Abstract

   Purpose: The objective of this study was to examine the expression and
   role of Centromere protein W (CENPW) in bladder cancer (BLCA), as well
   as its potential mechanistic impact on the progression of BLCA.

   Methods: In this study, we conducted a comparative analysis of the mRNA
   expression level of CENPW in BLCA tissues and adjacent normal tissues
   using data from the Cancer Genome Atlas (TCGA) and Gene Expression
   Omnibus (GEO) databases. Additionally, we investigated the association
   between CENPW expression and patient prognosis. Furthermore, we
   performed in vitro and in vivo experiments to assess the impact of
   CENPW knockdown on various tumor biological phenotypes in BLCA.
   Finally, we conducted an analysis to elucidate the underlying
   mechanisms responsible for the observed phenotypic alterations in BLCA.

   Results: The expression of CENPW was found to be upregulated in BLCA,
   and its higher expression was associated with a poorer disease-specific
   survival (DSS). CENPW was found to have close associations with the
   cell cycle, mitosis, and DNA replication. In vitro and in vivo
   experiments demonstrated that the inhibition of CENPW led to a
   suppression of BLCA progression. Specifically, the knockdown of CENPW
   resulted in cell cycle arrest phase and induced apoptosis in BLCA by
   potentially inactivating the signal transducer and activator of
   transcription3 (STAT3) signaling pathway.

   Conclusion: CENPW has the potential to function as a molecular marker
   indicating an unfavorable prognosis in BLCA. Additionally, CENPW
   exhibits promise as a novel therapeutic target for BLCA.

   Keywords: Bladder cancer, Disease-specific survival, Progression, Cell
   cycle, Apoptosis

Introduction

   Based on the 2020 global cancer statistics data, bladder cancer (BLCA)
   holds the tenth and thirteenth positions among 36 cancer types in terms
   of incidence and mortality, respectively [41]^1. In China, it is
   estimated that there were 91,893 new cases and 42,973 deaths attributed
   to bladder cancer in 2019, making it the second most prevalent
   malignancy within the urological system [42]^2. Approximately 90
   percent of BLCA cases are urothelial carcinomas, which can be further
   categorized as either superficial or muscle-invasive types [43]^3.
   Patients with BLCA may experience trans-urethral resection or radical
   cystectomy with urinary diversion due to frequent recurrence and rapid
   progression, leading to significant impacts on their quality of life
   and socioeconomic status [44]^4^, [45]^5.

   The advancement of high-throughput sequencing technology and
   bioinformatics has facilitated the identification of certain proteins
   that serve as potential biomarkers for cancer prognosis and therapeutic
   targets [46]^6. Consequently, the identification of oncogenes
   associated with BLCA holds considerable significance in the diagnosis
   and treatment of this disease.

   Centromere protein W (CENPW), previously referred to as cancer
   upregulated gene 2 (CUG2), exhibits significant upregulation in various
   cancer types such as ovarian, liver, colon, and lung cancers, and is
   implicated in tumorigenesis [47]^7^, [48]^8. Notably, CENPW has been
   recognized as a novel centromeric component essential for the accurate
   functioning of kinetochores during cellular division [49]^9^-[50]^11.
   Previous investigations have demonstrated the potential of centromere
   proteins as targeted therapies for mitosis-related antitumor
   interventions [51]^12^-[52]^14. Besides, CENPW involve in centromere
   microtubule attachment and meiotic progression of mouse oocytes
   [53]^15. Therefore, we hypothesized that CENPW might be involved in the
   mitosis of tumor cells. Nevertheless, the relationship between CENPW
   and BLCA has not been explored thus far.

   This study aims to examine the correlation between CENPW expression and
   clinicopathological characteristics of BLCA. Additionally, it was
   discovered that inhibiting CENPW resulted in the suppression of BLCA
   progression both in vitro and in vivo. Collectively, these findings
   provide evidence that CENPW could serve as a promising prognostic
   biomarker and therapeutic target for BLCA.

Materials and Methods

CENPW expression in pan-cancer

   The unified and standardized pan-cancer datasets can be downloaded from
   UCSC: TCGA TARGET GTEx (PANCAN, N = 19131, G = 60499). Within this
   dataset, we specifically extracted information regarding the expression
   of the CENPW gene in 13 human solid tumors. To analyze the data, we
   utilized R software (version 3.6.4) to calculate the expression
   differences between paired tumor and adjacent normal tissues.
   Furthermore, we employed an unpaired Wilcoxon Rank Sum and Signed Rank
   Test to assess the statistical significance of these differences.

Characteristics of CENPW in bladder cancer

   The Cancer Genome Atlas (TCGA) dataset was initially employed to
   identify differentially expressed genes (DEGs) in BLCA compared to
   normal tissues. To validate the mRNA expression in BLCA, microarray
   gene expression profiles of bladder urothelial carcinoma (UCB) tumor
   tissues and adjacent non-tumor tissues were obtained from the Gene
   Expression Omnibus (GEO) database ([54]GSE13507) [55]^16. The data
   processing and analysis were performed using Perl (5.30.1) and R
   (version 3.6.2) software. Two-group comparisons were conducted using
   either the unpaired Student's t-test or paired t-test. In this study,
   we conducted an analysis of the relationship between CENPW expression
   and DSS in two cohorts, namely TCGA-BLCA and [56]GSE13507. To determine
   the optimal threshold for CENPW expression in BLCA, we employed the
   "maxstat" R package. Additionally, we utilized the "survival" R package
   to assess the prognostic significance difference between the two groups
   using the logrank test method.

Gene set enrichment analysis and Gene correlation analysis

   Furthermore, we investigated the associations of CENPW with biological
   processes and signaling pathway gene sets using Gene set Enrichment
   Analysis (GSEA v4.2.1) [57]^17. Significant enrichment was
   operationally defined as the identification of KEGG pathways with false
   discovery rate (FDR) q-values less than 0.05 and an absolute normalized
   enrichment score (NES) greater than 1. Our research specifically
   focused on investigating the signaling pathways implicated in the
   development and progression of bladder cancer. Additionally, we
   conducted gene correlation analysis between the CENPW gene and the
   enriched genes using the GEPIA2 database [58]^18.

Tissue specimens

   A total of 14 fresh BLCA tissues and corresponding normal tissues were
   obtained from the Guizhou Provincial People's Hospital, with written
   informed consent provided by all patients. The inclusion and exclusion
   criteria for this study encompassed the diagnosis of bladder urothelial
   carcinoma in all pathological results, the absence of distant
   metastasis prior to surgery, and the absence of any concurrent
   malignancies. Detailed patient characteristics pertaining to the tissue
   samples can be found in [59]Table S1. The Institutional Research Ethics
   Committee of Guizhou Provincial People's Hospital approved this study
   (NO.2023-072).

Cells and transfection

   The human bladder urothelial carcinoma cell lines (5637, T24, RT4, J82,
   UM-UC-3) and an immortalized urothelial cell line (SV-HUC-1) were
   obtained from Procell Life Technology Co. Ltd (Wuhan, China). 10% fetal
   bovine serum (FBS) was added to RPMI 1640 medium to culture 5637 cells.
   The T24 and RT4 cells were cultured in Myc medium supplemented with 10%
   FBS. The J82 and UM-UC-3 cells were cultured in MEM medium supplemented
   with 10% FBS (Sijiqing, Hangzhou, China). All cells were incubated in a
   humidified air atmosphere at 37°C. The cells were transfected when they
   reached a confluence of 60-80%. Transfections with siRNA (GenePharma
   Co., Ltd.; Shanghai, China) were performed using Lipofectamine RNAiMAX
   reagent (Introgen, USA) following the manufacturer's instructions.
   After transfection, the cells were utilized for subsequent experiments
   within a time frame of 24-72 hours. Stable cell lines were generated
   using lentivirus and the lentiviral vector
   pHBLV-U6-MCS-CMV-ZsGreen-PGK-PURO obtained from Hanheng Biotechnology
   (Hanheng Biotechnology Co., Ltd., Shanghai, China). Puromycin was added
   to the cell pools at a concentration of 1mg/mL for a duration of 1
   week. The knockdown efficiency was assessed through Western blot
   analysis and RT-qPCR.

RNA preparation and qRT-PCR

   Total RNA was isolated following the manufacturer's instructions of the
   Total RNA Kit (YEASEN, Shanghai, China). The concentration and purity
   of the RNA were determined using a Nanodrop 2000 spectrophotometer
   (Thermo Fisher Scientific). cDNA was synthesized using the Hifair® Ⅲ RT
   SuperMix for qPCR (YEASEN, Shanghai, China), and the Hieff UNICON®
   SYBR-Green qPCR master mix (YEASEN, Shanghai, China) was employed for
   the qPCR. Real-time PCR analysis was conducted using the Bio-Rad CFX
   Connect Real-Time PCR Detection System. To standardize the measurement
   of gene expression, the β-actin gene was employed. The primer sequences
   utilized can be found in [60]Table S2. The relative mRNA expression was
   determined using the 2(^-ΔΔCT) method and adjusted based on the levels
   of β-actin mRNA.

Western blotting

   A western blot assay was performed using protein extracted from
   cultured cells and tissues. Protein samples were separated using 10%
   and 12% SDS-PAGE and subsequently transferred onto PVDF membranes with
   a pore size of 0.22μm. The membrane was blocked using 5% skim milk and
   a primary antibody was then incubated overnight at 4 °C on the
   membranes. The primary antibodies were used as follows: anti-CENPW
   (1:1,000, 1030382-3; Abcam, United Kingdom), anti-βactin (1:10,000;
   60004-1-Ig; Proteintech, Wuhan, China), anti-Cleavedcaspase 3 (1:1,000,
   #9661S; Zenbio, Chengdu, China), anti-CyclinB1 (1:1,000, 60186-1-Ig;
   Proteintech, Wuhan, China), anti-CDK1 (1:1,000, 11554-1-AP;
   Proteintech, Wuhan, China), anti-Bcl-2 (1:1,000, #2872T; CST, United
   States), anti-P21 (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China),
   anti-Bax (1:1,000, 50599-2-Ig; Proteintech, Wuhan, China), and
   anti-STAT3 (1:1,000, #9139; CST, United States), anti-p-STAT3 (Tyr705)
   (1:2000, #9145; CST, United States). A chemiluminescence system was
   employed to detect protein bands subsequent to the incubation of
   primary antibody probes with secondary antibodies. The scanned blots
   were subjected to analysis using ImageJ version 1.51 in order to
   ascertain the density of the bands. Protein expression was normalized
   by comparing it to the expression of β-actin.

Cell proliferation assay

   Following a 24-hour transfection with siRNA-CENPW, 5637 and UM-UC3
   cells were seeded onto a 96-well plate at a density of 3,000 cells per
   well. Subsequently, 10μl of cell counting kit-8 (CCK-8) solution
   (Apexbio, K1018, USA) was added to each well at 24, 48, 72, and 96
   hours. The optical density (OD) value for each well was measured at 450
   nm after a 2-hour incubation at 37°C using a microplate reader (BioTek,
   Winooski, VT, USA).

Edu staining

   5637 and UM-UC3 cells were transfected with siRNA-CENPW as previously
   described. After plating in a 96-well plate for 72 hours, cell
   proliferation staining was conducted using an Edu kit (C10310-1,
   RIBOBIO, EDU-567, Guangzhou, China) following the manufacturer's
   instructions. Cells were imaged using Olympus IX71 inverted
   fluorescence microscopes, and the quantification of Edu-positive cells
   was performed using ImageJ (ImageJ 1.52a, United States).

Clone formation assay

   Stable knockdown CENPW 5637 and UM-UC3 cells were seeded onto a 6-well
   plate (400 cells/well) and incubated for 14 days. Subsequently, the
   cells were treated with a 4% formaldehyde solution for a duration of 15
   minutes. Subsequently, the cells were subjected to staining using a
   0.1% crystal violet solution for a duration of 30 minutes and counted.
   Finally, the colonies were quantified using ImageJ software.

Immunofluorescence

   Frozen tissue sections (8umm thick) or cell sheets were fixed using a
   4% paraformaldehyde solution, followed by permeabilization using 0.2%
   Triton X-100 for 10 minutes. Subsequently, blocking was performed using
   a 5% bovine serum albumin (BSA) solution from Sigma for a duration of 2
   hours at room temperature. The primary antibodies were incubated
   overnight at 4 °C, followed by treatment with a fluorescein-conjugated
   anti-rabbit secondary antibody and Tubulin Tracker Green (Beyotime
   Biotechnology, Shanghai, China) were incubated for 1 hour at room
   temperature. After a 10-minute incubation period with DAPI, the
   fluorescence signal was visualized using either an Olympus IX70
   fluorescence microscope (Olympus, Japan) or a confocal imaging system
   (Zeiss, Germany).

Cell cycle and apoptosis analysis

   Following 48 hours of transfection, the cells were harvested through
   centrifugation at 1000 rpm for 4 minutes. The cells were then fixed
   overnight at 4°C in 70% ethanol. After 24 hours of fixation, the cells
   were washed three times with PBS. Subsequently, the cells were stained
   with a solution containing 500 μl of propidium iodide (Elabscience,
   Wuhan, China) and RNase A at a ratio of 9:1. The cells were subjected
   to flow cytometric analysis using a flow cytometer (BD Biosciences, San
   Jose, CA, United States) following a 45-minute incubation at room
   temperature. After 72 hours of transfection, the cells were harvested
   by centrifugation (1000 rpm, 4 minutes) and washed thrice with PBS.
   Subsequently, the cells were resuspended in 500μl of binding buffer and
   double stained with Annexin V and PI, in accordance with the
   instructions provided by the FITC Annexin V/PI Apoptosis kit
   (Elabscience, Wuhan, China). Flow cytometry was then performed on the
   samples using the flow cytometer (BD Biosciences) after a 15-minute
   incubation in the dark at room temperature.

TUNEL analysis

   Prior to analysis, the tumor xenografts were fixed and converted into
   frozen sections, which were subsequently washed twice with PBS for 5
   minutes each time to remove OCT. The apoptosis of the tumor xenografts
   was assessed using the TUNEL kit (Elabscience, Wuhan, China) according
   to the standard protocol. Finally, the fluorescence signal of sh-NC and
   sh-CENPW was observed using a fluorescent microscope.

Animal models

   Eight 4-week-old BALB/c nude mice, obtained from Vital River Laboratory
   Animal Technology Co, were used in xenograft experiments. All
   experiments were conducted at the animal center of Guizhou Medical
   University and were approved by the Animal Care and Use Committee
   (2305079). The mice were randomly assigned to two groups of four, and 1
   × 10^7 5637 cells in 200 μl saline were injected subcutaneously into
   the right armpit of each BALB/c nude mouse. Tumor measurements were
   taken every third day, starting on the fifth day after injection. The
   calculation of tumor volume was determined using the formula V =
   (length x width^2) / 2. Once the tumor xenografts met the experimental
   requirements, the nude mice were euthanized and the tumor xenografts
   were excised and imaged.

Statistical analysis

   Statistical analyses were conducted using IBM SPSS Statistics Version
   25.0, R Statistical Software Version 3.6.1, and GraphPad Prism 7
   (GraphPad Software, CA, United States). A two-tailed paired or unpaired
   t-student t-test was employed to compare two groups when variables were
   normally distributed, while a Mann-Whitney U-test was used for other
   variables. A two-way analysis of variance (ANOVA) with Sidak's test was
   employed for conducting multiple comparisons. Pearson's correlation
   analysis was utilized to examine gene correlations. Statistical
   significance was determined when the P-values were below 0.05 (*P <
   0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Results

CENPW expression in Pan-Cancer

   To identify differentially expressed CENPW genes between human tumors
   and normal tissues, a differential gene expression analysis was
   conducted using the TCGA and GTEx databases. The findings revealed that
   CENPW exhibited significantly higher expression levels in breast
   cancer, cervical cancer, lung cancer, stomach cancer, esophageal
   cancer, colon cancer, rectum cancer, head and neck squamous cell
   carcinoma, kidney renal clear cell carcinoma, liver hepatocellular
   carcinoma, thyroid carcinoma, glioblastoma multiforme, and bladder
   urothelial carcinoma compared to their respective normal tissues
   (Figure [61]1).

Figure 1.

   [62]Figure 1
   [63]Open in a new tab

   CENPW expression in pan-cancer. Differential CENPW expression analysis
   in human tumors and normal tissues in UCSC database.

Characteristics of CENPW in bladder cancer

   In order to investigate the biological functions of CENPW in BLCA, we
   conducted an analysis of both the TCGA-BLCA RNA-Seq databases and the
   GEO-BUC Gene-chip databases to assess CENPW expression. As anticipated,
   CENPW exhibited upregulation in BLCA tissues when compared to normal
   controls in TCGA (407 BLCA tissues versus 19 normal adjacent tissues, P
   < 0.001; 19 pairs of tumor tissues and normal adjacent tissues, P <
   0.001; Figure [64]2A, B). As CENPW was also found to be upregulated in
   BLCA tissues compared to adjacent tissues in the [65]GSE13507 cohort
   from GEO, these findings were consistent with the results obtained from
   the TCGA dataset (188 BLCA tissues versus 167 adjacent tissues, P <
   0.0001; 51 matched tumor tissues and adjacent tissues, P < 0.001;
   Figuer2 C, D). To confirm the expression of CENPW in BLCA, we assessed
   both mRNA and protein expression levels in 14 pairs of samples include
   bladder cancer tissues and normal adjacent tissues. In comparison to
   adjacent normal tissues, tumor tissues exhibited significantly elevated
   levels of CENPW mRNA in 14 pairs of tissues (P < 0.001; Figure [66]2E).
   Western blot results demonstrated higher levels of CENPW expression in
   BLCA tissues compared to adjacent normal tissues in 8 pairs of samples
   (P < 0.01; Figure [67]2F). Furthermore, a notable disparity DSS was
   observed between patients exhibiting high CENPW expression and those
   with lower CENPW expression in the TCGA-BLCA dataset (n=385, Hazard
   Ratio (HR) = 1.51, P (HR) = 0.02; Figure [68]2G). Subsequently, we
   validated the significant correlation between high CENPW expression and
   poorer DSS in BLCA patients within the [69]GSE13507 cohort (n=165, HR =
   4.13, P (HR) = 6.4e-5; Figure [70]2H), which aligns with the findings
   from the PrognoScan database [71]^19.

Figure 2.

   [72]Figure 2
   [73]Open in a new tab

   Features of CENPW expression in BLCA. (A, B). CENPW expression in
   TCGA-BLCA cases, unpaired and paired samples. (C, D). CENPW expression
   in [74]GSE13507 cases, unpaired and paired samples. (E, F). CENPW mRNA
   and protein levels in BLCA tissues. (G, H). High CENPW expression is
   associated with significantly poor DSS.

Pathway enrichment analysis of genes co-expressed with CENPW

   To explore the potential signaling pathways affected by differential
   CENPW expression in BLCA, we conducted Gene Set Enrichment Analysis
   (GSEA) on high and low expression CENPW datasets. The analysis revealed
   that the cell cycle-related pathways (normalized enrichment score [NES]
   = 2.58, nominal [NOM]-P < 0.0001, false discover rate [FDR]-q <
   0.0001), oocyte meiosis (NES = 2.38, NOM-P < 0.0001, FDR-q < 0.0001),
   and DNA replication (NES = 2.25, NOM-P =0, FDR-q= 5.022008E-4) were
   significantly enriched in the high CENPW expression dataset (Figure
   [75]3A-C). Subsequently, an examination of the associations between
   CENPW and cell cycle as well as cell proliferation genes was carried
   out using GEPIA. The findings demonstrated a positive correlation
   between CENPW gene expression and CDK1 (R=0.38, P =2.9e-15), CCNB1
   (R=0.41, P <0.0001), and PCNA (R=0.24, P =1.2e-06) (Figure [76]3D-F).
   Additionally, the correlation between CENPW expression and CDK1, CCNB1,
   and PCNA was further investigated in 10 bladder cancer tissue samples.
   The results consistently indicated a positive correlation between CENPW
   expression and CDK1 (R=0.8182, P =0.0058), CCNB1 (R=0.8424, P =0.0037),
   and PCNA [R=0.6606, P =0.0438] (Figure [77]3G-I). The high expression
   of CENPW might relate to the development of BLCA.

Figure 3.

   [78]Figure 3
   [79]Open in a new tab

   Upregulation of CENPW correlates with activation of cell cycle, mitosis
   and DNA replication in BLCA. (A-C). Gene set enrichment analysis (GSEA)
   of CENPW in BLCA. (D-F). Functional gene correlation analysis of CENPW
   with CDK1, CyclinB1 and PCNA. (G-I). Verified the correlation between
   CENPW and CDK1, CCNB1 and PCNA expression in bladder cancer samples.

Knockdown of CENPW inhibits cell proliferation and cell migration

   Initially, the expression of CENPW was examined in normal urothelial
   cells SV-HUC-1 and bladder urothelial cancer cells (RT4, T24, J82,
   UM-UC-3, 5637). It was observed that CENPW exhibited significantly
   higher expression levels in 5637 (P < 0.0001), UM-UC-3 (P < 0.01), and
   J82 (P < 0.05) compared to SV-HUC-1 (Figure [80]4A). T24 and RT4 did
   not show a significant difference in CENPW expression compared to
   SV-HUC-1. Subsequently, 5637 and UM-UC-3 were selected for further
   loss-of-function experiments. The knockdown efficiency was assessed
   after sequential transfection of siRNA, revealing a significant
   decrease in CENPW protein expression in si-CENPW transfected cells
   (5637, P < 0.001; UM-UC-3, P < 0.0001) (Figure [81]4B, C). The findings
   from the CCK8 assay demonstrate a significant reduction in the
   proliferation capacity of 5637 and UM-UC-3 cells following CENPW
   knockdown (5637, P < 0.001, UM-UC-3, P < 0.001) (Figure [82]4D, E).
   Additionally, the si-CENPW group of 5637 and UM-UC-3 cells exhibited
   abnormal cell morphology 96 hours after transfection ([83]Figure S1).
   The EDU results further indicate a significant decrease in DNA
   replication ability in the si-CENPW group compared to the control group
   (5637, P < 0.001, UM-UC-3, P < 0.0001) (Figure [84]4F, G). Moreover,
   the results of cell migration demonstrate a significant decrease in the
   migration ability of the si-CENPW group compared to the control group.
   (5637, P < 0.001, UM-UC-3, P < 0.0001) (Figure [85]4H, I).

Figure 4.

   [86]Figure 4
   [87]Open in a new tab

   Knockdown of CENPW inhibited bladder cancer cell progression. A. CENPW
   expression in Urothelial cell lines and bladder urothelial carcinoma
   cell lines. (B, C). Verification of the efficiency of knockdown of
   CENPW in 5637 and UM-UC-3 cells. (D-G). Depletion of CENPW inhibited
   bladder cancer cell proliferation. Bars, 500μm. (H, I). Depletion of
   CENPW inhabited bladder cancer cell migration. Bars, 100μm.

Knockdown of CENPW arrests the cell cycle and induces cell apoptosis

   The results of the cell cycle assay demonstrated that silencing of
   CENPW led to a significant increase in the proportions of G2/M phase in
   5637 (24.53 ± 3.455%, P = 0.0009) and UM-UC-3 (31.65 ± 2.554%, P =
   0.003) cells, compared to the 5637 and UM-UC-3 NC group where the
   percentages were (13.70 ± 0.9087%) and (18.85 ± 2.503%) respectively
   (Figure [88]5A, B). Following transfection for 72 hours, the apoptosis
   rates of 5637 and UM-UC-3 cells were measured and compared between the
   NC group and the si-CENPW group. The results demonstrated that the
   apoptosis rates of 5637 and UM-UC-3 cells in the NC group were 13.6 ±
   6.3% and6.76% ± 4.29%, respectively. Conversely, in the si-CENPW group,
   the apoptosis rates were 41.2 ± 10.32% and 17.62% ± 7.07% for 5637 and
   UM-UC-3 cells, respectively. Notably, transfection with si-CENPW
   significantly increased the apoptosis rates in both 5637 (P < 0.001)
   and UM-UC-3 (P < 0.05) cells (Figure [89]5C, D). Apoptosis tests
   further revealed that the knockdown of CENPW induced apoptosis in BLCA
   cells.

Figure 5.

   [90]Figure 5
   [91]Open in a new tab

   Knockdown of CENPW arrested cell cycle and induced apoptosis. (A, B).
   Cell cycle phase distribution in si-NC and si-CENPW group of 5637 and
   UM-UC-3 cells. (C, D). The percentage of apoptotic cells in si-NC and
   si-CENPW group of 5637 and UM-UC-3 cells.

Knockdown of CENPW suppresses tumorigenicity of BCLA cells in vitro and in
vivo

   To assess the long-term effects of CENPW knockdown on BLCA cells,
   stable knockdown cell lines were established using 5637 and UM-UC-3
   cells. The findings of this study indicate that CENPW protein levels
   was decreased in the sh-CENPW group compared to the sh-NC group (5637 P
   < 0.01, UM-UC-3 P < 0.001; Figure [92]6A, B). Furthermore, the
   knockdown of CENPW resulted in a significant reduction in both colony
   number and size in 5637 (P < 0.0001) and UM-UC-3 (P < 0.0001) cells, as
   compared to the sh-NC group (Figure [93]6C, D). In order to investigate
   the potential antitumor effects of CENPW knockdown in vivo, the tumor
   volume was measured every 3 days and a tumor growth curve was plotted.
   The results demonstrated that the tumor growth in the sh-CENPW group
   was significantly suppressed in comparison to the sh-NC group (P <
   0.001; Figure [94]6E). The Western blot (WB) analysis provided further
   confirmation that the protein level of CENPW in the sh-CENPW group was
   significantly lower than that in the sh-NC group (P < 0.05; Figure
   [95]6F). To investigate the underlying mechanism of tumor inhibition,
   we conducted a TUNEL assay on frozen sections of the tumors. The red
   fluorescence intensity of the sh-CENPW group (565.4 ± 102.1) was
   significantly higher compared to the sh-NC group (224.8 ± 31.39),
   indicating an increase in apoptosis in the sh-CENPW group (Figure
   [96]6G; P < 0.001).

Figure 6.

   [97]Figure 6
   [98]Open in a new tab

   The tumorigenicity was reduced in stable CENPW-knockdown cell lines of
   5637 and um-uc-3. (A, B). Verification of the efficiency of knockdown
   of CENPW in stable cell lines. (C, D). Poor clonogenicity in stable
   CENPW-knockdown cell lines. E. Weak tumor formation of stable
   CENPW-knockdown 5637 cell in nude mice. Bars, 1cm. F. The expression of
   CENPW was decreased in shCENP-W group. G. The higher proportion of
   apoptosis in the subcutaneous tumor of shCENP-W group. Bars, 100μm.

Knocking down CENPW inactivates the STAT3 pathway

   To further investigate the mechanism by which CENPW knockdown inhibits
   bladder cancer, we conducted a comparative analysis of the protein
   levels of STAT3 and P-STAT3 in both the si-NC group and si-CENPW group
   of 5637 and UM-UC-3 cells. The results suggest that the depletion of
   CENPW did not have an impact on the overall protein levels of STAT3 in
   both the si-NC and si-CENPW groups of 5637 and UM-UC-3 cells. However,
   the knockdown of CENPW did result in a decrease in the phosphorylated
   protein level of STAT3 (p-STAT3) in both the si-NC and si-CENPW group
   of 5637 and UM-UC-3 cells. The expression of cell cycle-promoting
   proteins CyclinB1 and CDK1, as well as the apoptosis suppressor protein
   Bcl2, was found to be inhibited compared to the si-NC group.
   Furthermore, the knockdown of CENPW resulted in an increase in cyclin
   inhibition protein P21 and the pro-apoptotic protein BAX, as well as
   Cleaved-caspase-3 in the si-CENPW group (Figure [99]7A, B). Therefore,
   we suggest that knockdown of CENPW inhibits bladder cancer probably by
   repressing phosphorylation of STAT3. Additionally, abnormal mitotic
   cases, such as multi-polarization of spindles and hypercondensed
   chromosomes, were observed in the si-CENPW group of 5637 cells (Figure
   [100]7C).

Figure 7.

   [101]Figure 7
   [102]Open in a new tab

   Potential mechanism to inhibit the development of bladder cancer cell.
   (A, B). Alteration in cell cycle arrest proteins, apoptosis-promoting
   proteins and apoptosis-inhibiting proteins. C. Abnormal spindle and
   misaligned chromosomes (white arrow) in si-CENPW group. Bars, 10μm.

Discussion

   BLCA is categorized into non-muscle-invasive and muscle-invasive types,
   each exhibiting distinct biological characteristics [103]^20.
   Approximately 40% of patients with non-muscle-invasive bladder cancer
   do not exhibit a response to BCG treatment, with some even progressing
   to muscle-invasive bladder cancer [104]^21^, [105]^22. Furthermore,
   even among those who undergo cystectomy, a subset of patients with
   muscle-invasive bladder cancer still experience metastasis and
   recurrence [106]^23. Despite significant advancements in neoadjuvant
   therapies for bladder cancer, such as immunotherapy, antibody-drug
   conjugates, and chemotherapy, their efficacy remains limited [107]^24.
   The search for novel therapeutic targets in the treatment of bladder
   cancer has been ongoing. Recent research suggests that centromeric
   protein molecules associated with mitosis could potentially serve as a
   promising avenue for tumor treatment [108]^12^, [109]^25. Specifically,
   CENPW, a member of the CCNA involved in mitosis, plays a crucial role
   in ensuring the accurate assembly of sister chromosomes prior to their
   division. Previous studies have demonstrated its high expression in
   various cancers, indicating its involvement in promoting cancer
   progression and resistance [110]^7^, [111]^9^, [112]^15^, [113]^26.

   The current study employed the TCGA-BLCA sequencing database and the
   [114]GSE13507 gene chip database to examine the upregulation of CENPW
   in BLCA. Our findings demonstrate a significant elevation in CENPW
   expression levels within BLCA tissues compared to normal bladder
   tissues, a result that was further validated using frozen tissue
   samples from bladder cancer patients. Moreover, patients with high
   CENPW expression exhibited poorer disease-specific survival rates in
   comparison to those with low expression levels. Furthermore, functional
   gene analysis revealed a positive correlation between CENPW expression
   and cell proliferation and cell cycle genes in BLCA. The expression
   level of CENPW was assessed in 5637, UM-UC-3, J82, T24, RT4 cells, and
   SV-HUC-1. The results indicated that CENPW expression was significantly
   higher in BLCA urothelial cells compared to normal urothelial cells.
   These findings suggest that CENPW may serve as a potential prognostic
   biomarker for BLCA and contribute to its development.

   In this study, transient interference and stable knockdown approaches
   were employed to treat BLCA cells. The expression of CENPW was notably
   reduced in both the si-CENPW and sh-CENPW groups when compared to the
   control group. A series of functional experiments conducted on BLCA
   cells provided evidence of a close association between CENPW and the
   progression of bladder cancer. The si-CENPW group exhibited significant
   reductions in cell proliferation, DNA synthesis, and cell migration
   when compared to the si-NC group. Additionally, an increasing number of
   cells in the si-CENPW and sh-CENPW groups displayed morphological
   changes indicative of apoptosis. To elucidate the underlying cause of
   the decreased cell proliferation capacity, we analyzed alterations in
   cell cycle and apoptosis. The results indicated a blockade of the cell
   cycle at the G2/M phase [115]^27. CDK1 and CyclinB1are known to be
   factors that promote the cell cycle, and their abnormal expression can
   disrupt the progression of the G2/M phase of the cell cycle [116]^28.
   Conversely, P21 is a protein that inhibits the cell cycle by
   deactivating CDK1, leading to cell cycle arrest [117]^29. Additionally,
   BCL2 and BAX play roles in regulating apoptosis, with BAX promoting
   apoptosis and BCL2 inhibiting it. Therefore, we conducted further
   analysis on the expression levels of proteins related to the cell cycle
   and apoptosis. Our findings revealed a decrease in the expression of
   CyclinB1, CDK1, and BCL2 in the si-CENPW group, while the expression of
   P21, BAX, and Cleaved-caspase3 increased. However, the specific
   mechanism by which CENPW knockdown influences cell cycle arrest remains
   unclear. we conducted a detailed analysis of mitosis in both the si-NC
   and si-CENPW groups. Interestingly, we observed an abnormal formation
   of the spindle during cell division in the si-CENPW group of 5637
   cells. This observation aligns with a previous study which demonstrated
   that the depletion of CENPW in mouse oocytes led to spindles with
   abnormal poles during the division phase and activated the spindle
   assembly checkpoint (SAC) [118]^15. Previous research had shown that
   tumor cells multi-polarization leaded to cell cycle arrest and
   apoptosis [119]^30. Based on these findings, we propose that CENPW
   plays a crucial role in the proper formation of the spindle in BLCA
   cells. Consequently, the aberrant spindle formation observed may result
   in the misalignment of sister chromosomes and subsequent cell mitosis
   failure. It is widely acknowledged that STAT3 plays a crucial role in
   various cellular processes such as cell proliferation, differentiation,
   and apoptosis. Previous studies have demonstrated that excessive
   activation of STAT3 contributes to the advancement of BLCA [120]^31^,
   [121]^32. Consequently, we conducted a further investigation into the
   protein expression profile of the STAT3 signaling pathway in both the
   si-NC group and the si-CENPW group. However, our findings indicated
   that the knockdown of CENPW resulted in a decrease in the
   phosphorylation protein expression of STAT3 in 5637 and UM-UC-3 cells.
   The potential relationship between the interference of CENPW expression
   and the inhibition of bladder cancer may be attributed to the
   inactivation of STAT3. Ultimately, the tumor formation capacity of
   sh-CENPW cells was examined in vivo. It was observed that the
   subcutaneous tumor growth was comparatively slower in the sh-CENPW
   group in comparison to the sh-NC group. Furthermore, the Tunel assay
   revealed a higher occurrence of apoptosis in the tumors of the sh-CENPW
   group.

Conclusion

   In conclusion, our findings demonstrate a significant upregulation of
   CENPW in both BLCA tissues and cell lines. Moreover, a high expression
   of CENPW is associated with tumor progression in BLCA and predicts a
   poorer DDS. Notably, the inhibition of BLCA progression can be achieved
   through the knockdown of CENPW. Consequently, CENPW exhibits potential
   as an innovative therapeutic target and biomarker for prognostic
   prediction in BLCA. However, it is important to acknowledge certain
   limitations in this study. Firstly, the cohort sample size is
   relatively small and should be expanded in future research. Secondly,
   further investigation is required to elucidate the underlying
   mechanisms by which knockdown of CENPW arrests cell cycle and promotes
   apoptosis in BLCA cells.

Supplementary Material

   Supplementary figure and tables.
   [122]Click here for additional data file.^ (608.4KB, pdf)

Acknowledgments