Abstract

Simple Summary

   In this study, we demonstrate that ubiquitin C-terminal hydrolase-L5
   (UCHL5), a member of the DUBs family, promotes the growth and migration
   of bladder cancer cells and facilitates tumor growth in vitro. Using
   shRNA-mediated knockdown and overexpression of UCHL5, we confirm that
   UCHL5 regulates c-Myc expression in bladder cancer cells. In addition,
   our data indicate that UCHL5 increases c-Myc expression by activating
   the AKT/mTOR signaling pathway. This newly defined UCHL5-AKT/mTOR-c-Myc
   axis represents a new therapeutic target for the treatment of bladder
   cancer.

Abstract

   Ubiquitin C-terminal hydrolase L5 (UCHL5) is a deubiquitinating enzyme
   (DUB) that removes ubiquitin from its substrates. Associations between
   UCHL5 and cancer have been reported in various tissues, but the effect
   of UCHL5 on bladder cancer has not been thoroughly investigated. This
   study investigates the expression and function of UCHL5 in bladder
   cancer. UCHL5 was shown to be abnormally expressed using IHC of tissue
   microarray and Western blotting. Several procedures were performed to
   assess the effect of UCHL5 overexpression or knockdown on bladder
   cancer, such as cell proliferation, colony formation, wound-healing,
   and Transwell assays. In addition, RNA-Seq and Western blotting
   experiments were used to verify the status of downstream signaling
   pathways. Finally, bladder cancers with knockdown or overexpression of
   UCHL5 were treated with either SC79 or LY294002 to examine the
   participation of the AKT/mTOR signaling pathway and the expression of
   downstream targets c-Myc, SLC25A19, and ICAM5. In contrast to adjacent
   tissue samples, we discovered that UCHL5 was substantially expressed in
   bladder cancer samples. We also found that UCHL5 downregulation
   significantly suppressed both tumor growth in vivo and cell
   proliferation and migration in vitro. According to RNA-Seq analyses and
   Western blotting experiments, the expression of c-Myc, SLC25A19, and
   ICAM5 was modified as a result of UCHL5 activating AKT/mTOR signaling
   in bladder cancer cells. All things considered, our findings show that
   increased UCHL5 expression stimulates AKT/mTOR signaling, subsequently
   triggering the expression of c-Myc, SLC25A19, and ICAM5, which in turn
   promotes carcinogenesis in bladder cancer. UCHL5 is therefore a
   potential target for therapy in bladder cancer patients.

   Keywords: UCHL5, bladder cancer, proliferation, migration, AKT, c-Myc

1. Introduction

   With 573,278 new cases reported in 2020, bladder cancer (BCa) is one of
   the most prevalent cancers worldwide and the most common malignancy
   affecting the urinary tract [[44]1]. Currently, urine exfoliative
   cytology and cystoscopy are the primary methods for identifying bladder
   cancer. The former is less sensitive, and the latter is an intrusive
   procedure associated with increased patient suffering
   [[45]2,[46]3,[47]4]. In clinical practice, bladder cancer is generally
   divided into non-muscle-invasive bladder cancer (NMIBC) and
   muscle-invasive bladder cancer (MIBC). Transurethral resection of
   bladder tumor (TURBT) in combination with intravesical instillation
   therapy is the principal approach for the treatment of NMIBC. Despite
   having a promising prognosis (five-year survival rate of around 85%),
   NMIBC is considered one of the most expensive tumors to manage due to
   frequent recurrence and high treatment costs [[48]5,[49]6]. The
   recommended course of treatment for MIBC is neoadjuvant chemotherapy
   (NAC) followed by radical cystectomy. However, the effectiveness of
   either NAC combined with radical cystectomy or a triple combination of
   surgery, radiation, and chemotherapy for MIBC or advanced bladder
   cancer remains inadequate, with a five-year survival rate of just 50%
   [[50]7,[51]8]. Therefore, further exploration of the pathogenesis of
   bladder cancer progression is necessary for identifying more effective
   targets and specific diagnostic molecules.

   Ubiquitin C-terminal hydrolase-L5 (UCHL5) belongs to the family of
   deubiquitinating enzymes (DUBs) that eliminate ubiquitin (Ub) chains
   from their protein substrates [[52]9]. DUBs participate in several
   biological functions, ranging from cell growth and differentiation to
   regeneration, translational modulation, and tumorigenesis. Notably, the
   interactions between these functions are dynamic and essential for the
   oncogenesis and evolution of tumors [[53]10,[54]11,[55]12]. UCHL5 is
   abnormally elevated in human cancer tissues or cell lines, including
   cervical carcinoma, epithelial ovarian cancer (EOC), esophageal
   squamous cell carcinoma (ESCC), lung cancer, and pancreatic carcinoma
   [[56]13,[57]14,[58]15,[59]16,[60]17]. Upregulation of transforming
   growth factor (TGF) signaling is the primary mechanism through which
   UCHL5 has been implicated in malignancies thus far
   [[61]17,[62]18,[63]19]. Chen Z et al. discovered that UCHL5 silencing
   might lead to cell death in A549 cells, a type of non-small-cell lung
   cancer cells, by activating the expression of Bax/Bcl-2, caspase-3, and
   caspase-9 [[64]16]. When UCHL5 is overexpressed in endometrial
   carcinoma (EC), Wnt/β-catenin signaling is stimulated, which results in
   decreased apoptosis and increased proliferation [[65]20]. Although
   UCHL5 is shown to be an oncogene in many malignancies, its function in
   bladder cancer remains unknown.

   In this study, we found that UCHL5 is substantially expressed in
   bladder cancer tissues and cells. Overexpression of UCHL5 enhances,
   while silencing of UCHL5 represses, cancer cell proliferation and
   migration by c-Myc, SLC25A19, and ICAM5 transformation via AKT/mTOR
   pathways. Our findings indicate that UCHL5 is a valuable diagnostic
   marker and potential therapeutic target for bladder cancer.

2. Materials and Methods

2.1. BCa Cohort Collection and Preprocessing

   We retrieved BCa expression cohort data from The Cancer Genome Atlas
   (TCGA) database. A total of 407 BCa samples and 19 normal samples were
   obtained from this database (TCGA-BLCA cohort). Another 9 normal
   samples were acquired from the GTEx database for subsequent analysis.
   The related clinical information, including living status, age, gender,
   stage, and grade, was also obtained to explore the association between
   UCHL5 expression and clinical features in determining the prognostic
   value of UCHL5. CIBERSORT, a computational approach developed by
   Alizadeh et al. [[66]21], aims for robust enumeration of cell subsets
   from expression profiles. CIBERSORT requires a specialized knowledge
   base of gene expression signatures, termed a “signature matrix”, for
   the deconvolution of cell types of interest. CIBERSORT implements a
   machine learning approach, called support vector regression (SVR), that
   improves deconvolution performance through a combination of feature
   selection and robust mathematical optimization techniques. CIBERSORT is
   a useful approach for high-throughput characterization of diverse cell
   types, such as tumor infiltrating leukocytes (TILs), from complex
   tissues. Thus, we used CIBERSORT for 22 immune cell infiltration levels
   estimation of BCa samples from the TCGA database by calculating the
   normalized enrichment score (NES). Then, Pearson analysis was performed
   to explore the association between UCHL5 and the 22 immune cell types.

2.2. Cell Culture

   The human bladder cancer cell lines EJ, T24, and UMUC3 were donated by
   the Stem Cell Bank, CASS, China. UMUC3 was incubated in Gibco’s DMEM
   sodium supplemented with 10% FBS, 100 mg/mL streptomycin sulfate, and
   100 U/mL penicillin-G sodium at 37 °C in 5% CO[2]; whereas EJ and T24
   were grown in Gibco’s RPMI 1640 media containing 10% fetal bovine serum
   (FBS; Gibco, Australia).

2.3. Antibodies and Reagents

   Antibodies against β-actin (Cat. AC038), UCHL5 (Cat. A7978), SLC25A29
   (Cat. A12373), ICAM5 (Cat. A4156), PI3K (Cat. A4860), p-PI3K (Cat.
   AP0854), AKT (Cat. 17909), p-AKT (S473, Cat. AP0637), mTOR (Cat.
   A11355), and P-mTOR (Cat. AP0115) were acquired from ABclonal
   (Abclonal, United States). Antibodies against c-Myc (Cat. #9402) and
   Ki67 (Cat. #9449) were obtained from Cell Signaling Technologies
   (Danvers, MA, United States). Sc79 (AKT activator) and LY294002 (PI3K
   inhibitor) were acquired from MedChemExpress (MedChemExpress, Monmouth
   Junction, NJ, USA).

2.4. Lentivirus-Mediated Stable Low Expression Cells

   Three targeted sequences, homologous to UCHL5, were designed using the
   lentiviral expression vector (pLKO.1-TRC-copGFP-2A-PURO, Tsingke
   Biotechnology Co., Ltd., Beijing, China). The sequences in human genes
   encoding UCHL5 were as follows: ShUCHL5-1 sense strand,
   5′-CCGGAGCCAGTTCATGGGTTAATTTCTCGAGAAATTAACCCATGAACTGGCTTTTTTG-3′,
   ShUCHL5-2 sense strand,
   5′-CCGGTGAAGGTGAAATTCGATTTAACTCGAGTTAAATCGAATTTCACCTTCATTTTTTG-3′,
   ShUCHL5-3 sense strand,
   5′-CCGGCTGGTTGTCTAACTACCATATCTCGAGATATGGTAGTTAGACAACCAGTTTTTTG-3. A
   negative control sequence with no homologous human protein was designed
   using the same method (ShControl sense strand, 5‘-TTCTCCGAACGTCGT-3,
   ShControl antisense strand, 5′-GTCGTGGATGAGTC-3). Using MAX
   transfection agent, 293T cells were infected with lentivirus vectors
   carrying the desired cDNA sequences, including psPAX2 (Addgene, Cat.
   #12260) and pMD2.G (Addgene, Cat. #12259). Then, EJ, T24, and UMUC3
   cells were infected with lentiviral particles containing ShControl or
   ShUCHL5. Puromycin (600 ng/mL) was used to select transfected cells
   after being infected with indicated shRNA for 5 days. Then, Western
   blotting was used to determine the transfection efficiency.

2.5. Lentivirus-Mediated Stable Overexpression

   Lentivirus production and the establishment of stable overexpression
   cell (EJ, T24, and UMUC3) lines were proceeded as previously described,
   with specific revisions [[67]22]. Briefly, 293T cells were incubated
   with expression plasmids (PHAGE-PURO, Tsingke Biotechnology Co., Ltd.)
   as well as the packaging vectors pMD2.G (Addgene, Cat. #12259) and
   psPAX2 (Addgene, Cat. #12260). For stable transfection with
   pHAGE-puro-UCHL5, puromycin was employed to eradicate the
   non-transfected cells for 5 days. Western blotting was used to assess
   the transfection efficiencies.

2.6. Cell Proliferation Assays

   Cell proliferation was examined using CCK-8 assay (MedChemExpress,
   China). In brief, bladder cancer cells (around 1000 cells/well) were
   added to 96-well plates and incubated for 1–5 days. Then, 10 μL of
   CCK-8 solvent (Sangon Biotech, Shanghai, China) was added into each
   well, and the cells were warmed for 2 h in a dark room. Finally,
   absorbance at 450 nm was measured using a microplate reader (cat. no.
   SpectraMaxM2; Molecular Devices, Sunnyvale, CA, USA).

2.7. Colony Formation Assays

   First, we inoculated 1000 cells/well in a 6-well culture dish followed
   by incubation for 10–14 days. then, colonies were stained for 20 min
   with 0.1% crystal violet after being fixed for 20 min with 4%
   paraformaldehyde. colonies containing over 50 cells were counted using
   imagej cell counter.

2.8. Wound-Healing Assay

   A wound-healing experiment was used to assess the impact on cell
   migration capacity. Cells were sown and grown on 6-well culture plates
   until they reached 80% confluency. Using a sterile 200 μL pipette tip,
   a straight wound was drawn in the center of the well, and the
   serum-free medium was subsequently replaced. The spread of wound
   closure was observed at different time points (0, 24 h) and
   photographed under a microscope. Using ImageJ, we computed the wound’s
   surface area. Cell migration results are shown as a proportion of the
   corresponding control group.

2.9. Transwell Assay

   Cell migration was also assessed using the Transwell assay. In general,
   cells (approximately 5 × 10^4) were planted into the top chamber at a
   volume of 100 μL of serum-free RPMI-1640 or DMEM medium. RPMI-1640 or
   DMEM containing 20% FBS (600 μL) was added to the lower chamber. The
   cells were stained with crystal violet at room temperature for 20 min
   after being cultured at 37 °C for 24 h. Five random fields were
   observed in each well, and cells were counted under a microscope.

2.10. Real-Time PCR

   Total RNA was extracted from bladder cancer cells using RaPure Total
   RNA Micro Kit (Magen, China) according to the manufacturer’s protocol.
   The RNA NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA,
   USA) was used to quantify RNA at 260 nm/280 nm. Following the package
   recommendations, 2 μg of total RNA was reverse transcribed to cDNA
   using ABScript II RT Master Mix (ABclonal, Wuhan, China). qRT-PCR was
   performed on a Bio-Rad (Hercules, CA, USA) CFX96 system to ascertain
   the mRNA levels of genes of interest based on SYBR green fluorescence
   levels. The primers used and their sequences are shown in [68]Table S1.
   The relative mRNA expression level of each target gene was estimated
   using the 2^−ΔΔCT method in conjunction with ACTB as an internal
   loading control.

2.11. Western Blotting Analysis

   Cells were sufficiently lysed in RIPA containing 1% protease inhibitor
   and 1% PMSF (all from Sigma-Aldrich, St. Louis, MO, USA). Total protein
   (40 μg) was separated by 10–12.5% SDS-PAGE electrophoresis and
   transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore,
   cat# IPVH00010, Shanghai, China). After blocking with 5% skim milk at
   room temperature for 2 h, the membrane was first treated with the
   primary antibody ([69]Table S2) at 4 °C for an overnight period,
   followed by an incubation with the secondary antibody—goat anti-rabbit
   IgG or goat anti-mouse IgG ([70]Table S3)—at room temperature for an
   additional two hours. The bands on the membrane were monitored on a
   Tanon-5200 ECL imager (Tanon, Shanghai, China) and visualized by an
   enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA,
   USA). The original Western Blots can be found at [71]Supplementary
   Materials.

2.12. Xenograft Assays

   Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing,
   China) supplied four-week-old male BALB/c nude mice.

   Animal experiments were conducted in the lab animal facility of
   Zhongnan Hospital at Wuhan University. Mice were subcutaneously
   injected with 4 × 10^6 shControl, shUCHL5-1, and shUCHL5-3 T24 cells (n
   = 5) following a week of the adapted diet. The tumors were removed and
   measured five weeks after the mice were euthanized by anesthesia
   overdose. Additionally, the tumor volume was assessed every three days.

2.13. Immunohistochemical (IHC)

   Immunohistochemical analysis was carried out as previously described
   [[72]22]. In brief, formalin-fixed, paraffin-embedded tissue sections
   were first deparaffinized. Endogenous peroxidase activity was then
   inhibited using H2O2. The indicated primary antibody ([73]Table S2) and
   secondary antibody ([74]Table S3) were added to the sections according
   to the recommended protocols provided by the manufacturer. All the
   slides were examined under an inverted microscope at 200×
   magnification.

2.14. Statistical Analysis

   All the experimental data are represented as the means ± standard
   errors. Student t-tests or one-way ANOVA were employed in assessment of
   the statistical analyses, with p < 0.05 regarded as indicating a
   statistically significant difference.

3. Results

3.1. The Expression of UCHL5 in Bladder Cancer and Its Role in Survival and
Clinical Applications

   Both TCGA and GTEx databases were used in this study to characterize
   the differences in UCHL5 expression between BCa and normal samples.
   UCHL5 was found to be overexpressed in BCa samples (n = 407) compared
   with normal samples (n = 28) using the TCGA and GTEx databases
   ([75]Figure 1A, p < 0.01), which was further validated using TCGA-BLCA
   data ([76]Figure 1B, p < 0.05). We further explored the clinical
   difference between UCHL5 high- and low-expression groups ([77]Figure
   1C). In the TCGA-BLCA cohort, patients classified in the UCHL5
   high-expression group occupied higher grades (p < 0.001) than those
   classified in the low-expression group, the latter of which is
   conducive to increased survival (p = 0.009). According to survival data
   from the TCGA database (p = 0.045, [78]Figure 1D), overall survival
   (OS) was significantly poorer in patients with high UCHL5 expression
   than in those with low UCHL5 expression. Then, we utilized tissue
   microarrays, including 63 bladder cancer tissue samples and 16 adjacent
   bladder tissue samples, using IHC analysis of UCHL5 to verify the
   earlier findings. The findings show substantially elevated UCHL5
   expression in the cancer tissue relative to adjacent bladder tissue
   ([79]Figure 1E,F). Next, we explored the association between UCHL5 and
   22 immune cell types defined by the CIBERSORT tool ([80]Figure 1G). We
   first measured the infiltration levels in the 22 immune cell types of
   BCa samples from the TCGA database by calculating the normalized
   enrichment score (NES) using CIBERSORT. UCHL5 was determined to be
   positively related to T cell CD4 memory activation, NK cell resting,
   macrophages M0, macrophages M1, dendritic cell activation, and mast
   cells activation (p < 0.05), but negatively related to plasma cells, T
   cell CD4 naïve, regulatory T cells, and monocytes (p < 0.05).

Figure 1.

   [81]Figure 1
   [82]Open in a new tab

   Expression of UCHL5 in bladder cancer. Differences in expression of
   UCHL5 between (A) ladder cancer (BLCA) samples (n = 407) and normal
   samples (n = 28) using TCGA (matched samples, n = 19) and GTEx
   databases (healthy tissue, n = 9). ** p < 0.01; (B) bladder cancer
   (BLCA) samples (n = 407) and matched samples (n = 19) using TCGA
   database. * p < 0.05. (C) The differences in clinical features (living
   status, age, gender, stage, and grade) across UCHL5 high- and
   low-expression groups in the TCGA cohort. (D) Overall survival curve
   across UCHL5 high- and low-expression groups in the TCGA cohort. The
   p-value is indicated. (E) Representative IHC images for UCHL5 in tissue
   microarrays containing tumor tissue samples and normal bladder tissue
   samples. (F) Tissue microarray data analysis of UCHL5 expression in 63
   bladder cancer tissue samples and 16 adjacent bladder tissue samples.
   ** p < 0.01. (G) Association of UCHL5 expression with 22 immune cell
   infiltration levels defined by CIBERSORT algorithm. Values are shown as
   mean ±  SD. * p < 0.05 and ** p < 0.01 compared with controls.

3.2. Knocking Down UCHL5 Inhibits Cell Proliferation and Migration

   First, in EJ, T24, and UMUC3 cells, we used lentiviral-mediated shRNA
   to knock down expression of the UCHL5 gene toward fully understanding
   its function in biological processes. Western blot analysis was used to
   confirm silencing of UCHL5 protein expression ([83]Figure 2A). To
   ascertain the function of UCHL5 in proliferation and colony formation
   abilities, CCK-8 and colony formation assays were conducted. In
   comparison to control cells, transfection of the UCHL5-shRNA
   significantly slowed the development of bladder cancer cells as seen in
   [84]Figure 2B,C. Additionally, the Transwell migration and the
   wound-healing assays revealed that downregulating UCHL5 significantly
   reduced cell movement ([85]Figure 2D,E).

Figure 2.

   [86]Figure 2
   [87]Open in a new tab

   UCHL5 depletion represses tumor formation in vitro. (A) UCHL5 silencing
   in bladder cancer cells confirmed via Western blotting assays. β-Actin
   is used as the loading control. (B) The proliferation of cells between
   two UCHL5 silencing groups and control group at 24, 48, 72, 96, and 120
   h by CCK-8 assay. (C) Colony formation assays. The number of clones was
   counted and plotted. (D,E) Migration ability evaluated using
   wound-healing (Scale bar = 100 μm) and Transwell migration assay (Scale
   bar = 200 μm). Values are shown as mean ±  SD of three independent
   experiments. * p < 0.05, ** p < 0.01, *** p <0.001, and **** p < 0.0001
   compared with controls.

3.3. UCHL5 Overexpression Increases Cell Proliferation and Migration in
Bladder Cancer

   In the following experiment, we upregulated the UCHL5 expression in
   bladder cancer cells through lentivirus infection. The protein levels
   of the UCHL5 overexpression were evaluated using Western blotting
   ([88]Figure 3A). The subsequent CCK-8 and colony formation assays
   demonstrate that UCHL5 overexpression enhances cell proliferation and
   colony formation capacity ([89]Figure 3B,C). The impact of cell
   migration could be measured using the wound-healing assay. The
   wound-healing assay results reveal that UCHL5 overexpression promotes
   cell migration, as seen in [90]Figure 3D. Similar results were observed
   in the Transwell migration analysis, which further support the results
   of the wound-healing assay and show that UCHL5 overexpression
   significantly enhances the number of migrating cells ([91]Figure 3E).

Figure 3.

   [92]Figure 3
   [93]Open in a new tab

   UCHL5 overexpression increases cancer cell proliferation and migration
   in vitro. (A) UCHL5 overexpression in bladder cancer cells confirmed by
   Western blotting. β-Actin is used as the loading control. (B) CCK-8
   assays to examine the proliferation ability of bladder cancer cells
   between the UCHL5 overexpression group and vector group at five time
   points (24, 48, 72, 96, and 120 h). (C) Representative images of cell
   colony formation and the number of colonies were counted. (D,E) Cell
   migration was characterized using wound-healing (Scale bar = 100 μm)
   and Transwell migration assay (Scale bar = 200 μm). Values are shown as
   mean ±  SD of three independent experiments. * p < 0.05, ** p < 0.01,
   and **** p < 0.0001 compared with controls.

3.4. UCHL5 Silencing Results in Inactivation of AKT/mTOR Signal Pathway and
Repressing the Downstream Expression of c-Myc, SLC25A19, and ICAM5

   To confirm which signaling pathways UCHL5 is involved in, we conducted
   RNA sequence analysis following UCHL5 downregulation with shRNA in the
   T24 cell line and a control cell line. Using KEGG pathway enrichment
   models, we performed gene set enrichment analysis (GSEA) and discovered
   that knocking down UCHL5 dramatically inhibited the AKT/mTOR signaling
   pathway as well as the downstream expression of c-Myc, SLC25A19, and
   ICAM5 ([94]Figure 4A,B). Consistent with these results, we also found
   that low expression of UCHL5 in the TCGA database is linked with
   inhibited activation of the AKT/mTOR pathway and the expression of
   c-Myc ([95]Figure S1D–F). We also attempted to validate the association
   between UCHL5 and AKT/mTOR pathway via bioinformatics analysis. The
   AKT/mTOR pathway score was first calculated using the single sample
   gene set enrichment analysis (ssGSEA) method. We found that BLCA
   patients categorized into UCHL5 high-expression group showed
   significantly higher enrichment scores for the AKT/mTOR pathway
   ([96]Figure 4E, p < 0.05) compared with BLCA patients in the UCHL5
   low-expression group. Moreover, UCHL5 was positively associated with
   the AKT/mTOR pathway (Spearman’s R = 0.13, p = 0.008, [97]Figure 4F).
   These results are consistent with those of our experiments.

Figure 4.

   [98]Figure 4
   [99]Open in a new tab

   Knockdown of UCHL5 results in inactivation of the AKT/mTOR signaling
   pathway and contributes to decreased c-Myc, SLC25A19, and ICAM5
   expression. (A) KEGG pathway enrichment analysis was used to assess
   RNA-Seq data containing shControl and shUCHL5 bladder cancer cells.
   (B,C) GSEA identified enrichment of AKT/mTOR signaling pathway and
   c-Myc gene sets. (D) Heat map of the differential expressed genes. (E)
   The difference in AKT/mTOR signaling pathway scores was defined using
   ssGSEA between the UCHL5 high- and UCHL5 low-expression groups. (F) The
   association of UCHL5 expression and AKT/mTOR signaling pathway score.
   (G) qRT-PCR determination of the mRNA levels of c-Myc, SLC25A19, and
   ICAM5 in bladder cancer cells transfected with shControl or shUCHL5.
   (H–K) Protein levels of the AKT/mTOR signal pathway proteins (t-PI3K,
   p-PI3K, t-AKT, p-AKT, t-mTOR, and p-mTOR), c-Myc, SLC25A19, and ICAM5
   in UCHL5 silenced and overexpressed bladder cancer cells shown by
   Western blotting. β-Actin is used as the loading control. Values are
   shown as mean ± SD. * p < 0.05 and **** p < 0.0001 compared with
   controls.

   Consequently, qRT-PCR was employed to reveal that mRNA levels of c-Myc,
   SLC25A19, and ICAM5 were significantly decreased after UCHL5 silencing
   ([100]Figure 4G). Finally, we reconfirmed that a decrease followed
   UCHL5 silencing in the p-PI3K, p-AKT, and p-mTOR protein expression
   model ([101]Figure 4H), and the same decrease in c-Myc, SLC25A19, and
   ICAM5 was observed in Western blot analysis([102]Figure 4J).
   Conversely, UCHL5 overexpression significantly enhanced the protein
   profiles of p-PI3K, p-AKT, p-mTOR, c-Myc, SLC25A19, and ICAM5 in
   Western blotting ([103]Figure 4I,K). Consistent with our earlier work,
   we also attempted to provide some bioinformatics evidence to support
   these relationships. We found that BLCA patients categorized into the
   UCHL5 high-expression group showed significantly higher c-Myc
   ([104]Figure S1G, p < 0.001), SLC25A19 ([105]Figure S1I, p < 0.001),
   and ICAM5 ([106]Figure S1K, p < 0.05) expression levels compared with
   BLCA patients in the UCHL5 low-expression group. Moreover, UCHL5 was
   positively associated with c-Myc (Spearman’s R = 0.33, p < 0.001,
   [107]Figure S1H), SLC25A19 (Spearman’s R = 0.21, p < 0.001, [108]Figure
   S1J), and ICAM5 (Spearman’s R = 0.11, p = 0.032, [109]Figure S1L).
   These results indicate that UCHL5 could regulate the expression of
   c-Myc, SLC25A19, and ICAM5 via the AKT/mTOR signaling pathway.

3.5. SC79 Reverses the Effect of UCHL5 Downregulation in Bladder Cancer Cells

   To further investigate whether the AKT/mTOR signaling pathway is
   required for UCHL5-mediated antitumor effects, AKT activator SC79 was
   applied for the treatment of EJ, T24, and UMUC3 cells transfected/not
   transfected with UCHL5 shRNA. The results show that SC79 completely
   reverses the diminished cell migration ability resulting from
   transfection of UCHL5 shRNA ([110]Figure 5A). As can be seen from
   [111]Figure 5B,C, the colony formation and CCK-8 assay results show
   that SC79 similarly reverses the attenuated cell colony formation and
   proliferation caused by UCHL5 silencing. Moreover, SC79 treatment
   elevates the phosphorylation of AKT (Ser473) and mTOR (Ser2448), as
   well as the levels of c-Myc, SLC25A19, and ICAM5 in cells with UCHL5
   knockdown.

Figure 5.

   [112]Figure 5
   [113]Open in a new tab

   SC79 recovers the effect of UCHL5 knockdown in bladder cancer cells.
   (A) Transwell assay (Scale bar = 200 μm) assessment of cell migration
   in shCtrl, shUCHL5-3, and shUCHL5-3 + SC79 groups. (B,C) Cell
   proliferation of three groups evaluated using CCK-8 and colony
   formation assays. (D) Western blotting analysis of the p-AKT, t-AKT,
   p-mTOR, t-mTOR, c-Myc, SLC25A19, and ICAM5 protein expression in
   bladder cancer cells. β-Actin is used as the loading control. Values
   are shown as mean ±  SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and
   **** p < 0.0001 compared with controls.

3.6. LY294002 Recovers the Effects of UCHL5 Upregulation in Bladder Cancer
Cells

   To further confirm the involvement of AKT/mTOR signaling in
   UCHL5-promoted tumor growth and migration, bladder cancer cells were
   transfected with pHAGE-puro-UCHL5 and then incubated with LY294002. We
   found that LY294002 blocked the effects of UCHL5 upregulation on cell
   migration ([114]Figure 6A) and proliferation ([115]Figure 6B,C).
   Furthermore, the elevated protein profiles of p-AKT and p-mTOR induced
   by UCHL5 upregulation were attenuated by LY294002 incubation and
   downstream levels of c-Myc, SLC25A19, and ICAM5 in cells.

Figure 6.

   [116]Figure 6
   [117]Open in a new tab

   LY294002 rescues the effect of UCHL5 upregulation in bladder cancer
   cells. (A) Transwell assay was used to assess cell migration of Vector,
   UCHL5, and UCHL5 + LY294002 (Scale bar = 200 μm). (B,C) Cell
   proliferation of three groups evaluated using CCK-8 and colony
   formation assays. (D) Western blotting analysis of the UCHL5, p-AKT,
   t-AKT, p-mTOR, t-mTOR, c-Myc, SLC25A19, and ICAM5 protein expression in
   bladder cancer cells. β-Actin is used as the loading control. Values
   are shown as mean ±  SD. * p < 0.05, ** p < 0.01, and **** p < 0.0001
   compared with controls.

3.7. Knocking Down UCHL5 Inhibits Bladder Cancer Growth In Vivo

   To further validate the UCHL5 function in vivo, we set up a xenograft
   model with four-week-old male BALB/c nude mice. T24 cells transfected
   with shControl, shUCHL5-1, and shUCHL5-3 were subcutaneously injected
   into three groups of mice. After 33 days, tumor-bearing mice were
   sacrificed, and the tumors were dissected and weighed. The volumes of
   the tumors from the shControl group were significantly higher than
   those from the shUCHL5-1 and shUCHL5-3 groups ([118]Figure 7A,B). The
   tumor weights also showed a substantial decrease in the two shUCHL5
   groups compared with the shControl group ([119]Figure 7C,D). These
   findings demonstrate that UCHL5 is crucial in controlling bladder
   cancer tumorigenesis in vivo. The immunohistochemistry (IHC) analysis
   of the paraffin-embedded dissected neoplasms demonstrates that the
   expression of Ki67 decreased in the two shUCHL5 groups ([120]Figure
   7E). Furthermore, the levels of UCHL5, c-Myc, and phospho-AKT were
   reduced in the two shUCHL5 groups ([121]Figure 7E), which demonstrates
   that the abolishment of UCHL5 suppresses the AKT/mTOR signaling
   pathway.

Figure 7.

   [122]Figure 7
   [123]Open in a new tab

   The depletion of UCHL5 inhibits bladder cancer tumorigenesis in vivo.
   (A) Xenograft models (n = 5) were established by subcutaneously
   inoculating shCtrl or shUCHL5 cells and allowing the cells to grow for
   five weeks. The mice were then sacrificed, and the tumors were removed
   and weighed. (B–D) The tumor volume and weight measurements are shown.
   (E) Representative H&E staining and immunohistochemical staining of the
   xenograft tumors from the tumor-bearing mice in the shCtrl and shUCHL5
   groups showed the expression of UCHL5, p-AKT, c-Myc, and Ki67 decreased
   in the shUCHL5 group compared with the shCtrl group. All scale bars =
   50 μm. Statistical analysis of the tumor volume and weight were
   performed using one-way ANOVA. Values are shown as mean ± SD. * p <
   0.05, ** p < 0.01, and **** p < 0.0001 compared with controls.

4. Discussion

   In this study, we showed that the deubiquitinating enzyme UCHL5 is
   abnormally elevated in bladder cancer. We verified that UCHL5 activates
   the AKT/mTOR pathway to enhance migration and proliferation. We also
   discovered important downstream molecules of AKT/mTOR signaling,
   including c-Myc, SLC25A19, and ICAM5. According to these results, we
   conclude that UCHL5 is significantly linked to the onset and spread of
   bladder cancer.

   Although many malignancies have revealed UCHL5 to be an oncogene, its
   function in bladder cancer is yet unknown. Immune cells (including NK
   cells, B cells, mast cells, etc.) represent the major components of
   antitumor immune reaction [[124]23]. Recent research has shown that
   immune cells are correlated with prognosis of cancers, which could also
   accelerate tumor initiation and progression [[125]24]. Thus, we also
   wanted to know whether the UCHL5 was associated with some immune cell
   types in bladder cancer. The result concluded that UCHL5 was positively
   related to T cells CD4 memory activated, NK cells resting, Macrophages
   M0, Macrophages M1, Dendritic cells activated, and Mast cells
   activated, meanwhile negatively related to Plasma cells, T cells CD4
   naïve, T cells regulatory, and monocytes. Polarization of immune cells
   is commonly observed in host responses associated with microbial
   immunity, inflammation, tumorigenesis, and tissue repair and fibrosis
   [[126]25]. In this process, immune cells adopt distinct programs and
   perform specialized functions in response to specific signals.
   Polarization of immune cells has been proven to play an important role
   in the development of cancers. It must be emphasized that this was only
   a preliminary exploration in the present study. Whether the UCHL5 is
   associated with immune polarization needs to be confirmed by in-depth
   studies. More exploration based on public data or experiments must be
   done in the near future.

   Excessive cell proliferation and migration are the hallmarks of cancer
   [[127]26]. Moreover, the role of UCHL5 in the regulation of cancer cell
   proliferation and migration has already been reported
   [[128]18,[129]27]. UCHL5 combined with DRAIC can increase the
   proliferation and metastasis of GC cells via ubiquitination [[130]28].
   Expanding the expression levels of UCHL5 has been demonstrated to
   accelerate the growth of endometrial cancer [[131]20]. Nevertheless,
   the exact roles of UCHL5 in bladder cancer cells remain unclear. In our
   study, UCHL5 downregulation significantly suppressed both tumor growth
   in vivo and cell proliferation and migration in vitro. By contrast,
   overexpression of UCHL5 in bladder cancer cell lines aggravated its
   proliferation and migration. We infer from these findings that UCHL5 is
   an essential determinant of bladder cancer progression.

   Currently, many investigations have concentrated on the potentially
   pathogenic mechanisms of UCHL5 in malignancies. Anthony J. et al.
   reported that UCHL5 could sustain early TGFβ pathway activation
   kinetics and significantly increase cell migration in pancreatic
   carcinoma [[132]17]. According to the research of Christina S. et al.,
   UCHL5 deubiquitinates E2 promoter activating factor 1 (E2F1) and
   stimulates its transcriptional activity, which in turn promotes the
   expression of E2F1 target genes that are proliferative and
   pro-apoptotic [[133]29]. Da Liu et al. reported that UCHL5 could
   accelerate endometrial cancer growth by triggering Wnt/β-catenin
   signaling [[134]20]. In urothelial carcinoma, the UCHL5 inhibitor
   b-AP15 increases protein polyubiquitination and endoplasmic reticulum
   (ER) stress, further inhibiting cancer stem cells and overcoming
   cisplatin resistance [[135]30]. To reveal how UCHL5 promotes
   carcinogenesis, we examined UCHL5-deficient T24 cell lines by RNA
   sequencing, and the results demonstrated that AKT/mTOR signaling is
   accordingly inactivated following knockdown of UCHL5. Then, SC79
   (activator of AKT) was used to treat bladder cancer cells transfected
   with UCHL5 shRNA, and Western blotting analysis showed rescue of
   p-AKT/p-mTOR protein expression affected by UCHL5 knockdown.
   Conversely, we found that LY294002 could repress the AKT/mTOR signaling
   pathway in cells overexpressing UCHL5, as evidenced by decreased
   expression of p-AKT/p-mTOR proteins. From these results, we infer that
   UCHL5 regulates the proliferation and migration of bladder cancer cells
   through the AKT/mTOR signaling pathway.

   In addition to discovering that the AKT/mTOR pathway is involved in
   regulating the progression of bladder cancer, we also identified
   downstream target proteins (c-Myc, SLC25A19, and ICAM5) of UCHL5 by
   RNA-Seq. The elevation of c-Myc expression has been implicated in
   various cancers. It is frequently linked to malignant, poorly
   differentiated tumors [[136]31], such as breast cancer, liver tumor,
   colorectal carcinoma, and prostatic neoplasia
   [[137]32,[138]33,[139]34,[140]35]. The superfamily of SLC transporters
   is a family of membrane proteins that participate in the cellular
   uptake of small molecules. Furthermore, evidence suggests that the
   abnormal expression of SLC transporters may play a vital role in the
   pathogenesis of human malignancies [[141]36]. Polly Zhang et al.
   illustrated that downregulating SLC25A19 limits the availability of the
   co-factor thiamine pyrophosphate (TPP), achieving additional control of
   prostate cancer metabolism [[142]37]. ICAM5, a member of the ICAM
   family of adhesion proteins, strongly stimulates neurite growth
   [[143]38]. S. I. Maruya et al. reported that ICAM5 might play an
   essential role in tumorigenesis and perineural invasion through the
   PI3K/AKT signaling pathway [[144]39]. These conclusions are consistent
   with the results of our current study. We showed, using Western
   blotting analysis, that the addition of SC79 rescued the low expression
   of c-Myc, SLC25A19, and ICAM5 proteins resulting from UCHL5 shRNA
   knockdown, and that LY294002 repressed the resultant production of
   c-Myc, SLC25A19, and ICAM5 proteins in cells overexpressing UCHL5.
   Thus, our results reveal that UCHL5 can modulate the expression of
   downstream target genes (c-Myc, SLC25A19, and ICAM5) through the
   AKT/mTOR pathway.

5. Conclusions

   In conclusion, we found that UCHL5 is overexpressed in bladder cancer
   patients, and that high UCHL5 expression is associated with aggressive
   tumor characteristics and a poor prognosis. Functionally, we propose
   that UCHL5 plays a newly discovered role in regulating
   AKT/mTOR-c-Myc-mediated downstream processes of cell proliferation and
   migration. Therefore, UCHL5 is a potential target for therapy in
   bladder cancer.

Acknowledgments