.

Importantly, the present study has limitations that should be carefully considered. Experiments were performed using a single high-grade bladder cancer cell line, which may limit generalizability across different molecular subtypes of BC. In addition, although statistically significant differences were observed in migration and invasion assays, effect sizes and confidence intervals were not calculated, which may limit the interpretation of the magnitude of biological impact. Incorporating these measures in future investigations may provide a more comprehensive assessment of the biological relevance of the observed effects. Furthermore, downstream molecular targets and signaling pathways were not experimentally validated, and in vivo models were not included, restricting mechanistic and translational conclusions. Future transcriptomic and proteomic studies may help identify direct and indirect targets of miRNA-23b and miRNA-27b and further elucidate the signaling pathways involved in their biological effects. In addition, other biological processes potentially regulated by miRNA-23b and miRNA-27b, including apoptosis and autophagy, were not evaluated in the present study and should be investigated in future research to provide a more comprehensive understanding of their biological functions in bladder cancer. Future studies should correlate miRNA-23b and miRNA-27b expression with clinicopathological characteristics, recurrence-free survival, and cancer-specific survival in bladder cancer cohorts. Such investigations may help determine whether these miRNAs could serve as prognostic biomarkers or potential therapeutic targets in patients with aggressive bladder cancer. Moreover, before clinical application can be considered, comprehensive toxicological and safety studies will be necessary to evaluate the tolerability and potential adverse effects of miRNA-based therapeutic approaches. Future investigations should also explore the interaction of miRNA-23b and miRNA-27b with other molecular factors involved in bladder cancer progression, including genetic alterations and epigenetic modifications, to better define their role within the broader regulatory network of the disease.

Taken together, our findings reinforce the tumor-suppressive role of the miRNA-23b/27b cluster in high-grade bladder cancer and support further investigation of these molecules as potential modulators of tumor invasiveness and metastatic progression.

Conclusions

Restoration of miRNA-23b and miRNA-27b expression significantly reduced migration and invasion in a high-grade bladder cancer cell line, without significantly affecting proliferation. These findings support the tumor-suppressive role of the miRNA-23b/27b cluster in high-grade BC and suggest that modulation of these miRNAs may represent a promising therapeutic strategy. Further mechanistic and in vivo studies are warranted.

Abbreviations

AJCC

American Joint Committee on Cancer

ATCC

American Type Culture Collection

BC

Bladder câncer

cDNA

Complementary DNA

Ct

Cycle threshold

EMT

Epithelial–mesenchymal transition

FBS

Fetal bovine sérum

MEM

Minimum Essential Medium

MIBC

Muscle-invasive bladder câncer

miRNA

MicroRNA

NMIBC

Non-muscle-invasive bladder câncer

PBS

Phosphate-buffered saline

qPCR

Quantitative real-time polymerase chain reaction

RNU48

Small nucleolar RNA, C/D box 48

SD

Standard deviation

TCGA

The Cancer Genome Atlas

TNM

Tumor, Node, Metastasis classification system

WHO

World Health Organization

Authors’ contributions

All authors contributed to the study conception and design.Conceptualization: Felipe Fakhouri, Patrícia Candido, Sabrina T. ReisData curation: Ruan PimentaFormal analysis: Felipe Fakhouri, Patrícia Candido, Ruan PimentaInvestigation: Felipe Fakhouri, Patrícia CandidoMethodology: Vanessa R. Guimarães, Guilherme NebóProject administration: Sabrina T. ReisResources: Carlos Eduardo da SilvaSoftware: Ruan PimentaSupervision: Kátia Ramos Moreira Leite, Sabrina T. ReisValidation: William C. Nahas, Kátia Ramos Moreira Leite, Carlo PasserottiVisualization: Kátia Ramos Moreira Leite, Sabrina T. ReisWriting – original draft: Felipe Fakhouri, Patrícia CandidoWriting – review and editing: Sabrina T. ReisAll authors read and approved the final manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable. This study was conducted exclusively using commercially available human bladder cancer cell lines and did not involve human participants, patient-derived samples, or animal experiments. Therefore, institutional ethics committee approval and informed consent were not required.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.