Abstract

Objective

   This study aimed to elucidate the expression profile and biological
   implications of peroxidase 5 (PRDX5) in bladder cancer (BC),
   specifically investigating its influence on BC progression through
   modulation of reactive oxygen species (ROS) levels and activation of
   ferroptosis pathways.

Methods

   We employed urine proteomics data and transcriptomic information from
   the Cancer Genome Atlas (TCGA) to identify differentially expressed
   genes in BC tissues, focusing on PRDX5. Using single-cell RNA
   sequencing (scRNA-seq), we assessed PRDX5 distribution across various
   cell types in the tumor microenvironment. We conducted in vitro
   experiments to analyze the impact of PRDX5 on BC cell proliferation,
   migration, and invasion, while exploring its mechanisms of modulating
   ROS levels and ferroptosis. In vivo experiments were performed to
   observe PRDX5's influence on ferroptosis signaling in tissue contexts.

Results

   We found significant upregulation of PRDX5 in BC tissues, with
   scRNA-seq revealing its enrichment in bladder epithelial cells,
   correlating with disease advancement and established BC markers. In
   vitro analyses showed that overexpressed PRDX5 enhanced proliferation,
   migration, and invasion of BC cells, while PRDX5 knockout produced
   opposing effects. Additionally, PRDX5 modulated ROS levels and impacted
   ferroptosis pathways. In vivo experiments confirmed that PRDX5 knockout
   inhibited tumor growth and activated ferroptosis signaling pathways in
   tissues.

Conclusion

   Our study highlights the elevated expression of PRDX5 in BC and its
   role in promoting tumor progression through regulation of ROS levels
   and ferroptosis. PRDX5 may serve as a promising target for BC
   treatment, supporting further exploration of its potential in clinical
   applications.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12885-025-13881-y.

   Keywords: Bladder cancer, PRDX5, Ferroptosis, Multi-omics, Urine,
   Progression, Single cell

Introduction

   Bladder cancer (BC) is one of the most common malignancies affecting
   the urinary system [[42]1, [43]2]. Global statistics indicate that
   approximately 540,000 new cases of BC are diagnosed each year,
   resulting in around 190,000 deaths attributed to the disease [[44]3].
   Clinically, BC is classified into two main types based on the depth of
   invasion into the bladder wall: muscle-invasive bladder cancer (MIBC)
   and non-muscle-invasive bladder cancer (NMIBC) [[45]4]. Patients with
   MIBC continue to face a high risk of distant metastasis, even after
   undergoing radical cystectomy and systemic chemotherapy, with an
   approximate 5-year survival rate of 32%[[46]5]. Despite the fact that
   the combination of gemcitabine and cisplatin is considered the
   first-line chemotherapy regimen for this disease [[47]6], the objective
   response rate for patients with advanced BC who are undergoing this
   treatment is only 40–60%, with an increase in overall survival of
   merely 5% [[48]7, [49]8]. Therefore, it is crucial to identify new
   therapeutic targets to develop precision treatment strategies.

   Recent studies have increasingly focused on the molecular mechanisms
   and therapeutic targets of BC [[50]9]. Redox processes have been
   observed in cells, and reactive oxygen species (ROS)-byproducts of
   metabolic activity [[51]10], play a critical role in cellular
   physiology [[52]11]. Ferroptosis, a form of programmed cell death, is
   initiated by iron-induced lipid hydro-peroxidation [[53]12, [54]13].
   Ferroptosis is regulated by oxidative damage to the cell membrane
   [[55]14], where abnormal accumulation of phospholipid hydroperoxides
   (lipid ROS or lipid LOOH) is regarded as a hallmark feature of
   ferroptosis [[56]15]. Considering that a key characteristic of
   ferroptosis is the accumulation of ROS leading to cell death, and given
   that the core function of the Peroxiredoxins (PRDX1-PRDX6, PRDXs) are
   the regulation of peroxides [[57]16], we hypothesize that a member of
   the PRDXs may modulate ROS levels, thereby regulating ferroptosis and
   ultimately influencing the progression of BC. PRDX5 is particularly
   significant due to its unique subcellular localization and its role in
   regulating ROS [[58]17]. It is widely accepted that the accumulation of
   iron ions, which results from lipid peroxidation, along with the
   subsequent increase in ROS levels, serves as a critical trigger for
   ferroptosis [[59]18]. A recent study by Fujita et al. [[60]19]
   highlighted that the deletion of PRDX6 can lead to decreased
   selenoprotein expression and enhance ferroptosis by inhibiting the
   expression of glutathione peroxidase 4 (GPX4). Although there is
   currently a lack of direct evidence linking PRDX5 to the regulation of
   ferroptosis, emerging research suggests that PRDX5 may play a crucial
   role in this process due to its known antioxidant properties and
   involvement in ROS regulation. Given this potential link, this study
   aims to investigate how PRDX5 modulates ferroptosis and assess its
   impact on the development and progression of BC.

Materials and methods

Multi-omics integration for differential gene identification

   This study collected urine samples from five patients diagnosed with BC
   and fresh urine samples from five healthy control individuals, in
   adherence to the Declaration of Helsinki and ethical guidelines set by
   the Institutional Medical Ethics Committee of Lanzhou University Second
   Hospital, all participants provided signed written informed consent
   prior to their inclusion in the study. The pathological diagnoses for
   each BC patient were independently confirmed by at least two certified
   pathologists. Furthermore, this investigation utilized the BC dataset
   from the Cancer Genome Atlas (TCGA,
   [61]https://portal.gdc.cancer.gov/), which includes gene expression
   profiles and survival data for 414 BC patients surviving more than
   60 days post-diagnosis, along with 19 adjacent normal tissue samples.

Transcriptome RNA sequencing and single-cell RNA-Seq data analysis

   UMUC-3 cells with a knockout of PRDX5, along with control UMUC-3 cells,
   were treated with TRIzol reagent (Invitrogen) and immediately to liquid
   nitrogen for cryopreservation. All subsequent procedures were conducted
   by Majorbio Bio-Pharm Technology Co., Ltd. in Shanghai, China. To
   analyze the single-cell dataset, we performed dimensionality reduction
   and clustering on samples from two datasets: [62]GSE135337, which
   included seven tumor samples and one normal sample, and [63]GSE129845,
   which encompassed three normal samples. Cell type annotations were
   assigned based on cell type-specific marker genes previously identified
   [[64]20]. The single-cell RNA sequencing (scRNA-seq) data underwent
   preprocessing utilizing the 'Seurat' R package, following detailed
   methodological protocols outlined in prior research. Principal
   component analysis was used to the dataset to reduce dimensionality,
   and the top 15 principal components were selected for further analysis
   and visualization. To refine the classification of cell populations,
   utilized the UMAP algorithm for dimensionality reduction and
   visualization, building upon the PCA results. Finally, we utilized the
   k-nearest neighbor algorithm to subdivide the cell populations, setting
   the resolution parameter to 0.1.

Tissue and cell lines

   Five pairs of fresh-frozen BC tissues, along with their corresponding
   adjacent normal tissues, were surgically obtained and immediately
   preserved in liquid nitrogen for subsequent analysis of PRDX5 protein
   expression. Additionally, 16 normal tissue samples and 16 BC tissue
   samples were collected to assess PRDX5 RNA expression levels. All
   clinical samples were collected with informed consent from the patients
   and the collection was approved by the Medical Ethics Committee of the
   Lanzhou University Second Hospital.

   SV-HUC-1 cells, which are immortalized human ureteral epithelial cells,
   were cultured in DMEM (L110KJ; BasalMedia, Shanghai, China). In
   contrast, BC cell lines, including UMUC-3, 5637, and J82, were
   maintained in RPMI-1640 medium (L210KJ; BasalMedia, Shanghai, China).
   All cell lines were procured from the Key Laboratory of Urinary System
   Diseases at the Lanzhou University Second Hospital, located in Lanzhou
   City, Gansu Province, China. Each culture medium was supplemented with
   10% fetal bovine serum (FBS) (ST30-3302; PAN-Biotech, Germany). All
   cultures were maintained at 37 °C in a humidified incubator containing
   5% CO[2].

Quantitative reverse transcription PCR (qRT-PCR)

   Total RNA was extracted following the manufacturer's instructions using
   TRIzol Reagent (AcuracyBio). After RNA extraction, reverse
   transcription was carried out, and real-time quantitative PCR (qPCR)
   analysis was conducted using Prime-Script RT Master Mix (Takara Bio)
   and the SYBR Premix Ex Taq kit (Takara Bio). The primer sequences used
   in this study are provided in Supplementary Table 1.

Western blotting

   Total protein was extracted using RIPA lysis buffer (Solarbio).
   Subsequently, 20 μg of protein was subjected to electrophoresis and
   transferred to polyvinylidene difluoride (PVDF) membranes, and the
   blots were cut prior to incubation with antibodies during blotting.
   Following transfer, the membranes were incubated with the appropriate
   primary and secondary antibodies, and protein signals were visualized
   using chemiluminescence on an ECL detection system. Densitometric
   analysis of protein band intensities was performed using ImageJ
   software. The antibodies utilized in this study included PRDX5
   (ab180587; Abcam, USA), GAPDH (HY-P80137; MedChemExpress, China), and
   the Ferroptosis Essentials Antibody Kit (PK30002; Proteintech, China).

CRISPR/Cas9 gene editing technology

   To establish the PRDX5 knockout cell line, two single-guide RNAs
   (sgRNAs) were designed: sg1-PRDX5 (5′-CACCGGGCTATATACTCGTCGGTGGTTT-3′)
   and sg2-PRDX5 (5′-CACCATTACTAGATGATTCGCGTTT-3′). These sgRNAs were
   synthesized using the Q5 Site-Directed Mutagenesis Kit (E0552S; New
   England Biolabs, USA) and subsequently cloned into the lenti-Rv2
   plasmid (Addgene). After co-transfecting HEK293T cells with the psPAX2
   and pMD2.G packaging plasmids, viral supernatants were harvested to
   infect UMUC-3 and J82 cells. Monoclonal cell lines were generated by
   limiting dilution in 96-well plates following selection with puromycin.
   The successful PRDX5 knockout was confirmed by Sanger sequencing and
   Western blot analysis.

Construction of overexpression cell line and lentiviral transfection

   The full-length coding sequence of human PRDX5 was cloned into the
   pLV3-CMV-3 × FLAG-mCherry-Puro vector (Miaoling Biological, China).
   HEK293T cells were co-transfected with the psPAX2 and pMD2.G vectors,
   and viral supernatants were collected to infect UMUC-3, J82, and T24
   cells. Stable cell lines overexpressing PRDX5 were generated after
   puromycin selection. Cell transfection was performed using Polybrene
   (Solaibao, China) following the manufacturer's protocol.

Cell counting kit (CCK) −8 and colony formation assay

   We initially seeded UMUC-3 and J82 cells, which were in the logarithmic
   growth phase, at a density of 2000 cells per well in 96-well plates.
   After allowing the cells to adhere, we added CCK-8 reagent (Biosharp)
   diluted in serum-free medium at a ratio of 9:1 to each well. Following
   a 2-h incubation period, the absorbance was measured at 450 nm (OD450)
   using a microplate reader. This process was repeated at regular
   intervals up to 96 h to comprehensively assess the cell proliferation
   rate. For the negative control, we used cell-free medium and similarly
   incubated it with the CCK-8 reagent. At the end of the incubation
   period, the absorbance was measured at 450 nm using a microplate
   reader. To determine the percentage of cell proliferation relative to
   the negative control, the absorbance values of each experimental group
   were divided by the absorbance value of the negative control (NC group)
   and then multiplied by 100. This calculation provided the cell
   proliferation rate as a percentage, thereby enabling the quantitative
   analysis of the proliferation rates of UMUC-3 and J82 cells.

   To further investigate long-term proliferation capability, we performed
   colony formation assays. Cells were seeded at low densities (500 cells
   per well) in 6-well plates and allowed to grow for 10–14 days until
   visible colonies formed. During this period, the culture medium was
   refreshed every three days to ensure optimal growth conditions. After
   the incubation period, the colonies were fixed with 4% paraformaldehyde
   for 30 min and stained with 0.1% crystal violet for 15 min. Colonies
   containing more than 50 cells were counted under a microscope. The
   number of colonies provided an indication of the cells' ability to
   proliferate and form colonies, thereby assessing their long-term
   proliferative potential.

Wound healing, migration, and invasion assays

   In the wound healing experiments, BC cells were cultured to confluence
   in 6-well plates. A sterile pipette tip was then used to create a
   uniform scratch in the center of each well, which served as a marker
   for subsequent analysis. After that, unattached cells were removed by
   washing with phosphate-buffered saline, and the remaining cells were
   incubated in serum-free medium to facilitate continued cultivation. The
   migratory capacity of the cells was evaluated by measuring the closure
   of the scratch at 0, 24, and 48 h after the injury. ImageJ software was
   employed to calculate the area of the scratch at both 0, 24, and 48 h
   after scratching. The migration rate of cells was then represented by
   the ratio of the reduced area over time.

   For the migration assays, 4 × 10^4 BC cells were seeded in the upper
   chamber of a transwell plate with 8 µm pores, and 700 µL of complete
   medium was added to the lower chamber. After a 24-h incubation period,
   the cells that migrated through the membrane were fixed with 4%
   paraformaldehyde and subsequently stained with crystal violet for
   visualization.

   In the invasion experiments, transwell plates with 8 µm pores were
   first coated with a layer of Matrigel (Corning, USA) to simulate the
   extracellular matrix environment. After the Matrigel solidified,
   8 × 10^4 BC cells were inoculated into the upper chamber, while the
   lower chamber was filled with complete medium. After a 24-h incubation,
   the invading cells that crossed the membrane were fixed in 4%
   paraformaldehyde and stained with crystal violet for quantification.

3D matrigel drop invasion assay and flow cytometry analysis

   BC cells (5 × 10^4) were resuspended in 10 μL of Matrigel (Corning,
   USA) and carefully added to each well of 24-well plates using a
   vertical dropwise technique. The plates were left undisturbed for
   20 min to allow the Matrigel to solidify into a droplet structure.
   After solidification, complete medium was added to each well. The
   invasive capacity of the cells was assessed by measuring the distance
   of migration from the edge of the Matrigel drops after 6 days of
   culture.

   To evaluate apoptosis, an Annexin V-FITC/PI Apoptosis Detection Kit
   (AT101; Multi Sciences) was employed. Following the manufacturer's
   protocol, cell aliquots were stained with FITC-Annexin V and propidium
   iodide (PI) to identify apoptotic cells, enabling a comprehensive
   analysis of cell viability and apoptosis.

MitoSOX, TMRE, JC-1, ROS assays

   BC cells were cultured in confocal dishes and treated with MitoSOX
   probes (MX4313, Mkbio) in addition to Hoechst 33,342 (C0031, Solaibao)
   for a duration of 30 min. After the treatment, the cells were examined
   using a Zeiss LSM 510 confocal microscope (Germany) equipped with a
   60 × 1.3 NA oil immersion objective. Initially, 1 × 10^6 cells were
   seeded into the confocal dishes. The following day, 0.5 mL of working
   solutions of JC-1 dye (C2005, Beyotime) and Hoechst 33,342 were added
   to each dish and incubated for 25 min at a temperature of 37 °C.
   Subsequently, the cells were observed under confocal microscopy.

   Furthermore, BC cells in confocal dishes were treated with TMRE probes
   (C2001S, Beyotime) and Hoechst 33,342 for an additional 30 min, after
   which imaging was conducted to assess mitochondrial membrane potential.
   Intracellular levels of ROS were evaluated using the DCFH-DA
   fluorescent probe (HY-D0940, MedChemExpress). For this analysis, BC
   cells were plated in six-well plates and treated with DCFH-DA at a
   final concentration of 25 μM, followed by incubation in the dark for
   30 min. Fluorescence intensity was quantified using a Beckman CytoFLEX
   flow cytometer.

Xenograft model

   All animal experiments and procedures were conducted with the approval
   of the Animal Welfare and Ethics Committee of the Second Hospital of
   Lanzhou University. Ten 4-week-old male nude mice (Huachuang Sino,
   China) were randomly allocated into two groups (KO-PRDX5 and NC-PRDX5),
   with five mice in each group. The cells for both the KO-PRDX5 and NC
   groups were collected and suspended in phosphate-buffered saline at a
   concentration of 2 × 10^7 cells/mL, then mixed with Matrigel (Corning,
   USA) at a 1:1 ratio. A total of 100 μL of this cell suspension was
   subcutaneously injected into the axillary region of the nude mice.
   Throughout the experimental period, the body weight and tumor volume of
   the mice were measured every 5 days. On day 15, the mice were
   euthanized via intraperitoneal injection of an overdose of
   pentobarbital sodium. Following euthanasia, the tumors were excised,
   weighed, their dimensions recorded, and photographed. Tumor volume was
   assessed using caliper measurements and calculated using the following
   formula: Volume = 0.5 × length × width^2.

Statistical analysis

   Data are presented as mean ± standard deviation from a minimum of three
   independent experiments, each conducted in triplicate. The following
   statistical tests were performed with Prism 9 software (GraphPad). The
   following statistical analyses were used: t test (unpaired and
   two-tailed) and one-way analysis of variance (ANOVA) with Dunnett’s
   multiple comparisons test. P-values < 0.05 were considered significant,
   represented by "*", P values less than 0.01 were denoted as "**", P
   values less than 0.001 were indicated as "***", while P values less
   than 0.0001 were represented by "#".

Results

Integrated multi-omics approach for identifying differentially expressed
genes in BC

   Our previous study successfully executed RNA extraction and LC–MS
   detection of urinary samples from BC patients and healthy controls
   [[65]21], Building upon this foundation, our objective was to identify
   noninvasive diagnostic markers for BC and identify highly
   differentially expressed genes. We initially screened for
   differentially expressed genes in TCGA using volcano plots
   (Fig. [66]1A). To enhance the reliability of the selected markers, we
   also incorporated data from urine proteomics. Using the urine
   proteomics volcano plot (Fig. [67]1B) and the combined multi-omics
   differential distribution plot (Fig. [68]1C), we identified
   differential molecules that were highly expressed in BC. Our focus was
   on molecules that are readily secreted into urine for early diagnosis.
   Thus, we prioritized markers showing elevated expression levels across
   multiple omics platforms. A Venn diagram (Fig. [69]1D) highlighted the
   overlap between the 3,895 highly expressed DEGs in TCGA-BC
   transcriptomics (left circle) and the 352 in urine proteomics (right
   circle), revealing 32 overlapping molecules (center intersection).
   Incorporating the 32 hub genes into a lasso regression analysis
   (Fig. [70]1E), we found that the model achieved its best fit when the
   Beta coefficient was set to 7. Subsequently, seven molecules were
   chosen for inclusion in the prognostic model. Among these, PRDX5
   emerged as the most significant (Fig. [71]1F), with higher expression
   levels associated with more accurate prognostic predictions. Further
   exploration of PRDX5 expression in various cancers, derived from the
   TCGA database, indicated a high expression level in the majority of
   cancer types (Fig. [72]1G).

Fig. 1.

   [73]Fig. 1
   [74]Open in a new tab

   Selection of differential genes in BC through multi-omics integration.
   A Volcano plot of differentially expressed genes in the TCGA-BLCA gene
   expression profile; (B) Volcano plot of differentially expressed genes
   in the urinary proteomics gene expression profile; (C) Rank versus
   expression abundance curve for differentially expressed genes combining
   TCGA-BLCA and urinary proteomics data; (D) A Venn diagram illustrating
   the intersection of highly expressed differential genes from urine
   proteomics data and the TCGA-BLCA gene expression profile, along with
   their respective top-ranked differential genes; (E) Screening of Lasso
   regression coefficients for 32 hub genes; (F) 1-year, 3-year, and
   5-year nomograms used to predict overall survival in BC; (G) Expression
   levels of PRDX5 across different cancer types

Expression characteristics of PRDX5 in BC and its correlation with clinical
and pathological features

   To investigate the expression profile of PRDX5 in BC and its potential
   biological relevance, we initially analyzed urine samples from five
   healthy volunteers and five BC patients. The analysis revealed a
   significant upregulation of PRDX5 in the BC cohort (Fig. [75]2A). This
   observation suggests that PRDX5 may serve as a noninvasive early
   diagnostic biomarker for BC.

Fig. 2.

   [76]Fig. 2
   [77]Open in a new tab

   Differences in PRDX5 expression levels in BC and their correlation with
   clinical pathological features. A PRDX5 expression levels in urine
   proteomics from 5 BC patients and 5 normal individuals; (B) Comparison
   of PRDX5 expression levels between BC and normal tissues; (C) Paired
   comparison of PRDX5 expression levels between BC and normal tissues;
   (D) Comparison of PRDX5 expression levels among different histological
   grades; (E) Comparison of PRDX5 expression levels among different
   subtypes; (F) Relationship between PRDX5 expression levels and overall
   survival of BC patients; (G) Immunohistochemical staining of PRDX5 in
   adjacent non-cancerous tissues; (H) Immunohistochemical staining of
   PRDX5 in BC tissues

   To validate these preliminary findings, we used data from the TCGA-BLCA
   dataset. The analysis of this dataset demonstrated a marked increase in
   PRDX5 expression in BC tissues compared to normal tissue controls
   (Fig. [78]2B). Importantly, this significant difference persisted when
   cancer samples were matched to their adjacent non-tumor tissues
   (Fig. [79]2C). We further explored the expression patterns of PRDX5
   across different pathological grades of BC. Notably, we observed a
   significant upward trend in PRDX5 expression in both low-grade and
   high-grade tumors (Fig. [80]2D). In cases of papillary BC, PRDX5
   expression reached its highest level (Fig. [81]2E), highlighting its
   potential role in BC progression.

   While there were no significant differences observed in overall
   survival (Fig. [82]2F) between the groups classified based on high and
   low PRDX5 expression, it is worth noting that patients with high levels
   of PRDX5 exhibited a trend towards a poorer prognosis. Taking this
   cumulative evidence into account, we believe that PRDX5 is a promising
   candidate biomarker for the early noninvasive diagnosis of BC. In
   addition, immunohistochemical data obtained from the Human Protein
   Atlas (HPA) database confirmed that the staining intensity of PRDX5 in
   adjacent normal tissues (H-score: 25.6 ± 5.2) (Fig. [83]2G) was
   significantly lower than that in BC tissues (H-score: 188.3 ± 46.2)
   (Fig. [84]2H), and the staining intensity and the proportion of
   positive cells in tumor tissues were significantly higher than those in
   normal tissues.

Single-cell sequencing reveals the role of PRDX5 in the bladder tumor
microenvironment

   In this study, we explored the functional characteristics of PRDX5
   across different cell types within the tumor microenvironment of BC
   using scRNA-seq technology. Subsequently, we applied dimensionality
   reduction techniques to analyze the single-cell BC data, allowing us to
   visualize spatial structural information (Figs. [85]3A, B). We further
   examined the levels of PRDX5 enrichment across different subcellular
   types (Figs. [86]3C-H). Notably, our results indicated significant
   enrichment of PRDX5 in bladder urothelial cells (Fig. [87]3D), a trend
   that was corroborated by findings from the independent [88]GSE130001
   single-cell dataset (Figs. [89]3G, H).

Fig. 3.

   [90]Fig. 3
   [91]Open in a new tab

   ScRNA-seq analysis of cell type distribution in the bladder tumor
   microenvironment and its relationship with PRDX5 expression. A t-SNE
   plot showing the distribution of different cell types in the bladder
   tumor microenvironment; (B) UMAP plot illustrating the distribution of
   various cell types in the bladder tumor microenvironment; (C) PRDX5
   expression levels among different cell types within the bladder tumor
   microenvironment; (D) PRDX5 expression levels across different cell
   clusters in the bladder tumor microenvironment; (E) Dimensionality
   reduction analysis of different cell types in the bladder tumor
   microenvironment; (F) Cluster analysis of PRDX5 expression levels among
   various cell types in the BC microenvironment; (G) Cluster analysis of
   different cell types in the bladder tumor microenvironment from the
   single-cell dataset [92]GSE130001; (H) PRDX5 expression levels in
   different cell types within the bladder tumor microenvironment from the
   single-cell dataset [93]GSE130001

   These observations indicate that the expression pattern of PRDX5 within
   the BC microenvironment is specific to certain cells, with notably
   elevated expression levels in bladder epithelial cells. This suggests
   that PRDX5 may play a crucial role in the development of BC and
   provides valuable insights for a more comprehensive understanding of BC
   pathogenesis, as well as the potential advancement of targeted
   treatment strategies.

Single-cell sequencing investigates the localization and role of PRDX5 in
bladder tumors

   To investigate the function of PRDX5 in bladder epithelial cells, we
   used scRNA-seq data for cluster analysis (Fig. [94]4A). We identified
   six distinct cell types: endothelial cells, epithelial cells,
   fibroblasts, macrophages, and plasma cells (Fig. [95]4B) based on
   previously established cell type-specific marker genes from our
   research group [[96]20]. The results indicated that PRDX5 was more
   prevalent and abundant in tumor cells compared to normal tissue cells
   (Fig. [97]4C). Importantly, PRDX5 showed significant enrichment in
   bladder epithelial cells (Fig. [98]4D).

Fig. 4.

   [99]Fig. 4
   [100]Open in a new tab

   Analysis of scRNA-seq data in BC tissues. A Eight clusters were
   annotated based on cell type-specific marker genes following scRNA-seq
   of BC samples; (B) Cells were classified into five main types based on
   transcriptomic characteristics: endothelial cells, epithelial cells,
   fibroblasts, macrophages, and plasma cells; (C) Differences in PRDX5
   expression between single cells from normal tissues and tumor tissues;
   (D) Expression levels of PRDX5 in various cell types, with notably
   higher expression in bladder epithelial cells; (E) Relationship between
   tumor T staging and PRDX5 expression in single-cell BC tissues; (F)
   Positive correlation of PRDX5 with common epithelial malignancy-related
   genes in BC, such as GATA3, KRT7, S100P, TP63, and UPK2

   Additionally, we examined the relationship between PRDX5 expression and
   bladder tumor T stage at the single-cell level. Our findings revealed
   that PRDX5 expression significantly increases with advancing T stage
   (Fig. [101]4E). This suggests that PRDX5 may play a crucial role in the
   progression of BC.

   Further analysis demonstrated that in BC samples, PRDX5 shows a
   positive correlation with several epithelial malignancy-associated
   genes (GATA3, KRT7, S100P, TP63, and UPK2) (Fig. [102]4F). This
   correlation indicates that PRDX5 might also be involved in regulating
   the development of BC.

   In summary, our scRNA-seq analysis has uncovered a significant
   upregulation of PRDX5 expression in bladder epithelial cells as the
   disease progresses. More importantly, the strong correlation between
   PRDX5 and specific BC markers suggests that PRDX5 not only plays a
   pivotal role in the onset of BC but could also serve as an important
   indicator for assessing patient prognosis. These findings provide new
   insights into the mechanisms by which PRDX5 contributes to BC and lay
   the groundwork for developing diagnostic or therapeutic strategies
   based on PRDX5.

Analysis of PRDX5 expression in BC cell lines and tissues

   To investigate the differential expression of PRDX5 between BC cell
   lines (UMUC-3, J82, T24, 5637) and normal bladder epithelial cells
   (SV-HUC-1), we employed two validation methods: RT-PCR (Fig. [103]5A)
   and Western blot analysis (Fig. [104]5B). The results indicated that
   PRDX5 expression levels were significantly higher in BC cells (UMUC-3,
   J82, T24) compared to normal bladder epithelial cells (SV-HUC-1).
   Furthermore, analysis of data from the CELL database revealed that
   PRDX5 exhibited high expression levels in most BC cell lines, although
   the selected lines (UMUC-3, J82, T24) had relatively lower expression
   levels (Fig. [105]5C).

Fig. 5.

   [106]Fig. 5
   [107]Open in a new tab

   Expression of PRDX5 in BC cell lines and clinical patients. A mRNA
   expression levels of PRDX5 in normal bladder epithelial cell line
   SV-HUC-1 and BC cell lines UMUC-3, J82, T24, and 5637; (B) Protein
   expression levels of PRDX5 in normal bladder epithelial cell line
   SV-HUC-1 and BC cell lines UMUC-3, J82, T24, and 5637; (C) mRNA
   expression profile of PRDX5 in BC cell lines; (D) mRNA expression
   levels of PRDX5 in BC tissues and adjacent non-cancerous tissues; (E)
   Protein expression levels of PRDX5 in BC tissues and adjacent
   non-cancerous tissues; (F) mRNA expression levels following PRDX5
   overexpression in BC cell lines; (G) Protein expression levels
   following PRDX5 overexpression in BC cell lines; (H) mRNA expression
   level assessment of KO-PRDX5 efficiency in BC cell lines; (I) Protein
   expression level assessment of KO-PRDX5 efficiency in BC cell lines.
   Pa, adjacent non-cancerous tissue; Ca, BC tissue; ns, not significant;
   *, p < 0.05; **, p < 0.01; ***, p < 0.001; #, p < 0.0001

   Additionally, RT-PCR validation was performed using 16 pairs of BC and
   adjacent tissue samples collected from the Lanzhou University Second
   Hospital. This analysis confirmed that the expression of PRDX5 was
   significantly higher in BC tissues compared to adjacent normal tissues
   (Fig. [108]5D). Furthermore, examination of tissue proteins from five
   BC patients showed a similar trend, with significantly higher levels of
   PRDX5 expression in cancerous tissue compared to adjacent normal
   tissues (Fig. [109]5E). Taken together, these findings support our
   hypothesis that PRDX5 may have a role in the development of BC.

   To further investigate the potential impact of PRDX5 in BC, we
   introduced lentiviral vectors overexpressing PRDX5 into BC cell lines
   (UMUC-3, J82, T24). The efficacy of overexpression was assessed through
   RT-PCR (Fig. [110]5F) and Western blot analysis (Fig. [111]5G), both
   confirming high levels of PRDX5 overexpression. Conversely, we employed
   CRISPR-Cas9 gene editing technology to knockout PRDX5 in the BC cell
   lines (UMUC-3, J82) that had relatively high expression levels.
   Monoclonal cell lines were selected from these cultures, and two clonal
   knockout fragments were validated. Analysis through RT-PCR
   (Fig. [112]5H) and Western blot (Fig. [113]5I) confirmed the successful
   establishment of KO-PRDX5 cell lines from BC cell lines UMUC-3 and J82.

Impact of PRDX5 on the biological behavior of BC cells

   To systematically explore the potential role of PRDX5 in the biological
   behavior of BC cell lines (UMUC-3, J82, T24), we employed a series of
   in vitro experimental methods for comprehensive assessment. Initially,
   a CCK-8 cell proliferation assay (Fig. [114]6A) revealed a significant
   association between PRDX5 overexpression and an increased proliferation
   rate of BC cells. This finding was further supported by colony
   formation assays, which showed that the PRDX5-overexpressing group
   exhibited denser and larger clonal colonies compared to the control
   group (Fig. [115]6B), highlighting the role of PRDX5 in promoting BC
   cell proliferation and growth.

Fig. 6.

   [116]Fig. 6
   [117]Open in a new tab

   Effects of PRDX5 overexpression in BC cell lines (UMUC-3, J82, and
   T24). A Comparison of cell proliferation ability over time in BC cell
   lines overexpressing PRDX5; (B) Cloning formation assays demonstrating
   enhanced growth and proliferative capacity in BC cell lines with PRDX5
   overexpression; (C) Scratch migration assays indicating increased
   migration ability in cell lines overexpressing PRDX5; (D) Transwell
   migration assays showing enhanced migration capability in cell lines
   with PRDX5 overexpression; (E) Transwell invasion assays revealing
   increased invasive ability in cell lines with PRDX5 overexpression; (F)
   Enhanced invasive capability in 3D spheroid models of
   PRDX5-overexpressing UMUC-3; (G) Enhanced invasive capability in 3D
   spheroid models of PRDX5-overexpressing J82

   Subsequent experiments aimed to evaluate alterations in cell migration
   and invasion. Cell scratch assays (Fig. [118]6C) demonstrated that the
   overexpression of PRDX5 considerably augmented the migratory activity
   of BC cells. Transwell migration assays (Fig. [119]6D) not only
   validated this enhancement but also revealed that PRDX5 overexpression
   was associated with a noteworthy rise in cell invasion (Fig. [120]6E),
   thereby offering more profound insights into the invasive capability of
   BC cells.

   To assess the long-term effects, we employed a 3D sphere-forming
   invasion model (Figs. [121]6F, 6G), which consistently demonstrated
   that PRDX5-overexpressing cells exhibited a sustained increase in
   invasive capacity over time, thereby supporting the idea that PRDX5
   functions as a positive regulator of BC progression.

   In contrast, we examined the impact of PRDX5 deletion on BC cell
   behavior using CRISPR-Cas9 knockout technology. The CCK-8 proliferation
   assay (Fig. [122]7A) revealed that KO-PRDX5 led to a decreased
   proliferation rate in BC cells. Colony formation assays (Fig. [123]7B)
   further confirmed this finding, indicating that both the number and
   size of colonies in the knockout group were smaller than those in the
   control group. This suggests that PRDX5 is crucial for maintaining
   proliferative activity in BC cells.

Fig. 7.

   [124]Fig. 7
   [125]Open in a new tab

   Effects of KO-PRDX5 in BC cell lines (UMUC-3 and J82). A Comparison of
   cell proliferation ability over time in BC cell lines with KO-PRDX5;
   (B) Cloning formation assays indicating reduced growth and
   proliferative capacity in BC cell lines with KO-PRDX5; (C) Scratch
   migration assays demonstrating decreased migration ability in cell
   lines with KO-PRDX5; (D) Transwell migration assays showing reduced
   migration capability in cell lines with KO-PRDX5; (E) Transwell
   invasion assays revealing decreased invasive ability in cell lines with
   KO-PRDX5; (F) Reduced invasive capability in 3D spheroid models of
   KO-PRDX5 UMUC-3; (G) Reduced invasive capability in 3D spheroid models
   of KO-PRDX5 J82

   Cell scratch assays (Fig. [126]7C) and transwell assays (Figs. [127]7D,
   E) demonstrated significant decreases in cell migration and invasion
   following PRDX5 knockout, thereby providing additional evidence of its
   involvement in facilitating invasive behavior. Ultimately, the results
   from the 3D sphere-forming invasion model (Figs. [128]7F, G) further
   supported these findings, indicating a gradual decline in invasion over
   time in KO-PRDX5 BC cells.

PRDX5 regulates cell death in BC cell lines and its potential mechanisms

   In the UMUC-3 cell line, PRDX5 overexpression was significantly
   associated with a reduction in cell death compared to the control cells
   (2.15% vs. 3.82%). This phenomenon was also observed in the J82 cell
   line (2.13% vs. 4.84%) (Fig. [129]8A). Conversely, PRDX5 knockout led
   to a significant increase in cell death within the UMUC-3 cell line
   (3.19% vs. 10.48%), with a similar trend observed in the J82 cell line
   (3.67% vs. 10.47%) (Fig. [130]8B). These findings highlight the crucial
   role of PRDX5 in modulating cell death in BC.

Fig. 8.

   [131]Fig. 8
   [132]Open in a new tab

   Impact of PRDX5 expression levels on apoptosis, gene expression, and
   ROS levels in BC cell lines. A Comparison of apoptosis rates between
   PRDX5 overexpressing cell lines and control groups; (B) Comparison of
   apoptosis rates between KO-PRDX5 cell lines and control groups; (C)
   Differences in the expression of classic apoptosis pathway genes
   between PRDX5 overexpressing and control groups in UMUC-3; (D)
   Differences in the expression of classic apoptosis pathway genes
   between PRDX5 overexpressing and control groups in J82; (E) Differences
   in the expression of classic apoptosis pathway genes between KO-PRDX5
   and control groups in UMUC-3; (F) Differences in the expression of
   classic apoptosis pathway genes between KO-PRDX5 and control groups in
   J82; (G) Comparison of ROS fluorescence levels between PRDX5
   overexpressing cell lines and control groups; (H) Comparison of ROS
   fluorescence levels between KO-PRDX5 cell lines and control groups; (I)
   Flow cytometry analysis comparing ROS levels between KO-PRDX5 cell
   lines and control groups

   To further elucidate the mechanisms underlying PRDX5's regulatory
   effects on apoptosis, we assessed the mRNA expression levels of three
   key genes involved in classical apoptotic pathways in cell lines
   overexpressing PRDX5 and KO-PRDX5 cell lines. Notably, there were no
   significant alterations in the expression of markers associated with
   the mitochondrial apoptotic pathway or the death receptor pathway. In
   contrast, we observed a marked upregulation of markers related to the
   endoplasmic reticulum stress pathway in cell lines overexpressing PRDX5
   (Figs. [133]8C, D). Meanwhile, while the expression levels of markers
   for the mitochondrial and death receptor pathways remained largely
   unchanged in KO-PRDX5 cell lines, we noted a pronounced downregulation
   of markers related to the ER stress pathway (Figs. [134]8E, F).
   Collectively, these results suggest that PRDX5-mediated cell death may
   function independently of classical apoptotic pathways.

   Considering that the primary function of PRDX5 is the regulation of
   ROS, and given that ROS accumulation is a pivotal feature of
   ferroptosis [[135]15], we hypothesize that PRDX5 may influence cell
   death by modulating ROS levels and impacting ferroptosis signaling
   pathways. To test this hypothesis, we first evaluated ROS levels in
   both PRDX5-overexpressing and knockout cell lines. Our results
   indicated that overexpression of PRDX5 significantly decreased ROS
   levels in BC cells (Fig. [136]8G), whereas knockout of PRDX5 resulted
   in a substantial increase in ROS levels (Fig. [137]8H). Flow cytometry
   assays further confirmed that KO-PRDX5 enhanced ROS production
   (Fig. [138]8I). Taken together, these findings suggest that PRDX5
   regulates cell death through the modulation of ROS levels rather than
   through conventional apoptotic pathways. This discovery provides
   valuable insights into the mechanisms by which PRDX5 influences BC
   biology.

PRDX5 influences ferroptosis in BC by regulating lipid metabolism and
mitochondrial function

   To thoroughly investigate the role of PRDX5 in BC, we conducted a
   transcriptome sequencing analysis comparing KO-PRDX5 lines with control
   strains in UMUC-3 cells. The findings revealed that the functional
   enrichment of differentially expressed genes between PRDX5-depleted
   cell lines and their controls predominantly associated with
   cancer-related pathways (Fig. [139]9A). Additionally, KEGG pathway
   enrichment analysis highlighted significant involvement of lipid
   metabolism and immune system pathways (Fig. [140]9B). Given the
   critical link between aberrant lipid accumulation and ferroptosis
   [[141]15], we hypothesize that PRDX5 may influence ferroptosis through
   its regulatory effects on lipid metabolism, thereby modulating BC cell
   death.

Fig. 9.

   [142]Fig. 9
   [143]Open in a new tab

   Impact of KO-PRDX5 on gene expression profiles and mitochondrial
   function in BC cell lines. A Disease-related functional enrichment
   analysis of differentially expressed genes between KO-PRDX5 cell lines
   and control groups; (B) KEGG pathway enrichment analysis of
   differentially expressed genes between KO-PRDX5 cell lines and control
   groups; (C) Comparison of mitochondrial superoxide (MitoSOX) levels
   between KO-PRDX5 and control groups in the UMUC-3 cell line; (D)
   Comparison of mitochondrial superoxide (MitoSOX) levels between
   KO-PRDX5 and control groups in the J82 cell line; (E) Comparison of
   mitochondrial membrane potential between KO-PRDX5 and control groups in
   the UMUC-3 cell line; (F) Comparison of mitochondrial membrane
   potential between KO-PRDX5 and control groups in the J82 cell line; (G)
   Mitochondrial fluorescent probe (TMRE) staining showing KO-PRDX5 and
   control groups in UMUC-3 cells; (H) Mitochondrial fluorescent probe
   (TMRE) staining showing KO-PRDX5 and control groups in J82 cells

   To validate this hypothesis, we first assessed the impact of KO-PRDX5
   on mitochondrial superoxide levels (as measured by MitoSOX) in the BC
   cell lines UMUC-3 and J82. Our results indicated a substantial increase
   in mitochondrial superoxide levels in the KO-PRDX5 cell lines compared
   to controls (Fig. [144]9C, D). Furthermore, the evaluation of
   mitochondrial membrane potential using the JC-1 assay demonstrated a
   marked decline in potential (as indicated by diminished red
   fluorescence and increased green fluorescence) in the PRDX5-depleted
   cell lines (Fig. [145]9E, F), suggesting compromised mitochondrial
   functionality. Complementary experiments employing TMRE fluorescent
   probes further substantiated these findings, revealing a significant
   decrease in mitochondrial membrane potential (as indicated by reduced
   red fluorescence) following KO-PRDX5 in both UMUC-3 and J82 cell lines
   (Fig. [146]9G, [147]H).

   In summary, our data suggest that PRDX5 plays a pivotal role in
   regulating ferroptosis and, consequently, cell death in BC by
   modulating lipid metabolism and mitochondrial functions. These findings
   offer valuable insights into mechanisms underlying PRDX5's contribution
   to the progression of BC.

The role of PRDX5 in regulating ferroptosis during BC progression and its in
vivo validation

   To investigate the involvement of PRDX5 in the regulation of the
   ferroptosis pathway, we initially compared the transcriptomic profiles
   of control and KO-PRDX5 group (Fig. [148]10A). Notably, our analysis
   revealed alterations in signaling molecules essential for ferroptosis
   that were activated following KO-PRDX5 (Fig. [149]10B-E). To further
   evaluate this hypothesis, we examined the impact of KO-PRDX5 on both
   UMUC-3 and J82. Our findings showed that PRDX5 depletion led to the
   activation of the ferroptosis signaling pathway, as evidenced by
   elevated levels of acyl-CoA synthetase long-chain family member 4
   (ACSL4) and kelch-like ECH associated protein 1 (KEAP1) proteins, along
   with a corresponding decrease in the levels of NRF2 (Nuclear Factor
   Erythroid 2–related Factor 2), GPX4, and SLC7A11 proteins
   (Fig. [150]10F).

Fig. 10.

   [151]Fig. 10
   [152]Open in a new tab

   The function of PRDX5 in BC progression. A Volcano plot showing
   differential gene expression between the NC group and the KO-PRDX5
   group; (B) Distribution of relative expression TPM values for ACSL4 in
   the NC group versus the KO-PRDX5 group; (C) Distribution of relative
   expression TPM values for KEAP1 in the NC group versus the KO-PRDX5
   group; (D) Distribution of relative expression TPM values for NRF2 in
   the NC group versus the KO-PRDX5 group; (E) Distribution of relative
   expression TPM values for GPX4 in the NC group versus the KO-PRDX5
   group; (F) Changes in the levels of ferroptosis marker proteins due to
   PRDX5 knock-out in cell lines; (G) Curve graph illustrating changes in
   tumor volume over time, highlighting differences in tumor growth rates
   between the NC-PRDX5 and KO-PRDX5 groups; (H) Comparison of tumor
   weights, demonstrating differences between the NC-PRDX5 and KO-PRDX5
   groups; (I) Appearance of tumors between the NC-PRDX5 and KO-PRDX5
   groups; (J) Size comparison of tumors between the NC-PRDX5 and KO-PRDX5
   groups; (K) Changes in the levels of ferroptosis marker proteins due to
   PRDX5 knock-out in tumor tissues from nude mice

   To corroborate the in vivo function of PRDX5 in BC progression, we
   conducted a subcutaneous xenograft model using nude mice. The results
   demonstrated that the KO-PRDX5 group exhibited a significantly slower
   increase in tumor volume over time compared to the control group
   (Fig. [153]10G). Moreover, the final tumor weight in the KO-PRDX5 group
   was significantly reduced relative to the control group
   (Fig. [154]10H). Visual observations of the subcutaneous tumors in the
   nude mice (Fig. [155]10I) and the quantification of tumor volumes
   (Fig. [156]10J) further confirmed that KO-PRDX5 effectively inhibited
   tumor growth. Additionally, analysis of subcutaneous tumor tissues from
   the xenograft model verified the activation of ferroptosis signaling
   pathways induced by KO-PRDX5 (Fig. [157]10K).

   In conclusion, our study elucidates a critical role for PRDX5 in the
   progression of BC. The knockout of PRDX5 in BC cell lines activated the
   ferroptosis pathway, as evidenced by increased ACSL4 and KEAP1 protein
   levels and decreased levels of NRF2, GPX4, and SLC7A11. Moreover, PRDX5
   depletion significantly diminished tumor volume and weight, effectively
   inhibiting tumor growth in the subcutaneous xenograft model. These
   findings highlight the potential of PRDX5 as a therapeutic target for
   BC treatment.

Discussion

   In our study, we observed that PRDX5 was significantly upregulated in
   BC tissues, with the highest expression levels detected specifically in
   cases of papillary BC. Although our analysis did not reveal a
   statistically significant correlation between elevated PRDX5 expression
   and overall or disease-specific survival rates in patients, a
   discernible trend suggestive of poorer prognosis emerged among those
   with high PRDX5 levels. This observation aligns with prior research
   that identifies PRDX5 as a negative prognostic marker in various cancer
   types, further highlighting its potential relevance in the context of
   BC outcomes [[158]22, [159]23]. Additionally, our study represents the
   first report of PRDX5 expression levels in urine samples from BC
   patients, suggesting its potential as a noninvasive diagnostic
   biomarker for future clinical applications. Through the application of
   scRNA-seq, we identified a significant enrichment of PRDX5 in the
   epidermal cells of BC, indicating its potential critical role in the
   disease’s pathogenesis. This cell-specific expression pattern offers a
   fresh perspective on the functional implications of PRDX5 within the
   context of BC.

   Moreover, we observed that as BC progresses, PRDX5 expression levels
   are notably elevated and closely correlated with specific markers of
   BC, such as GATA3, KRT7, S100P, TP63, and UPK2, all of which are
   implicated in the malignant transformation of bladder epidermal cells.
   This underscores the relevance of PRDX5 in the disease's trajectory and
   its potential role in facilitating BC progression. Through a
   comprehensive series of in vitro and in vivo experiments, our study
   demonstrated that the overexpression of PRDX5 in BC cell lines markedly
   enhanced cellular proliferation, migration, and invasion. Conversely,
   silencing PRDX5 expression resulted in a significant reduction in these
   tumorigenic behaviors. These results corroborate earlier research
   highlighting the role of PRDX5 in cancer biology, reinforcing its
   influence as a pivotal factor in BC progression dynamics [[160]22].
   These findings further underscore the critical role of PRDX5 in the
   development of BC. Prior research has established that PRDX5 acts as an
   oncogene across various cancer types. For instance, Lee et al.
   [[161]24] reported elevated levels of PRDX5 in oral cancer,
   highlighting its involvement in promoting tumor cell proliferation.
   Likewise, Xie et al.[[162]23] found that PRDX5 expression was
   significantly increased in hepatocellular carcinoma, where it was
   associated with tumor invasion and metastasis. Our study builds upon
   this existing body of knowledge, specifically illuminating the
   mechanisms by which PRDX5 exerts its effects in the context of BC
   progression.

   Further studies revealed that PRDX5 impacts cell death by regulating
   ROS levels rather than through traditional apoptotic pathways. This
   finding aligns with existing literature on the role of PRDX5 in redox
   balance [[163]25]. Given that members of the PRDX family play
   significant roles in ferroptosis progression, Cui et al.[[164]26]
   revealed that PRDX3 undergoes specific hyperoxidation during
   ferroptosis, establishing it as a novel biomarker for ferroptosis.
   Additionally, a recent study by Fujita et al. [[165]27] indicated that
   loss of PRDX6 could promote ferroptosis onset. Considering that ROS
   accumulation is a hallmark of ferroptosis and PRDX5 primarily regulates
   ROS levels[[166]28], our study provides the first evidence that PRDX5
   influences the ferroptosis pathway by regulating lipid metabolism. This
   mechanism significantly affects the fate of BC cells, highlighting the
   intricate relationship between PRDX5, lipid metabolism, and ferroptosis
   in BC. We observed alterations in ferroptosis-associated markers
   following PRDX5 knockout, indicating its influence on this pathway.
   Additionally, our in vivo experiments demonstrated that the activation
   of ferroptosis effectively inhibited tumor growth. These findings are
   consistent with literature highlighting the role of ferroptosis in BC
   and other malignancies, further underscoring the potential therapeutic
   implications of targeting this pathway in cancer [[167]29, [168]30].
   The role of PRDX5 in BC indicates its potential as a therapeutic
   target. By influencing cell fate through the regulation of the
   ferroptosis pathway, targeting PRDX5 may represent a novel therapeutic
   strategy. Research has shown that modifications to ferroptosis
   regulators can significantly affect tumor responses to therapy.
   Repurposing drugs and administering various vitamins, such as vitamin
   D, as prophylactics can have a modulatory effect and positively impact
   redox potential in cancer [[169]31]. For instance, the pharmacological
   inhibition of GPX4 activity has been shown to induce ferroptosis,
   leading to reduced tumor growth [[170]32]. This finding enhances the
   understanding of ferroptosis in cancer, offering valuable insights into
   the mechanisms by which PRDX5 influences BC development. It is
   noteworthy that the expression of PRDX5, as an antioxidant enzyme, is
   often influenced by various proteins, particularly NRF2 [[171]25]. NRF2
   is a key regulatory factor in cellular antioxidant responses,
   initiating the expression of a series of antioxidant genes by binding
   to antioxidant response elements. Existing studies have shown that NRF2
   can directly regulate the expression of multiple antioxidant enzymes,
   including PRDX5 [[172]33]. Evidence indicates that NRF2 not only
   influences the expression of antioxidant enzymes through
   transcriptional regulation but can also interact directly with PRDX5,
   thereby enhancing their activity and stability[[173]34].

   Although there is currently no direct evidence to demonstrate a similar
   interaction between PRDX5 and NRF2 in BC, understanding the potential
   interaction between PRDX5 and NRF2 could not only elucidate the reasons
   behind the high expression of PRDX5 in BC but also provide a basis for
   developing new therapeutic strategies. Inhibitors targeting the NRF2
   signaling pathway may represent a potential method for reducing PRDX5
   expression and enhancing chemotherapy sensitivity. This study
   represents the first systematic investigation into the expression
   characteristics of PRDX5 in BC and its specific mechanisms in disease
   progression, while also establishing a relationship between PRDX5 and
   other tumor markers, thereby paving new avenues for BC treatment.
   Future research should focus on exploring the relationship between
   PRDX5 and NRF2 to provide theoretical support for targeted therapy
   strategies.

Conclusion

   In this study, we demonstrated that PRDX5 is highly expressed in BC and
   confirmed its role in promoting tumor progression by regulating ROS
   levels and the ferroptosis pathway. These findings not only enhance our
   understanding of BC pathogenesis but also establish a theoretical basis
   for developing novel therapeutic strategies targeting PRDX5. Future
   research should investigate the efficacy and safety of PRDX5 as a
   potential therapeutic target in the context of BC, paving the way for
   innovative treatment approaches to improve patient outcomes.

Supplementary Information

   [174]Supplementary Material 1^ (4.4MB, docx)
   [175]Supplementary Material 2^ (14.2KB, docx)

Abbreviations

   BC
          Bladder cancer

   PRDX5
          Peroxidase 5

   ROS
          Reactive oxygen species

   TCGA
          The Cancer Genome Atlas

   scRNA-seq
          Single-cell RNA sequencing

   MIBC
          Muscle-invasive bladder cancer

   NMIBC
          Non-muscle-invasive bladder cancer

   GPX4
          Glutathione peroxidase 4

   CCK-8
          Cell Counting Kit-8

   NRF2
          Nuclear Factor Erythroid 2–related Factor 2

   ACSL4
          Acyl-CoA synthetase long-chain family member 4

   KEAP1
          Kelch-like ECH associated protein 1

Authors' contributions

   Shun Wan, Kun-Peng Li, and Si-Yu Chen are mainly responsible for
   writing manuscripts, later revision of language and grammar, and
   article format. Shan-hui Liu and Li Yang are mainly responsible for
   writing manuscripts and article format. Chen-Yang Wang and Kun Cheng
   are accountable for this article's leading writing and structure
   arrangement. Jian-Wei Yang, Li-yun Ding, and Tuan-jie Che are mainly
   responsible for data access. They have made corresponding contributions
   to this article. All authors have read and approved the final
   manuscript.

Funding

   This study was supported by Cuiying Scientific and Technological
   Innovation Program of Lanzhou University Second Hospital (Grant numbers
   CY2021-MS-A12); Investigation of the Effects and Mechanisms of HTRA1 on
   Bladder Tumors Through Combined Non-invasive Urinary Metabolomics
   (Grant numbers 22YF7FA090); Exploring the Accuracy and Mechanisms of
   Non-Invasive Urinary HTRA1 Molecular Detection in BC Diagnosis (Grant
   numbers 23JRRA1006); Multimodal Urinary Analysis for the Identification
   of Bladder Tumor Biomarkers and Investigation into the Molecular
   Mechanisms of HTRA1 (Grant numbers 2023–4-34).

Data availability

   Data were download from the TCGA database- BLCA
   ([176]https://portal.gdc.cancer.gov). All data generated or analyzed
   during this study are included in this published article.

Declarations

Ethics approval and consent to participate

   Our study adhered to the Declaration of Helsinki and ethical guidelines
   established by the Institutional Medical Ethics Committee of Lanzhou
   University Second Hospital. All clinical samples were collected with
   informed consent from the patients and the collection was approved by
   the Medical Ethics Committee of the Lanzhou University Second Hospital
   (D2024-774).

Consent for publication

   Not applicable.

Competing Interest

   The authors declare no competing interests.

Footnotes

   Publisher's Note

   Springer Nature remains neutral with regard to jurisdictional claims in
   published maps and institutional affiliations.

   Shun Wan, Kun-peng Li and Si-yu Chen contributed equally to this work.

Contributor Information

   Shan-hui Liu, Email: 1106074594@qq.com.

   Li Yang, Email: ery_yangli@lzu.edu.cn.

References