MG53 Suppresses the Progression of Bladder Cancer Through E3 Ubiquitin Ligase-Mediated Degradation of AMPKα

MG53 Suppresses the Progression of Bladder Cancer Through E3 Ubiquitin Ligase-Mediated Degradation of AMPKα | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article MG53 Suppresses the Progression of Bladder Cancer Through E3 Ubiquitin Ligase-Mediated Degradation of AMPKα Xiangfei He, He Zhang, Yanping Zhang, Junwei Wu, Tianen Wang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9590464/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background MG53/TRIM72 is a member of the tripartite motif family of E3 ubiquitin ligases that regulates metabolic homeostasis and tumor-associated signaling; however, its role in bladder cancer remains undefined. Methods We investigated the expression, function, and molecular mechanism of MG53 in bladder cancer. MG53 expression was analyzed in bladder cancer tissues and cell lines. Functional assays including proliferation, invasion, and glycolytic metabolism were performed. The interaction between MG53 and AMPKα was examined using co-immunoprecipitation and ubiquitination assays. In vivo tumor growth was assessed in xenograft models. Results MG53 was significantly downregulated in bladder cancer tissues compared to adjacent normal tissues, and its expression was inversely correlated with AMPKα levels. Overexpression of MG53 suppressed cell proliferation, invasion, and glycolytic metabolism in bladder cancer cells. Mechanistically, MG53 promoted the ubiquitination and proteasomal degradation of AMPKα. In vivo experiments demonstrated that MG53 overexpression inhibited tumor growth in xenograft models. Conclusions Our findings reveal that MG53 functions as a tumor suppressor in bladder cancer by promoting AMPKα degradation through its E3 ubiquitin ligase activity, thereby attenuating the Warburg effect. MG53 represents a potential therapeutic target for bladder cancer treatment. bladder cancer MG53/TRIM72 AMPKα E3 ubiquitin ligase Warburg effect tumor suppressor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Bladder cancer (BC) represents one of the most prevalent malignancies of the urinary system worldwide. According to contemporary epidemiological data, bladder cancer ranks among the most frequently diagnosed malignancies in both men and women, and its global disease burden remains substantial [ 1 ]. Bladder carcinogenesis and progression are driven by a complex interplay of risk factors, encompassing tobacco exposure, occupational and environmental carcinogens, genetic susceptibility, and metabolic dysregulation [ 2 ]. High-grade invasive bladder cancer is characterized by high rates of recurrence, metastasis, and therapeutic resistance. Despite continuous advances in surgery, radiotherapy, chemotherapy, and immune checkpoint inhibition, the prognosis of patients with advanced bladder cancer remains unsatisfactory, with a 5-year survival rate below 50% [ 3 ]. Therefore, elucidating the molecular mechanisms underlying bladder cancer progression and identifying novel diagnostic biomarkers and therapeutic targets are of considerable clinical importance. Mitsugumin 53 (MG53), also designated as tripartite motif protein 72 (TRIM72), is a member of the tripartite motif (TRIM) family protein. As an E3 ubiquitin ligase, MG53 primarily functions by catalyzing the ubiquitination of target proteins, thereby governing protein turnover and modulating intracellular signaling pathways [ 4 , 5 ]. Previous studies have shown that MG53 is predominantly expressed in striated muscle and has been established as an essential mediator of plasma membrane repair, oxidative stress responses, and metabolic homeostasis [ 6 ]. In addition to its physiological functions, MG53 has been increasingly implicated in the pathogenesis of metabolic diseases, including diabetes and insulin resistance [ 7 ]. More recently, emerging evidence has positioned MG53 as a tumor suppressor in several malignancies, including hepatocellular carcinoma [ 8 ], ovarian cancer [ 9 ], and other solid tumors [ 10 ]; however, its role in bladder cancer remains largely unexplored. AMP-activated protein kinase α (AMPKα) functions as a master sensor of cellular energy status that regulates metabolic reprogramming in response to changes in the intracellular AMP/ATP or ADP/ATP ratio. In tumor cells, AMPKα is frequently aberrantly activated and has been shown to sustain tumor progression by enhancing glucose uptake and aerobic glycolysis, thereby providing energy and biosynthetic precursors required for rapid proliferation [ 11 , 12 ]. Accumulating evidence indicates that AMPKα plays a context-dependent oncogenic role in bladder cancer, contributing to tumor proliferation, migration, and therapeutic resistance [ 13 , 14 ]. Importantly, a prior study by Jiang et al. demonstrated that MG53 mediates AMPKα ubiquitination and degradation under high-glucose conditions in metabolic disease models [ 15 ], raising the possibility that a similar regulatory axis may operate in cancer. In the present study, we characterized the expression profile of MG53 in human bladder cancer tissues, evaluated its functional impact on key malignant phenotypes including proliferation and invasion, and delineated the molecular mechanism by which MG53 regulates AMPKα through ubiquitin-dependent proteasomal degradation. We further examined the downstream consequences of this regulatory axis for glycolytic metabolism and tumor growth in vivo. Our findings establish a novel MG53–AMPKα signaling axis that links ubiquitin-mediated proteostasis to metabolic reprogramming and tumor suppression in bladder cancer. 2. Materials and Methods 2.1. Human tissue samples 2.1.1. Ethics approval and patient enrollment This study was reviewed and approved by the Institutional Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval No.: 2022-KY-1274; approval date: 09-15-2022). All procedures were conducted in accordance with the Declaration of Helsinki and applicable national guidelines governing biomedical research involving human subjects. Patients with histopathologically confirmed high-grade invasive bladder cancer who underwent radical cystectomy at our institution between January 2024 and December 2024 were prospectively enrolled. Paired specimens of primary tumor tissue and adjacent morphologically normal urothelium were collected from each patient. Adjacent normal tissues were obtained at least 2 cm from the tumor margin and subsequently verified to be free of malignant involvement by independent histopathological examination. All patients were pathologically diagnosed with high-grade invasive urothelial carcinoma and had received no prior antitumor treatment, including radiotherapy, chemotherapy, or intravesical therapy. Complete clinicopathological data, including tumor stage, pathological grade, tumor size, and lymph node status, were available for all patients. Written informed consent was obtained from all participants. 2.1.2. Inclusion and exclusion criteria The inclusion criteria were as follows: (1) histopathologically confirmed high-grade invasive bladder cancer (T2–T4) with urothelial carcinoma histology; (2) primary bladder cancer without history of prior antitumor treatment; (3) availability of complete clinical and follow-up data; (4) absence of severe dysfunction of major organs, including the heart, liver, or kidneys, and no severe infection or autoimmune disease; and (5) availability of sufficient fresh-frozen and formalin-fixed tissue specimens to permit immunohistochemistry, immunoblotting, transcriptome sequencing, and other downstream analyses. The exclusion criteria were as follows: (1) non-urothelial histological variants, including but not limited to squamous cell carcinoma and adenocarcinoma of the bladder; (2) receipt of any neoadjuvant or preoperative antitumor therapy, including radiotherapy, chemotherapy, targeted therapy, or immunotherapy before surgery; (3) presence of synchronous or metachronous malignancies of other organ sites; (4) inadequate tissue preservation, tissue necrosis, or contamination that precluded experimental analysis; and (5) incomplete or missing clinicopathological documentation. 2.1.3. Sample processing Fresh tissue samples were bisected immediately upon surgical resection under sterile conditions. One portion was snap-frozen in liquid nitrogen within 30 min and stored at − 80°C until used for Western blotting, qPCR, and transcriptome sequencing. The other portion was fixed in 10% neutral buffered formalin for 4–6 h at room temperature, dehydrated, and paraffin-embedded. Paraffin sections (4 µm) were prepared for hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and immunofluorescence analyses. All specimens were collected, processed, and stored in accordance with standard biobank operating procedures to ensure sample quality. 2.1.4. Transcriptome sequencing Tumor tissues and matched adjacent normal tissues from a subset of five patients were subjected to transcriptome sequencing. Total RNA was extracted and subjected to quality control. Strand-specific cDNA libraries were prepared and subjected to paired-end sequencing on an Illumina NovaSeq platform by Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). Differentially expressed genes (DEGs) between tumor and matched normal tissues were identified using established bioinformatics pipelines with thresholds of |log₂ fold change| ≥ 1 and adjusted P-value < 0.05. The transcriptional expression profile of MG53 was specifically examined within the DEG dataset. 2.2. Cell lines and culture conditions Human bladder cancer cell lines T24 and J82, and the immortalized normal human bladder epithelial cell line SV-HUC-1, were procured from the Cell Bank of the Chinese Academy of Sciences. Cell line authentication was confirmed by short tandem repeat profiling, and mycoplasma-free status was routinely confirmed using a PCR-based mycoplasma detection kit prior to experimental use. T24 and J82 cells were cultured in RPMI-1640 medium (Gibco, 12633012) supplemented with 10% fetal bovine serum (FBS; Gibco, A5256901), 100 U/mL penicillin, and 100 µg/mL streptomycin. SV-HUC-1 cells were cultured in DMEM (Gibco, 12491015) supplemented with 10% FBS. All cells were maintained at 37°C in a humidified incubator with 5% CO₂. The culture medium was replaced every 24–48 h. Cells were passaged with 0.25% trypsin (Gibco, 25300054) when they reached 80%–90% confluence. All experiments were performed using cells in the logarithmic growth phase within 20 passages of receipt from the repository. 2.3. Animals and tumor models 2.3.1. Animals and housing MG53 knockout (mg53−/−) mice on a C57BL/6N background were generated using CRISPR/Cas9 technology, and littermate wild-type (WT) C57BL/6N mice were used as controls for the establishment of the orthotopic bladder cancer model. Athymic nude mice (BALB/c-nu/nu) were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. and used for the human bladder cancer xenograft model. All mice were 6–8 weeks old and weighed 20–25 g. An equal number of male and female mice were used. Mice were housed under specific-pathogen-free conditions at Zhengzhou University at 22 ± 2°C and 50 ± 5% humidity, under a 12-h light/dark cycle, with free access to sterile food and water. All animal experiments were reviewed and approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University and conducted in accordance with institutional guidelines for animal care and use (Approval No.: 2022-KY-1274; approval date: 09-15-2022). 2.3.2. Experimental grouping and model establishment For the orthotopic bladder cancer model, 50 mg53−/− mice and 50 WT mice were randomly assigned to five groups corresponding to 0, 7, 14, and 21 days after tumor inoculation, with 10 mice per group (5 males and 5 females). The luciferase-labeled mouse bladder cancer cell line MB49-Luc was used to establish the orthotopic model. Cells were resuspended in a 1:1 mixture of PBS and Matrigel to achieve a final concentration of 5 × 10^6 cells/mL, and 20 µL of cell suspension was orthotopically injected into the bladder wall of each mouse. For the subcutaneous xenograft model, 40 nude mice were randomly divided into four groups (n = 10 per group, including 5 males and 5 females): Ad-MG53 group (T24 cells infected with Ad-MG53), Ad-Vector group (T24 cells infected with Ad-Vector), Ad-shMG53 group (T24 cells infected with Ad-shMG53), and Ad-shVector group (T24 cells infected with Ad-shVector). T24 cells were infected with the indicated adenoviruses for 12 h, and 1 × 10^5 cells in 20 µL suspension were injected subcutaneously into the right dorsal flank of each mouse. 2.3.3. Tumor monitoring and sample collection In the orthotopic mg53−/− mouse model, bioluminescence intensity was monitored using an in vivo imaging system (PerkinElmer) at 0, 7, 14, and 21 days post-inoculation. Tumor growth was recorded over time. Mice were euthanized at the indicated time points, and bladder and tumor tissues were harvested. Surface blood and fluid were gently removed with filter paper. The wet weight of tumors was measured using a precision electronic balance (0.1 mg sensitivity), and tumor length and width were measured with a vernier caliper. In the nude mouse subcutaneous xenograft model, tumor length and width were measured every 3 days beginning on day 7 post-implantation, and tumor volume was calculated to generate growth curves. On day 22 post-inoculation, mice were humanely euthanized, subcutaneous tumors were surgically excised, and tumor wet weight was recorded. 2.4. Reagents Adenoviral vectors (Ad-MG53, Ad-Vector, Ad-shMG53, and Ad-shVector) and plasmids encoding Myc-AMPKα, Flag-MG53, and HA-Ub were synthesized and packaged by Shandong Weizhen Biotechnology (Shandong, China). Primary antibodies against MG53 (Cat# 22151-1-AP), AMPKα (Cat# 10929-2-AP), HA tag (Cat# 51064-2-AP), Myc tag (Cat# 16286-1-AP), Flag tag (Cat# 66008-4-Ig), KIF11 (Cat# 23333-1-AP), Ki67 (Cat# 27309-1-AP), and GAPDH (Cat# 60004-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). Anti-CK20 antibody (Cat# GB112050) was obtained from Servicebio (Wuhan, China). HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat# 7074S) and HRP-conjugated horse anti-mouse IgG secondary antibody (Cat# 7076S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Glucose Uptake Probe-Red kit (Cat# UP03) was purchased from Dojindo (Kumamoto, Japan); the L-lactate assay kit (Cat# A019-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); the proteasome inhibitor MG132 (Cat# HY-13259) was purchased from MedChemExpress (Monmouth Junction, NJ, USA); the Protein A + G Agarose Co-immunoprecipitation Kit (Cat# P2055) was purchased from Beyotime (Shanghai, China); D-luciferin potassium salt (Cat# Luc-1G) was purchased from GoldBio (St. Louis, MO, USA); and primers were synthesized by Sangon Biotech (Shanghai, China). 2.5. Experimental procedures 2.5.1. Adenoviral infection and cell treatment T24 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 5 × 10^5 cells/well. After 24 h, when cell confluence reached approximately 80%, cells were infected with the indicated adenoviral vectors (Ad-MG53, Ad-Vector, Ad-shMG53, or Ad-shVector) at a multiplicity of infection (MOI) of 100. Following a 12 h infection period, the medium was replaced with fresh complete medium, and cells were cultured for an additional 48 h before subsequent assays. For the doxycycline (DOX)-inducible overexpression model, cells infected with Ad-MG53 were treated with 2 µg/mL DOX for 48 h, and MG53 expression was confirmed by Western blotting analysis. For proteasome inhibition experiments, cells were treated with MG132 at 10 µmol/L for 12 h. Plasmid transfection was performed using Lipofectamine 3000 (Invitrogen), and experiments were conducted 48 h after transfection. 2.5.2. Cell function assays Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. Briefly, T24 cells from each group were seeded into 96-well plates at a density of 1 × 10^3 cells/well, with six replicate wells per group. At 0, 1, 2, 3, 4, and 5 days after seeding, 10 µL of CCK-8 reagent was added to each well, and the plates were incubated at 37°C for 2 h. Absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA), and cell proliferation curves were generated accordingly. Cell invasion capacity was evaluated using the Transwell invasion assay. Briefly, Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was diluted 1:8 and uniformly applied to the upper chamber of Transwell inserts, then incubated at 37°C for 30 min to solidify. T24 cells were harvested and resuspended in serum-free medium. A total of 200 µL of cell suspension (1 × 10^5 cells/mL) was added to the upper chamber, and 600 µL of complete medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation at 37°C in a humidified atmosphere, invaded cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Stained cells were imaged and counted in five randomly selected fields per insert under a light microscope at 100× magnification. Colony formation ability was assessed using the colony formation assay. Briefly, T24 cells were seeded into 6-well plates at a density of 800 cells/well, with three replicate wells per group, and cultured for 14 days. The culture medium was replaced every 3 days to maintain optimal growth conditions. At the end of the culture period, colonies were washed twice with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet for 20 min. Excess stain was removed by gentle washing with distilled water, and plates were air-dried prior to imaging. Colonies containing at least 50 cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and colony formation efficiency was calculated as the number of colonies formed divided by the number of cells initially seeded, expressed as a percentage. 2.5.3. Detection of cellular metabolism Glucose uptake was measured using the Glucose Uptake Probe-Red kit (Cat# UP03; Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, T24 cells were seeded into 6-well plates at an appropriate density and allowed to adhere overnight. Prior to the assay, cells were washed twice with PBS and subjected to serum and glucose starvation in serum-free, glucose-free medium for 6 h at 37°C to eliminate residual intracellular glucose. After starvation, the Glucose Uptake Probe-Red reagent was added to a final concentration of 10 µmol/L, and cells were incubated at 37°C for 30 min. After incubation, cells were washed twice with ice-cold PBS, harvested by trypsinization, and resuspended in PBS for flow cytometric analysis. Fluorescence intensity was measured by flow cytometry (excitation, 570 nm; emission, 590 nm), and glucose uptake capacity was quantified based on the mean fluorescence intensity (MFI) of each experimental group. Extracellular lactate levels were determined using the L-lactate assay kit (Cat# A019-2-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. Briefly, T24 cells from each experimental group were seeded into 6-well plates at an appropriate density and cultured under standard conditions. At the designated time point, culture supernatants were carefully collected and centrifuged at 1,000 × g for 5 min at 4°C to remove cellular debris. The clarified supernatants were then processed according to the kit protocol, and absorbance was measured at 530 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Lactate concentration was calculated from a standard curve generated with kit-provided lactate standards and normalized to total protein content or cell number in the corresponding wells to account for differences in cell density across groups. 2.5.4. Western blotting and co-immunoprecipitation For Western blotting, total protein was extracted using RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit according to the manufacturer’s instructions. Equal amounts of protein (30 µg/lane) were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat milk for 2 h, incubated with primary antibodies overnight at 4°C, and then incubated with the corresponding secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an internal loading control to ensure equal protein loading across all lanes. For endogenous co-immunoprecipitation (Co-IP), cells infected with the indicated adenoviral vectors were lysed, and total protein was extracted at a concentration of 1 µg/µL. Protein lysates were pre-cleared with Protein A/G agarose beads for 1 h at 4°C to reduce nonspecific binding, then incubated with 2 µg anti-MG53 or anti-AMPKα antibody overnight at 4°C, followed by incubation with 50 µL Protein A/G agarose beads for 4 h at 4°C. The immunoprecipitates were collected by centrifugation at 5000 rpm for 5 min, washed five times with TBST, and eluted by denaturation in 5× loading buffer at 95°C for 5 min. The resulting samples were analyzed by Western blotting. Normal rabbit or mouse IgG was used as a negative control to assess nonspecific antibody binding. To assess exogenous protein interactions and ubiquitination, T24 cells were co-transfected with plasmids encoding Myc-AMPKα, Flag-MG53, and HA-Ub. At 36 h post-transfection, cells were treated with MG132 (10 µmol/L) for 12 h. Total protein was then extracted under denaturing conditions, and immunoprecipitation was performed using anti-Myc antibody to capture Myc-AMPKα and its associated protein complexes. The resulting immunoprecipitates were subjected to Western blot analysis to detect co-precipitated Flag-MG53 and HA-labeled polyubiquitin chains, thereby assessing the ubiquitination status of AMPKα by MG53. 2.5.5. Immunohistochemistry and immunofluorescence staining Immunohistochemical staining was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections. Briefly, sections were deparaffinized in xylene and rehydrated through a graded ethanol series to distilled water. Antigen retrieval was performed using citrate buffer (pH 6.0) with high-temperature heating for 10 min, followed by cooling to room temperature. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min at room temperature, followed by three PBS washes. Nonspecific binding was blocked with 5% BSA for 30 min in a humidified chamber. After three PBS washes, sections were incubated overnight at 4°C with primary antibodies, then with secondary antibodies for 1 h at room temperature. Signals were developed using 3,3′-diaminobenzidine (DAB) substrate solution, and the reaction was monitored under a light microscope to ensure consistent staining intensity across sections. Nuclei were counterstained with hematoxylin, followed by bluing in running tap water. Sections were then dehydrated through a graded ethanol series, cleared in xylene, and mounted with neutral resin mounting medium. The integrated optical density (IOD) of positively stained areas was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Double immunofluorescence staining was performed on FFPE tissue sections as described above for deparaffinization, rehydration, and antigen retrieval. Following antigen retrieval, sections were blocked with 5% BSA for 30 min at room temperature in a humidified chamber to minimize nonspecific background. Sections were incubated overnight at 4°C with anti-MG53 (1:200) and anti-KIF11 (1:200) antibodies. After three PBS washes, sections were incubated with the corresponding fluorescent secondary antibodies for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI, and sections were mounted using an antifade fluorescence mounting medium. Fluorescence signals were captured using a laser scanning confocal microscope. Fluorescence intensity was quantified using the accompanying imaging software. 2.5.6. Statistical analysis All experiments were performed at least three times independently, and results are presented as the mean ± standard deviation (SD). Statistical analyses were performed using SPSS 22.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8.0 (San Diego, CA, USA). Prior to inferential testing, the normality of data distribution was assessed using the Shapiro-Wilk test, and homogeneity of variance was assessed using Levene’s test. For normally distributed data with equal variance, comparisons between two groups were performed using the independent-samples Student’s t-test, whereas comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. For data that were not normally distributed or did not show equal variance, the Mann-Whitney U test was used for two-group comparisons and the Kruskal-Wallis H test for multiple-group comparisons. Categorical variables were analyzed using the chi-square (χ²) test, with Fisher’s exact test applied when expected cell frequencies were below five. Correlations were analyzed using Pearson’s or Spearman’s correlation analysis, as appropriate. All statistical tests were two-sided, and a p-value < 0.05 was considered statistically significant. 3. Results 3.1. MG53 is downregulated in bladder cancer tissues and inversely correlated with AMPKα expression To characterize the expression pattern and clinical relevance of MG53 in bladder cancer, we analyzed five pairs of high-grade invasive bladder cancer tissues and matched adjacent normal tissues. H&E staining, together with IHC, confirmed the characteristic histopathological features of high-grade invasive urothelial carcinoma. The H-scores of the proliferation marker Ki67 and the urothelial marker CK20 were significantly elevated in tumor tissues compared with adjacent normal tissues (both p < 0.001), consistent with postoperative pathological diagnosis and supporting the reliability of the tissue samples (Fig. 1 a, b). IHC analysis revealed that MG53 was predominantly localized in the cytoplasm and cell membrane. Compared with adjacent normal tissues, bladder cancer tissues exhibited a markedly reduced MG53-positive area and substantially weaker staining intensity, resulting in a significantly lower H-score (p < 0.001) (Fig. 1 a, b). Double immunofluorescence staining further confirmed that MG53 fluorescence was markedly decreased in bladder cancer tissues, whereas KIF11 fluorescence was significantly increased, indicating enhanced proliferative activity in tumor cells (Fig. 1 c, d). Notably, prior studies have shown that MG53 can suppress tumor growth through transcriptional inhibition of KIF11 [ 16 ]. Transcriptome sequencing demonstrated that MG53 mRNA expression was significantly downregulated in bladder cancer tissues compared with matched adjacent tissues (p < 0.001) (Fig. 1 e, f). Consistent with these transcriptional data, Western blot analysis showed that MG53 protein expression was significantly lower in bladder cancer tissues than in adjacent normal tissues (p < 0.01), whereas AMPKα protein expression was markedly increased (Fig. 1 g, h). Correlation analysis further revealed a significant inverse relationship between MG53 and AMPKα protein expression levels (r = − 0.6844, p < 0.05) (Fig. 1 i). Together, these data demonstrate that MG53 is significantly downregulated in bladder cancer at both the mRNA and protein levels and is inversely associated with AMPKα expression, suggesting that loss of MG53 may contribute to bladder cancer progression. 3.2. MG53 suppresses the proliferation and invasion of bladder cancer cells To evaluate the functional consequences of MG53 modulation in bladder cancer cells, we established doxycycline (DOX)-inducible MG53 overexpression and adenovirus-mediated MG53 knockdown systems in T24 cells. Western blot analysis confirmed that MG53 protein expression was significantly elevated in the DOX + Ad-MG53 group compared with the control, Ad-Vector, DOX, and DOX + Ad-Vector groups (all p < 0.001), confirming robust induction of MG53 overexpression by DOX (Fig. 2 a, b). In contrast, MG53 protein expression was significantly reduced in the Ad-shMG53 group relative to the control and Ad-shVector groups (both p < 0.001), confirming efficient knockdown of MG53 (Fig. 2 h, i). CCK-8 assays revealed that MG53 overexpression significantly inhibited T24 cell proliferation starting on day 2, with the effect most pronounced by day 5 compared with the DOX + Ad-Vector group (p < 0.001) (Fig. 2 c). Conversely, MG53 knockdown significantly enhanced T24 cell proliferation from day 2 onward, with the strongest effect on day 5 compared with the Ad-shVector group (p < 0.001) (Fig. 2 j). These data indicate that MG53 exerts a potent inhibitory effect on bladder cancer cell proliferation. Transwell assays showed that MG53 overexpression markedly decreased the numbers of migratory and invasive cells (Fig. 2 d, e). Colony formation assays further showed that MG53 overexpression significantly reduced clonogenic potential, as evidenced by fewer and smaller colonies compared with the DOX + Ad-Vector group (Fig. 2 f, g). In contrast, MG53 knockdown increased colony formation in T24 cells relative to the Ad-shVector group (Fig. 2 m, n), indicating enhanced self-renewal capacity. Taken together, these results demonstrate that MG53 suppresses the proliferation, migration, invasion, and self-renewal of bladder cancer cells, whereas MG53 depletion produces the opposite effects, supporting a tumor-suppressive role for MG53 in bladder cancer. 3.3. MG53 attenuates glycolytic metabolism in bladder cancer cells Given that metabolic reprogramming toward aerobic glycolysis is a hallmark of bladder cancer, we next examined whether MG53 regulates glucose metabolism in bladder cancer cells. Glucose uptake assays showed that MG53 overexpression significantly reduced glucose uptake in T24 cells, as evidenced by a marked decrease in fluorescence intensity in the DOX + Ad-MG53 group compared with the DOX + Ad-Vector group (p < 0.001) (Fig. 3 a, b). Consistent with this finding, analysis of metabolic output demonstrated that lactate production in the culture supernatant was significantly decreased following MG53 overexpression (p < 0.01) (Fig. 3 c), indicating reduced glycolytic flux. Together, these data suggest that MG53 suppresses glucose utilization and downstream lactate generation, key features of glycolysis. These results support a role for MG53 as a negative regulator of metabolic reprogramming in bladder cancer cells, attenuating the Warburg phenotype and thereby limiting the bioenergetic and biosynthetic capacity required for tumor growth. 3.4. MG53 promotes the ubiquitination and proteasomal degradation of AMPKα To elucidate the molecular mechanism by which MG53 regulates bladder cancer progression, we examined its effects on AMPKα protein stability and the potential interaction between MG53 and AMPKα. Western blot analysis demonstrated that Ad-MG53-Dox induction significantly increased MG53 protein levels while concomitantly reducing AMPKα protein expression in T24 cells (Fig. 4 a, b), consistent with findings from Jiang et al. [ 15 ], who reported that MG53 negatively regulates AMPKα abundance. Given the inverse correlation between MG53 and AMPKα expression observed in bladder cancer tissues, we next examined whether MG53 physically interacts with AMPKα. Endogenous co-immunoprecipitation (Co-IP) assays showed that AMPKα was specifically co-precipitated by anti-MG53 antibody in T24 cells, and reciprocally, MG53 was co-precipitated by anti-AMPKα antibody, whereas no corresponding bands were detected in the IgG control group (Fig. 4 c, d). These findings confirm an endogenous interaction between MG53 and AMPKα in bladder cancer cells. To determine whether MG53 regulates AMPKα through ubiquitin-mediated protein turnover, we performed Co-IP and ubiquitination assays in T24 cells co-transfected with Myc-AMPKα, Flag-MG53, and HA-Ub plasmids. Immunoprecipitation with anti-Myc antibody, followed by immunoblotting, confirmed the presence of Flag-MG53 in the AMPKα-containing complex. Notably, HA-Ub immunoblotting revealed a pronounced ubiquitin smear associated with AMPKα in the presence of MG53 (Fig. 4 e), indicating enhanced polyubiquitination of AMPKα. Taken together, these findings demonstrate that MG53 directly interacts with AMPKα and functions as an E3 ubiquitin ligase to promote its ubiquitination and proteasomal degradation. This MG53–AMPKα axis provides a mechanistic link between ubiquitin-mediated protein turnover and metabolic reprogramming and likely underlies the tumor-suppressive effects of MG53 in bladder cancer. 3.5. MG53 suppresses bladder cancer growth in vivo To extend our in vitro findings and establish the physiological relevance of MG53 in bladder cancer progression, we employed complementary in vivo models that interrogate both host-dependent and tumor cell-intrinsic functions of MG53. Specifically, we utilized an orthotopic bladder cancer model in mg53−/− mice to assess the contribution of endogenous MG53 in the tumor microenvironment, together with a subcutaneous xenograft model in nude mice to evaluate the cell-autonomous effects of MG53 modulation in tumor cells. In the orthotopic model, in vivo bioluminescence imaging revealed a progressive increase in tumor burden following tumor cell inoculation in mg53−/− mice, with significantly higher signal intensity compared to wild-type (WT) controls by day 21 (p < 0.001) (Fig. 5 a, c). Consistent with these imaging data, gross examination demonstrated markedly larger tumors in mg53−/−mice, accompanied by a significant increase in tumor wet weight relative to WT mice (p < 0.001) (Fig. 5 b, d). These findings indicate that loss of endogenous MG53 creates a permissive environment for bladder tumor growth, supporting a tumor-suppressive role for MG53 in vivo. To further dissect tumor cell-intrinsic effects, we evaluated MG53 gain- and loss-of-function in a nude mouse subcutaneous xenograft model. Tumor growth kinetics showed that xenografts derived from MG53-overexpressing T24 cells exhibited significantly reduced growth compared with vector controls, with a marked difference observed by day 22 post-implantation (p < 0.001). In contrast, tumors derived from MG53-silenced cells grew more rapidly and reached significantly larger sizes than those in the knockdown control group (p < 0.001) (Fig. 5 e, f). Consistent with these observations, endpoint tumor weights were significantly decreased in the MG53 overexpression group (p < 0.05) and increased in the MG53 knockdown group (p < 0.01) relative to their respective controls (Fig. 5 g). Together, these complementary in vivo approaches demonstrate that MG53 suppresses bladder cancer growth through both tumor cell-intrinsic and microenvironment-dependent mechanisms. These findings provide in vivo validation of MG53 as a tumor suppressor and reinforce the MG53–AMPKα axis as a key regulatory pathway governing bladder cancer progression. 4. Discussion In this study, we identify MG53/TRIM72 as a previously unrecognized tumor suppressor in bladder cancer and define a mechanistic link between MG53-mediated ubiquitination and metabolic reprogramming. We demonstrate that MG53 is significantly downregulated at both the mRNA and protein levels in human bladder cancer tissues, and that its loss is associated with increased expression of AMPKα, enhanced proliferative signaling, and aggressive tumor behavior. These findings are consistent with prior reports in pancreatic, lung, and breast cancers, supporting the concept that MG53 functions as a broadly acting tumor suppressor across multiple malignancies [ 16 – 18 ]. Importantly, our combined in vitro and in vivo data establish that restoration of MG53 suppresses proliferation, invasion, and tumor growth, whereas its depletion promotes malignant progression. In the context of high-grade invasive bladder cancer, characterized by high recurrence rates, metastatic potential, and limited targeted treatment options, these findings highlight MG53 as a candidate biomarker and therapeutic target with potential clinical relevance [ 19 , 20 ]. Metabolic reprogramming is a defining feature of bladder cancer, with tumor cells exhibiting a strong dependence on aerobic glycolysis to sustain rapid growth and survival under stress conditions [ 21 , 22 ]. While current metabolic interventions have largely focused on targeting individual glycolytic enzymes such as GLUT1, these approaches are often limited by metabolic plasticity and compensatory pathway activation [ 23 ]. Our study provides an alternative perspective by identifying MG53 as an upstream regulator of tumor metabolism. Specifically, we show that MG53 overexpression reduces glucose uptake and lactate production, thereby attenuating glycolytic flux and suppressing the Warburg phenotype. These findings suggest that targeting regulatory nodes that coordinate metabolic pathways, rather than individual enzymes, may represent a more effective strategy to disrupt tumor metabolism. A key mechanistic insight from our study is the identification of AMPKα as a direct downstream target of MG53. AMPKα serves as a central sensor of cellular energy status, but its role in cancer is highly context-dependent. In bladder cancer, accumulating evidence indicates that AMPKα is upregulated and contributes to tumor proliferation, migration, and therapeutic resistance [ 24 , 25 ]. Consistent with these observations, we found that AMPKα expression is elevated in bladder cancer tissues and inversely correlated with MG53 levels. Mechanistically, MG53 directly interacts with AMPKα and promotes its ubiquitination and proteasomal degradation, thereby limiting its abundance and downstream signaling. As a member of the TRIM family of E3 ubiquitin ligases [ 26 ], MG53 appears to exert its tumor-suppressive effects, at least in part, by regulating protein turnover of key metabolic and signaling mediators. Notably, previous work by Jiang et al. [ 15 ] demonstrated that MG53 mediates AMPKα ubiquitination under high-glucose conditions in metabolic disease models. Our findings extend this mechanism to bladder cancer and suggest that MG53-dependent regulation of AMPKα represents a conserved pathway that integrates metabolic control with disease-specific signaling contexts. Together, these data establish the MG53–AMPKα axis as a critical regulatory node linking ubiquitin-mediated proteostasis to metabolic reprogramming in cancer. The in vivo studies further strengthen the biological and translational significance of our findings. Using both an orthotopic bladder cancer model in mg53−/− mice and a xenograft model with MG53-modulated tumor cells, we demonstrate that MG53 deficiency accelerates tumor growth, whereas MG53 overexpression suppresses tumor progression. The use of complementary models allows us to distinguish tumor cell-intrinsic effects from microenvironmental contributions of MG53. The observation that systemic loss of MG53 enhances tumor growth suggests that MG53 may also influence the tumor microenvironment, potentially through modulation of metabolic stress responses, inflammation, or stromal interactions. These results provide strong preclinical evidence supporting MG53 as a tumor suppressor and reinforce the therapeutic relevance of targeting the MG53–AMPKα pathway. Collectively, our findings support a model in which MG53 restrains bladder cancer progression by promoting ubiquitin-mediated degradation of AMPKα, thereby suppressing glycolytic metabolism and limiting the metabolic flexibility required for tumor growth. This work expands the functional repertoire of MG53 beyond its established roles in membrane repair and metabolic homeostasis, positioning it as a key regulator of cancer cell metabolism and signaling. From a translational perspective, strategies aimed at restoring MG53 expression or activity, such as gene delivery, recombinant protein administration, or small-molecule modulators, may represent promising approaches for bladder cancer therapy. Despite these advances, several limitations should be considered. First, although both T24 and J82 cell lines were introduced, most functional studies were performed in T24 cells; validation across additional bladder cancer models will be important to confirm the generalizability of our findings. Second, the specific ubiquitination sites and chain topology (e.g., K48- versus K63-linked ubiquitination) of AMPKα targeted by MG53 remain undefined and warrant further investigation using site-directed mutagenesis, linkage-specific antibodies, and proteomic approaches. Third, the potential roles of MG53-regulated cell death, including pyroptosis and ferroptosis, remain to be explored. Fourth, the clinical cohort analyzed in this study was limited in size and derived from a single center; larger, multicenter studies will be required to establish the prognostic and diagnostic value of MG53 in bladder cancer. Finally, the present work is preclinical, and further studies are needed to evaluate the feasibility, safety, and efficacy of MG53-based therapeutic strategies in translational and clinical settings. 5. Conclusions In summary, our study identifies MG53 as a critical tumor suppressor in bladder cancer and reveals a novel MG53–AMPKα signaling axis that links ubiquitin-mediated protein degradation to metabolic reprogramming. These findings provide new mechanistic insights into bladder cancer biology and lay a foundation for developing MG53-targeted therapeutic strategies. Declarations CRediT Author Contribution Statement Xiangfei He: Conceptualization, Supervision, Funding acquisition, Writing – original draft. He Zhang: Methodology, Software, Investigation, Data curation, Visualization, Writing – original draft. Yanping Zhang: Validation. Junwei Wu: Formal analysis. Tianen Wang: Resources. Yunlong Liu: Resources. Ke Jin: Validation. Weixiang Tang: Validation. Qinghua Ye: Validation. Qingwei Wang: Conceptualization, Writing – review and editing, Project administration, Supervision. Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Generative AI and AI-Assisted Technologies in the Manuscript Preparation Process (I used the SCISPACE AI tool to format the layout of the paper.) Funding This research was funded by the Medical Science and Technology Research Program of Henan Province, Joint Project (grant number: LHGJ20220398). The funding source had no role in the study design, data collection, analysis, interpretation, or decision to submit the article for publication. Ethics Approval and Consent to Participate This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval No.: 2022-KY-1274; approval date: 09-15-2022). Animal experiments were approved by the Institutional Animal Care and Use Committee of Zhengzhou University (Approval No.: 2022-KY-1274; approval date: 09-15-2022). Written informed consent was obtained from all human subjects involved in the study. Data Availability Statement All data generated or analyzed in this study are included in this article. Further inquiries regarding the data supporting the findings of this study can be directed to the corresponding authors upon reasonable request. Acknowledgements The authors thank the staff of the Department of Pathology at the First Affiliated Hospital of Zhengzhou University for their assistance with tissue processing and histological analyses. 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Front Oncol 2020;10. https://doi.org/10.3389/fonc.2020.00281 Lu H, Mei C, Yang L, Zheng J, Tong J, Duan F, et al. PPM-18, an analog of vitamin K, induces autophagy and apoptosis in bladder cancer cells through ROS and AMPK signaling pathways. Front Pharmacol. 2021;12. https://doi.org/10.3389/fphar.2021.639250 . Gan D, He W, Yin H, Gou X. β-elemene enhances cisplatin-induced apoptosis in bladder cancer cells through the ROS-AMPK signaling pathway. Oncol Lett. 2020;19:291–300. https://doi.org/10.3892/ol.2019.11107 . Song R, Peng W, Zhang Y, Lv F, Wu HK, Guo J, et al. Central role of E3 ubiquitin ligase MG53 in insulin resistance and metabolic disorders. Nature. 2013;494:375–9. https://doi.org/10.1038/nature11834 . Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9590464","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":641658130,"identity":"fad50ccf-96b7-4c04-a145-2960817879d3","order_by":0,"name":"Xiangfei He","email":"data:image/png;base64,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","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Xiangfei","middleName":"","lastName":"He","suffix":""},{"id":641658133,"identity":"66fd5a80-9adc-4537-bb8a-a0501ea9f0c2","order_by":1,"name":"He Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Zhang","suffix":""},{"id":641658139,"identity":"37a6cecf-f778-4674-89b2-34b9c01a35d1","order_by":2,"name":"Yanping Zhang","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yanping","middleName":"","lastName":"Zhang","suffix":""},{"id":641658143,"identity":"786db971-ba65-46a8-ba5d-98a591a264c6","order_by":3,"name":"Junwei Wu","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Junwei","middleName":"","lastName":"Wu","suffix":""},{"id":641658144,"identity":"69c6e4a1-233e-47f9-bbba-3c12259128e6","order_by":4,"name":"Tianen Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Tianen","middleName":"","lastName":"Wang","suffix":""},{"id":641658146,"identity":"effe25a8-888f-4e84-961d-8294760c2a51","order_by":5,"name":"Yunlong Liu","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yunlong","middleName":"","lastName":"Liu","suffix":""},{"id":641658151,"identity":"6f8344d6-82a6-4a1e-afb0-d8193029df53","order_by":6,"name":"Ke Jin","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Ke","middleName":"","lastName":"Jin","suffix":""},{"id":641658154,"identity":"e99e253a-4023-4407-84b1-94d16de18489","order_by":7,"name":"Weixiang Tang","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Weixiang","middleName":"","lastName":"Tang","suffix":""},{"id":641658160,"identity":"a319d796-5541-4c9b-8922-8386fcf5cf88","order_by":8,"name":"Qinghua Ye","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Qinghua","middleName":"","lastName":"Ye","suffix":""},{"id":641658161,"identity":"4811e960-76b3-488f-82ae-9a22e6f53002","order_by":9,"name":"Qingwei Wang","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Qingwei","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-05-02 04:08:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9590464/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9590464/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109474089,"identity":"6cd55ad2-f0b9-445d-98c3-340c612e9b41","added_by":"auto","created_at":"2026-05-18 13:41:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":24988941,"visible":true,"origin":"","legend":"\u003cp\u003eMG53 is downregulated in bladder cancer tissues and inversely correlated with AMPKα expression. (a) Representative H\u0026amp;E staining and IHC staining for CK20, Ki67, MG53, and AMPKα in matched adjacent normal and bladder cancer tissues (scale bar = 400 µm). (b) Quantitative analysis of H-scores for CK20, Ki67, MG53, and AMPKα (n = 5 pairs). (c) Representative double immunofluorescence staining for MG53 and KIF11 in adjacent normal and bladder cancer tissues, with DAPI used for nuclear counterstaining (scale bar = 100 µm). (d) Quantitative analysis of the positive area fractions of MG53 and KIF11. (e) Volcano plot of transcriptome sequencing data showing differential gene expression between bladder cancer tissues and matched adjacent normal tissues, highlighting MG53. (f) Relative MG53 mRNA expression based on transcriptome sequencing analysis (n = 5 pairs). (g) Representative Western blot analysis of MG53 and AMPKα protein expression in paired adjacent normal and bladder cancer tissues. GAPDH was used as a loading control. (h) Quantification of MG53 and AMPKα protein expression normalized to GAPDH. (i) Correlation analysis between MG53 and AMPKα protein expression in bladder cancer tissues. Data are presented as mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9590464/v1/3d9fea4eb06d5c70886d4f32.png"},{"id":109760371,"identity":"5c8213bc-aa9b-49f0-abfe-5d1e604371b1","added_by":"auto","created_at":"2026-05-22 07:28:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":10273710,"visible":true,"origin":"","legend":"\u003cp\u003eMG53 suppresses the proliferation, migration, invasion, and self-renewal of bladder cancer cells. (a) Representative Western blot showing DOX-inducible MG53 overexpression in T24 cells. (b) Quantification of MG53 protein expression in the indicated groups. (c) CCK-8 assay showing the effect of MG53 overexpression on T24 cell proliferation. (d, e) Representative Transwell images and quantitative analysis of cell migration and invasion following MG53 overexpression (scale bar = 200 µm). (f, g) Representative colony formation images and quantitative analysis showing the effect of MG53 overexpression on self-renewal capacity. (h, i) Representative Western blot and quantification of MG53 knockdown efficiency in T24 cells. (j) CCK-8 assay showing the effect of MG53 knockdown on T24 cell proliferation. (k, l) Representative Transwell images and quantitative analysis of cell migration and invasion following MG53 knockdown (scale bar = 200 µm). (m, n) Representative colony formation images and quantitative analysis showing the effect of MG53 knockdown on self-renewal capacity. Data are presented as mean ± SD. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9590464/v1/813a13931c890c960af8eae0.png"},{"id":109906630,"identity":"370ba5e9-cd22-40e8-942a-844dca1a1e9d","added_by":"auto","created_at":"2026-05-25 06:40:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":606889,"visible":true,"origin":"","legend":"\u003cp\u003eMG53 suppresses the Warburg effect, increases ROS accumulation, and induces apoptosis in bladder cancer cells. (a, b) Representative glucose uptake histograms and quantitative analysis showing the effect of MG53 overexpression on glucose uptake in T24 cells. (c) Quantification of lactate production in the indicated groups. Data are presented as mean ± SD. ns: p \u0026gt; 0.05, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9590464/v1/17947e988840107cd8d9a6e1.png"},{"id":109474095,"identity":"928e62f3-312d-4171-9343-cc14ea17d5e3","added_by":"auto","created_at":"2026-05-18 13:41:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2055733,"visible":true,"origin":"","legend":"\u003cp\u003eMG53 promotes the ubiquitination and proteasomal degradation of AMPKα. (a, b) Representative Western blot images and quantitative analysis showing the effects of MG53 overexpression on MG53 and AMPKα protein expression in T24 cells. (c, d) Endogenous co-immunoprecipitation assays demonstrating the interaction between MG53 and AMPKα in T24 cells. IgG was used as a negative control. (e) Exogenous co-immunoprecipitation and ubiquitination assays in T24 cells co-transfected with Myc-AMPKα, Flag-MG53, and HA-Ub plasmids, showing that MG53 promotes AMPKα ubiquitination. Data are presented as mean ± SD (n = 3). ns: p \u0026gt; 0.05, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9590464/v1/445425ea314272401313c20c.png"},{"id":109474092,"identity":"d99b5e74-f2e3-44d7-a3de-219acce84855","added_by":"auto","created_at":"2026-05-18 13:41:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":10687055,"visible":true,"origin":"","legend":"\u003cp\u003eMG53 suppresses bladder cancer growth in vivo. (a) Representative in vivo bioluminescence images of orthotopic bladder tumors in WT and mg53−/− mice at the indicated time points. (b) Representative gross images of dissected tumors from WT and mg53−/− mice at 7, 14, and 21 days after inoculation. (c) Quantification of tumor bioluminescence intensity in WT and mg53−/− mice over time. (d) Quantification of tumor wet weight in WT and mg53−/− mice. (e) Representative images of subcutaneous xenograft tumors derived from T24 cells with MG53 overexpression or knockdown and their respective control groups. (f) Tumor growth curves of subcutaneous xenografts in nude mice. (g) Quantification of tumor wet weight in the indicated xenograft groups. Data are presented as mean ± SD. ns: p \u0026gt; 0.05, *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9590464/v1/daa6a5741385ab4417e35dd2.png"},{"id":109908973,"identity":"26a5cfd2-29bd-4d8f-a0e0-f020d296a264","added_by":"auto","created_at":"2026-05-25 06:50:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":47328696,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9590464/v1/39633ec0-442a-402d-9beb-4a297c1968a3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"MG53 Suppresses the Progression of Bladder Cancer Through E3 Ubiquitin Ligase-Mediated Degradation of AMPKα","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eBladder cancer (BC) represents one of the most prevalent malignancies of the urinary system worldwide. According to contemporary epidemiological data, bladder cancer ranks among the most frequently diagnosed malignancies in both men and women, and its global disease burden remains substantial [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Bladder carcinogenesis and progression are driven by a complex interplay of risk factors, encompassing tobacco exposure, occupational and environmental carcinogens, genetic susceptibility, and metabolic dysregulation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. High-grade invasive bladder cancer is characterized by high rates of recurrence, metastasis, and therapeutic resistance. Despite continuous advances in surgery, radiotherapy, chemotherapy, and immune checkpoint inhibition, the prognosis of patients with advanced bladder cancer remains unsatisfactory, with a 5-year survival rate below 50% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, elucidating the molecular mechanisms underlying bladder cancer progression and identifying novel diagnostic biomarkers and therapeutic targets are of considerable clinical importance.\u003c/p\u003e \u003cp\u003eMitsugumin 53 (MG53), also designated as tripartite motif protein 72 (TRIM72), is a member of the tripartite motif (TRIM) family protein. As an E3 ubiquitin ligase, MG53 primarily functions by catalyzing the ubiquitination of target proteins, thereby governing protein turnover and modulating intracellular signaling pathways [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Previous studies have shown that MG53 is predominantly expressed in striated muscle and has been established as an essential mediator of plasma membrane repair, oxidative stress responses, and metabolic homeostasis [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In addition to its physiological functions, MG53 has been increasingly implicated in the pathogenesis of metabolic diseases, including diabetes and insulin resistance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. More recently, emerging evidence has positioned MG53 as a tumor suppressor in several malignancies, including hepatocellular carcinoma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], ovarian cancer [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and other solid tumors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]; however, its role in bladder cancer remains largely unexplored.\u003c/p\u003e \u003cp\u003eAMP-activated protein kinase α (AMPKα) functions as a master sensor of cellular energy status that regulates metabolic reprogramming in response to changes in the intracellular AMP/ATP or ADP/ATP ratio. In tumor cells, AMPKα is frequently aberrantly activated and has been shown to sustain tumor progression by enhancing glucose uptake and aerobic glycolysis, thereby providing energy and biosynthetic precursors required for rapid proliferation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Accumulating evidence indicates that AMPKα plays a context-dependent oncogenic role in bladder cancer, contributing to tumor proliferation, migration, and therapeutic resistance [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Importantly, a prior study by Jiang et al. demonstrated that MG53 mediates AMPKα ubiquitination and degradation under high-glucose conditions in metabolic disease models [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], raising the possibility that a similar regulatory axis may operate in cancer.\u003c/p\u003e \u003cp\u003eIn the present study, we characterized the expression profile of MG53 in human bladder cancer tissues, evaluated its functional impact on key malignant phenotypes including proliferation and invasion, and delineated the molecular mechanism by which MG53 regulates AMPKα through ubiquitin-dependent proteasomal degradation. We further examined the downstream consequences of this regulatory axis for glycolytic metabolism and tumor growth in vivo. Our findings establish a novel MG53\u0026ndash;AMPKα signaling axis that links ubiquitin-mediated proteostasis to metabolic reprogramming and tumor suppression in bladder cancer.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Human tissue samples\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.1.1. Ethics approval and patient enrollment\u003c/h2\u003e \u003cp\u003e This study was reviewed and approved by the Institutional Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval No.: 2022-KY-1274; approval date: 09-15-2022). All procedures were conducted in accordance with the Declaration of Helsinki and applicable national guidelines governing biomedical research involving human subjects. Patients with histopathologically confirmed high-grade invasive bladder cancer who underwent radical cystectomy at our institution between January 2024 and December 2024 were prospectively enrolled. Paired specimens of primary tumor tissue and adjacent morphologically normal urothelium were collected from each patient. Adjacent normal tissues were obtained at least 2 cm from the tumor margin and subsequently verified to be free of malignant involvement by independent histopathological examination. All patients were pathologically diagnosed with high-grade invasive urothelial carcinoma and had received no prior antitumor treatment, including radiotherapy, chemotherapy, or intravesical therapy. Complete clinicopathological data, including tumor stage, pathological grade, tumor size, and lymph node status, were available for all patients. Written informed consent was obtained from all participants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.1.2. Inclusion and exclusion criteria\u003c/h2\u003e \u003cp\u003eThe inclusion criteria were as follows: (1) histopathologically confirmed high-grade invasive bladder cancer (T2\u0026ndash;T4) with urothelial carcinoma histology; (2) primary bladder cancer without history of prior antitumor treatment; (3) availability of complete clinical and follow-up data; (4) absence of severe dysfunction of major organs, including the heart, liver, or kidneys, and no severe infection or autoimmune disease; and (5) availability of sufficient fresh-frozen and formalin-fixed tissue specimens to permit immunohistochemistry, immunoblotting, transcriptome sequencing, and other downstream analyses.\u003c/p\u003e \u003cp\u003eThe exclusion criteria were as follows: (1) non-urothelial histological variants, including but not limited to squamous cell carcinoma and adenocarcinoma of the bladder; (2) receipt of any neoadjuvant or preoperative antitumor therapy, including radiotherapy, chemotherapy, targeted therapy, or immunotherapy before surgery; (3) presence of synchronous or metachronous malignancies of other organ sites; (4) inadequate tissue preservation, tissue necrosis, or contamination that precluded experimental analysis; and (5) incomplete or missing clinicopathological documentation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.1.3. Sample processing\u003c/h2\u003e \u003cp\u003eFresh tissue samples were bisected immediately upon surgical resection under sterile conditions. One portion was snap-frozen in liquid nitrogen within 30 min and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until used for Western blotting, qPCR, and transcriptome sequencing. The other portion was fixed in 10% neutral buffered formalin for 4\u0026ndash;6 h at room temperature, dehydrated, and paraffin-embedded. Paraffin sections (4 \u0026micro;m) were prepared for hematoxylin and eosin (H\u0026amp;E) staining, immunohistochemistry (IHC), and immunofluorescence analyses. All specimens were collected, processed, and stored in accordance with standard biobank operating procedures to ensure sample quality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.1.4. Transcriptome sequencing\u003c/h2\u003e \u003cp\u003eTumor tissues and matched adjacent normal tissues from a subset of five patients were subjected to transcriptome sequencing. Total RNA was extracted and subjected to quality control. Strand-specific cDNA libraries were prepared and subjected to paired-end sequencing on an Illumina NovaSeq platform by Majorbio Bio-pharm Technology Co., Ltd (Shanghai, China). Differentially expressed genes (DEGs) between tumor and matched normal tissues were identified using established bioinformatics pipelines with thresholds of |log₂ fold change| \u0026ge; 1 and adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The transcriptional expression profile of MG53 was specifically examined within the DEG dataset.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell lines and culture conditions\u003c/h2\u003e \u003cp\u003eHuman bladder cancer cell lines T24 and J82, and the immortalized normal human bladder epithelial cell line SV-HUC-1, were procured from the Cell Bank of the Chinese Academy of Sciences. Cell line authentication was confirmed by short tandem repeat profiling, and mycoplasma-free status was routinely confirmed using a PCR-based mycoplasma detection kit prior to experimental use. T24 and J82 cells were cultured in RPMI-1640 medium (Gibco, 12633012) supplemented with 10% fetal bovine serum (FBS; Gibco, A5256901), 100 U/mL penicillin, and 100 \u0026micro;g/mL streptomycin. SV-HUC-1 cells were cultured in DMEM (Gibco, 12491015) supplemented with 10% FBS. All cells were maintained at 37\u0026deg;C in a humidified incubator with 5% CO₂. The culture medium was replaced every 24\u0026ndash;48 h. Cells were passaged with 0.25% trypsin (Gibco, 25300054) when they reached 80%\u0026ndash;90% confluence. All experiments were performed using cells in the logarithmic growth phase within 20 passages of receipt from the repository.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Animals and tumor models\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. Animals and housing\u003c/h2\u003e \u003cp\u003eMG53 knockout (mg53\u0026minus;/\u0026minus;) mice on a C57BL/6N background were generated using CRISPR/Cas9 technology, and littermate wild-type (WT) C57BL/6N mice were used as controls for the establishment of the orthotopic bladder cancer model. Athymic nude mice (BALB/c-nu/nu) were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd. and used for the human bladder cancer xenograft model. All mice were 6\u0026ndash;8 weeks old and weighed 20\u0026ndash;25 g. An equal number of male and female mice were used. Mice were housed under specific-pathogen-free conditions at Zhengzhou University at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and 50\u0026thinsp;\u0026plusmn;\u0026thinsp;5% humidity, under a 12-h light/dark cycle, with free access to sterile food and water. All animal experiments were reviewed and approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University and conducted in accordance with institutional guidelines for animal care and use (Approval No.: 2022-KY-1274; approval date: 09-15-2022).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Experimental grouping and model establishment\u003c/h2\u003e \u003cp\u003eFor the orthotopic bladder cancer model, 50 mg53\u0026minus;/\u0026minus; mice and 50 WT mice were randomly assigned to five groups corresponding to 0, 7, 14, and 21 days after tumor inoculation, with 10 mice per group (5 males and 5 females). The luciferase-labeled mouse bladder cancer cell line MB49-Luc was used to establish the orthotopic model. Cells were resuspended in a 1:1 mixture of PBS and Matrigel to achieve a final concentration of 5 \u0026times; 10^6 cells/mL, and 20 \u0026micro;L of cell suspension was orthotopically injected into the bladder wall of each mouse.\u003c/p\u003e \u003cp\u003eFor the subcutaneous xenograft model, 40 nude mice were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;10 per group, including 5 males and 5 females): Ad-MG53 group (T24 cells infected with Ad-MG53), Ad-Vector group (T24 cells infected with Ad-Vector), Ad-shMG53 group (T24 cells infected with Ad-shMG53), and Ad-shVector group (T24 cells infected with Ad-shVector). T24 cells were infected with the indicated adenoviruses for 12 h, and 1 \u0026times; 10^5 cells in 20 \u0026micro;L suspension were injected subcutaneously into the right dorsal flank of each mouse.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Tumor monitoring and sample collection\u003c/h2\u003e \u003cp\u003eIn the orthotopic mg53\u0026minus;/\u0026minus; mouse model, bioluminescence intensity was monitored using an in vivo imaging system (PerkinElmer) at 0, 7, 14, and 21 days post-inoculation. Tumor growth was recorded over time. Mice were euthanized at the indicated time points, and bladder and tumor tissues were harvested. Surface blood and fluid were gently removed with filter paper. The wet weight of tumors was measured using a precision electronic balance (0.1 mg sensitivity), and tumor length and width were measured with a vernier caliper. In the nude mouse subcutaneous xenograft model, tumor length and width were measured every 3 days beginning on day 7 post-implantation, and tumor volume was calculated to generate growth curves. On day 22 post-inoculation, mice were humanely euthanized, subcutaneous tumors were surgically excised, and tumor wet weight was recorded.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Reagents\u003c/h2\u003e \u003cp\u003eAdenoviral vectors (Ad-MG53, Ad-Vector, Ad-shMG53, and Ad-shVector) and plasmids encoding Myc-AMPKα, Flag-MG53, and HA-Ub were synthesized and packaged by Shandong Weizhen Biotechnology (Shandong, China). Primary antibodies against MG53 (Cat# 22151-1-AP), AMPKα (Cat# 10929-2-AP), HA tag (Cat# 51064-2-AP), Myc tag (Cat# 16286-1-AP), Flag tag (Cat# 66008-4-Ig), KIF11 (Cat# 23333-1-AP), Ki67 (Cat# 27309-1-AP), and GAPDH (Cat# 60004-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). Anti-CK20 antibody (Cat# GB112050) was obtained from Servicebio (Wuhan, China). HRP-conjugated goat anti-rabbit IgG secondary antibody (Cat# 7074S) and HRP-conjugated horse anti-mouse IgG secondary antibody (Cat# 7076S) were purchased from Cell Signaling Technology (Danvers, MA, USA). The Glucose Uptake Probe-Red kit (Cat# UP03) was purchased from Dojindo (Kumamoto, Japan); the L-lactate assay kit (Cat# A019-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); the proteasome inhibitor MG132 (Cat# HY-13259) was purchased from MedChemExpress (Monmouth Junction, NJ, USA); the Protein A\u0026thinsp;+\u0026thinsp;G Agarose Co-immunoprecipitation Kit (Cat# P2055) was purchased from Beyotime (Shanghai, China); D-luciferin potassium salt (Cat# Luc-1G) was purchased from GoldBio (St. Louis, MO, USA); and primers were synthesized by Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Experimental procedures\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1. Adenoviral infection and cell treatment\u003c/h2\u003e \u003cp\u003eT24 cells in the logarithmic growth phase were seeded in 6-well plates at a density of 5 \u0026times; 10^5 cells/well. After 24 h, when cell confluence reached approximately 80%, cells were infected with the indicated adenoviral vectors (Ad-MG53, Ad-Vector, Ad-shMG53, or Ad-shVector) at a multiplicity of infection (MOI) of 100. Following a 12 h infection period, the medium was replaced with fresh complete medium, and cells were cultured for an additional 48 h before subsequent assays. For the doxycycline (DOX)-inducible overexpression model, cells infected with Ad-MG53 were treated with 2 \u0026micro;g/mL DOX for 48 h, and MG53 expression was confirmed by Western blotting analysis. For proteasome inhibition experiments, cells were treated with MG132 at 10 \u0026micro;mol/L for 12 h. Plasmid transfection was performed using Lipofectamine 3000 (Invitrogen), and experiments were conducted 48 h after transfection.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.5.2. Cell function assays\u003c/h2\u003e \u003cp\u003eCell proliferation was assessed using the Cell Counting Kit-8 (CCK-8) assay. Briefly, T24 cells from each group were seeded into 96-well plates at a density of 1 \u0026times; 10^3 cells/well, with six replicate wells per group. At 0, 1, 2, 3, 4, and 5 days after seeding, 10 \u0026micro;L of CCK-8 reagent was added to each well, and the plates were incubated at 37\u0026deg;C for 2 h. Absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA), and cell proliferation curves were generated accordingly.\u003c/p\u003e \u003cp\u003eCell invasion capacity was evaluated using the Transwell invasion assay. Briefly, Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) was diluted 1:8 and uniformly applied to the upper chamber of Transwell inserts, then incubated at 37\u0026deg;C for 30 min to solidify. T24 cells were harvested and resuspended in serum-free medium. A total of 200 \u0026micro;L of cell suspension (1 \u0026times; 10^5 cells/mL) was added to the upper chamber, and 600 \u0026micro;L of complete medium containing 10% FBS was added to the lower chamber as a chemoattractant. After 24 h of incubation at 37\u0026deg;C in a humidified atmosphere, invaded cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Stained cells were imaged and counted in five randomly selected fields per insert under a light microscope at 100\u0026times; magnification.\u003c/p\u003e \u003cp\u003eColony formation ability was assessed using the colony formation assay. Briefly, T24 cells were seeded into 6-well plates at a density of 800 cells/well, with three replicate wells per group, and cultured for 14 days. The culture medium was replaced every 3 days to maintain optimal growth conditions. At the end of the culture period, colonies were washed twice with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet for 20 min. Excess stain was removed by gentle washing with distilled water, and plates were air-dried prior to imaging. Colonies containing at least 50 cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and colony formation efficiency was calculated as the number of colonies formed divided by the number of cells initially seeded, expressed as a percentage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.5.3. Detection of cellular metabolism\u003c/h2\u003e \u003cp\u003eGlucose uptake was measured using the Glucose Uptake Probe-Red kit (Cat# UP03; Dojindo, Kumamoto, Japan) according to the manufacturer\u0026rsquo;s instructions. Briefly, T24 cells were seeded into 6-well plates at an appropriate density and allowed to adhere overnight. Prior to the assay, cells were washed twice with PBS and subjected to serum and glucose starvation in serum-free, glucose-free medium for 6 h at 37\u0026deg;C to eliminate residual intracellular glucose. After starvation, the Glucose Uptake Probe-Red reagent was added to a final concentration of 10 \u0026micro;mol/L, and cells were incubated at 37\u0026deg;C for 30 min. After incubation, cells were washed twice with ice-cold PBS, harvested by trypsinization, and resuspended in PBS for flow cytometric analysis. Fluorescence intensity was measured by flow cytometry (excitation, 570 nm; emission, 590 nm), and glucose uptake capacity was quantified based on the mean fluorescence intensity (MFI) of each experimental group.\u003c/p\u003e \u003cp\u003eExtracellular lactate levels were determined using the L-lactate assay kit (Cat# A019-2-1; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer\u0026rsquo;s instructions. Briefly, T24 cells from each experimental group were seeded into 6-well plates at an appropriate density and cultured under standard conditions. At the designated time point, culture supernatants were carefully collected and centrifuged at 1,000 \u0026times; g for 5 min at 4\u0026deg;C to remove cellular debris. The clarified supernatants were then processed according to the kit protocol, and absorbance was measured at 530 nm using a microplate reader (Bio-Rad, Hercules, CA, USA). Lactate concentration was calculated from a standard curve generated with kit-provided lactate standards and normalized to total protein content or cell number in the corresponding wells to account for differences in cell density across groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.5.4. Western blotting and co-immunoprecipitation\u003c/h2\u003e \u003cp\u003e For Western blotting, total protein was extracted using RIPA lysis buffer supplemented with protease and phosphatase inhibitors, and protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit according to the manufacturer\u0026rsquo;s instructions. Equal amounts of protein (30 \u0026micro;g/lane) were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% nonfat milk for 2 h, incubated with primary antibodies overnight at 4\u0026deg;C, and then incubated with the corresponding secondary antibodies for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system, and band intensity was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). GAPDH was used as an internal loading control to ensure equal protein loading across all lanes.\u003c/p\u003e \u003cp\u003eFor endogenous co-immunoprecipitation (Co-IP), cells infected with the indicated adenoviral vectors were lysed, and total protein was extracted at a concentration of 1 \u0026micro;g/\u0026micro;L. Protein lysates were pre-cleared with Protein A/G agarose beads for 1 h at 4\u0026deg;C to reduce nonspecific binding, then incubated with 2 \u0026micro;g anti-MG53 or anti-AMPKα antibody overnight at 4\u0026deg;C, followed by incubation with 50 \u0026micro;L Protein A/G agarose beads for 4 h at 4\u0026deg;C. The immunoprecipitates were collected by centrifugation at 5000 rpm for 5 min, washed five times with TBST, and eluted by denaturation in 5\u0026times; loading buffer at 95\u0026deg;C for 5 min. The resulting samples were analyzed by Western blotting. Normal rabbit or mouse IgG was used as a negative control to assess nonspecific antibody binding.\u003c/p\u003e \u003cp\u003eTo assess exogenous protein interactions and ubiquitination, T24 cells were co-transfected with plasmids encoding Myc-AMPKα, Flag-MG53, and HA-Ub. At 36 h post-transfection, cells were treated with MG132 (10 \u0026micro;mol/L) for 12 h. Total protein was then extracted under denaturing conditions, and immunoprecipitation was performed using anti-Myc antibody to capture Myc-AMPKα and its associated protein complexes. The resulting immunoprecipitates were subjected to Western blot analysis to detect co-precipitated Flag-MG53 and HA-labeled polyubiquitin chains, thereby assessing the ubiquitination status of AMPKα by MG53.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.5.5. Immunohistochemistry and immunofluorescence staining\u003c/h2\u003e \u003cp\u003eImmunohistochemical staining was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections. Briefly, sections were deparaffinized in xylene and rehydrated through a graded ethanol series to distilled water. Antigen retrieval was performed using citrate buffer (pH 6.0) with high-temperature heating for 10 min, followed by cooling to room temperature. Endogenous peroxidase activity was blocked with 3% H₂O₂ for 10 min at room temperature, followed by three PBS washes. Nonspecific binding was blocked with 5% BSA for 30 min in a humidified chamber. After three PBS washes, sections were incubated overnight at 4\u0026deg;C with primary antibodies, then with secondary antibodies for 1 h at room temperature. Signals were developed using 3,3\u0026prime;-diaminobenzidine (DAB) substrate solution, and the reaction was monitored under a light microscope to ensure consistent staining intensity across sections. Nuclei were counterstained with hematoxylin, followed by bluing in running tap water. Sections were then dehydrated through a graded ethanol series, cleared in xylene, and mounted with neutral resin mounting medium. The integrated optical density (IOD) of positively stained areas was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003cp\u003eDouble immunofluorescence staining was performed on FFPE tissue sections as described above for deparaffinization, rehydration, and antigen retrieval. Following antigen retrieval, sections were blocked with 5% BSA for 30 min at room temperature in a humidified chamber to minimize nonspecific background. Sections were incubated overnight at 4\u0026deg;C with anti-MG53 (1:200) and anti-KIF11 (1:200) antibodies. After three PBS washes, sections were incubated with the corresponding fluorescent secondary antibodies for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI, and sections were mounted using an antifade fluorescence mounting medium. Fluorescence signals were captured using a laser scanning confocal microscope. Fluorescence intensity was quantified using the accompanying imaging software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e2.5.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll experiments were performed at least three times independently, and results are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using SPSS 22.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8.0 (San Diego, CA, USA). Prior to inferential testing, the normality of data distribution was assessed using the Shapiro-Wilk test, and homogeneity of variance was assessed using Levene\u0026rsquo;s test. For normally distributed data with equal variance, comparisons between two groups were performed using the independent-samples Student\u0026rsquo;s t-test, whereas comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s post hoc test. For data that were not normally distributed or did not show equal variance, the Mann-Whitney U test was used for two-group comparisons and the Kruskal-Wallis H test for multiple-group comparisons. Categorical variables were analyzed using the chi-square (χ\u0026sup2;) test, with Fisher\u0026rsquo;s exact test applied when expected cell frequencies were below five. Correlations were analyzed using Pearson\u0026rsquo;s or Spearman\u0026rsquo;s correlation analysis, as appropriate. All statistical tests were two-sided, and a p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.1. MG53 is downregulated in bladder cancer tissues and inversely correlated with AMPKα expression\u003c/h2\u003e \u003cp\u003eTo characterize the expression pattern and clinical relevance of MG53 in bladder cancer, we analyzed five pairs of high-grade invasive bladder cancer tissues and matched adjacent normal tissues. H\u0026amp;E staining, together with IHC, confirmed the characteristic histopathological features of high-grade invasive urothelial carcinoma. The H-scores of the proliferation marker Ki67 and the urothelial marker CK20 were significantly elevated in tumor tissues compared with adjacent normal tissues (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), consistent with postoperative pathological diagnosis and supporting the reliability of the tissue samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003eIHC analysis revealed that MG53 was predominantly localized in the cytoplasm and cell membrane. Compared with adjacent normal tissues, bladder cancer tissues exhibited a markedly reduced MG53-positive area and substantially weaker staining intensity, resulting in a significantly lower H-score (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Double immunofluorescence staining further confirmed that MG53 fluorescence was markedly decreased in bladder cancer tissues, whereas KIF11 fluorescence was significantly increased, indicating enhanced proliferative activity in tumor cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, d). Notably, prior studies have shown that MG53 can suppress tumor growth through transcriptional inhibition of KIF11 [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTranscriptome sequencing demonstrated that MG53 mRNA expression was significantly downregulated in bladder cancer tissues compared with matched adjacent tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee, f). Consistent with these transcriptional data, Western blot analysis showed that MG53 protein expression was significantly lower in bladder cancer tissues than in adjacent normal tissues (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas AMPKα protein expression was markedly increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eg, h). Correlation analysis further revealed a significant inverse relationship between MG53 and AMPKα protein expression levels (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.6844, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ei).\u003c/p\u003e \u003cp\u003eTogether, these data demonstrate that MG53 is significantly downregulated in bladder cancer at both the mRNA and protein levels and is inversely associated with AMPKα expression, suggesting that loss of MG53 may contribute to bladder cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.2. MG53 suppresses the proliferation and invasion of bladder cancer cells\u003c/h2\u003e \u003cp\u003eTo evaluate the functional consequences of MG53 modulation in bladder cancer cells, we established doxycycline (DOX)-inducible MG53 overexpression and adenovirus-mediated MG53 knockdown systems in T24 cells. Western blot analysis confirmed that MG53 protein expression was significantly elevated in the DOX\u0026thinsp;+\u0026thinsp;Ad-MG53 group compared with the control, Ad-Vector, DOX, and DOX\u0026thinsp;+\u0026thinsp;Ad-Vector groups (all p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), confirming robust induction of MG53 overexpression by DOX (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). In contrast, MG53 protein expression was significantly reduced in the Ad-shMG53 group relative to the control and Ad-shVector groups (both p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), confirming efficient knockdown of MG53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eh, i).\u003c/p\u003e \u003cp\u003eCCK-8 assays revealed that MG53 overexpression significantly inhibited T24 cell proliferation starting on day 2, with the effect most pronounced by day 5 compared with the DOX\u0026thinsp;+\u0026thinsp;Ad-Vector group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Conversely, MG53 knockdown significantly enhanced T24 cell proliferation from day 2 onward, with the strongest effect on day 5 compared with the Ad-shVector group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ej). These data indicate that MG53 exerts a potent inhibitory effect on bladder cancer cell proliferation.\u003c/p\u003e \u003cp\u003eTranswell assays showed that MG53 overexpression markedly decreased the numbers of migratory and invasive cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, e). Colony formation assays further showed that MG53 overexpression significantly reduced clonogenic potential, as evidenced by fewer and smaller colonies compared with the DOX\u0026thinsp;+\u0026thinsp;Ad-Vector group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef, g). In contrast, MG53 knockdown increased colony formation in T24 cells relative to the Ad-shVector group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003em, n), indicating enhanced self-renewal capacity.\u003c/p\u003e \u003cp\u003eTaken together, these results demonstrate that MG53 suppresses the proliferation, migration, invasion, and self-renewal of bladder cancer cells, whereas MG53 depletion produces the opposite effects, supporting a tumor-suppressive role for MG53 in bladder cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.3. MG53 attenuates glycolytic metabolism in bladder cancer cells\u003c/h2\u003e \u003cp\u003eGiven that metabolic reprogramming toward aerobic glycolysis is a hallmark of bladder cancer, we next examined whether MG53 regulates glucose metabolism in bladder cancer cells. Glucose uptake assays showed that MG53 overexpression significantly reduced glucose uptake in T24 cells, as evidenced by a marked decrease in fluorescence intensity in the DOX\u0026thinsp;+\u0026thinsp;Ad-MG53 group compared with the DOX\u0026thinsp;+\u0026thinsp;Ad-Vector group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003eConsistent with this finding, analysis of metabolic output demonstrated that lactate production in the culture supernatant was significantly decreased following MG53 overexpression (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), indicating reduced glycolytic flux. Together, these data suggest that MG53 suppresses glucose utilization and downstream lactate generation, key features of glycolysis.\u003c/p\u003e \u003cp\u003eThese results support a role for MG53 as a negative regulator of metabolic reprogramming in bladder cancer cells, attenuating the Warburg phenotype and thereby limiting the bioenergetic and biosynthetic capacity required for tumor growth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.4. MG53 promotes the ubiquitination and proteasomal degradation of AMPKα\u003c/h2\u003e \u003cp\u003eTo elucidate the molecular mechanism by which MG53 regulates bladder cancer progression, we examined its effects on AMPKα protein stability and the potential interaction between MG53 and AMPKα. Western blot analysis demonstrated that Ad-MG53-Dox induction significantly increased MG53 protein levels while concomitantly reducing AMPKα protein expression in T24 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b), consistent with findings from Jiang et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], who reported that MG53 negatively regulates AMPKα abundance.\u003c/p\u003e \u003cp\u003eGiven the inverse correlation between MG53 and AMPKα expression observed in bladder cancer tissues, we next examined whether MG53 physically interacts with AMPKα. Endogenous co-immunoprecipitation (Co-IP) assays showed that AMPKα was specifically co-precipitated by anti-MG53 antibody in T24 cells, and reciprocally, MG53 was co-precipitated by anti-AMPKα antibody, whereas no corresponding bands were detected in the IgG control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d). These findings confirm an endogenous interaction between MG53 and AMPKα in bladder cancer cells.\u003c/p\u003e \u003cp\u003eTo determine whether MG53 regulates AMPKα through ubiquitin-mediated protein turnover, we performed Co-IP and ubiquitination assays in T24 cells co-transfected with Myc-AMPKα, Flag-MG53, and HA-Ub plasmids. Immunoprecipitation with anti-Myc antibody, followed by immunoblotting, confirmed the presence of Flag-MG53 in the AMPKα-containing complex. Notably, HA-Ub immunoblotting revealed a pronounced ubiquitin smear associated with AMPKα in the presence of MG53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), indicating enhanced polyubiquitination of AMPKα.\u003c/p\u003e \u003cp\u003eTaken together, these findings demonstrate that MG53 directly interacts with AMPKα and functions as an E3 ubiquitin ligase to promote its ubiquitination and proteasomal degradation. This MG53\u0026ndash;AMPKα axis provides a mechanistic link between ubiquitin-mediated protein turnover and metabolic reprogramming and likely underlies the tumor-suppressive effects of MG53 in bladder cancer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section2\"\u003e \u003ch2\u003e3.5. MG53 suppresses bladder cancer growth in vivo\u003c/h2\u003e \u003cp\u003eTo extend our in vitro findings and establish the physiological relevance of MG53 in bladder cancer progression, we employed complementary in vivo models that interrogate both host-dependent and tumor cell-intrinsic functions of MG53. Specifically, we utilized an orthotopic bladder cancer model in mg53\u0026minus;/\u0026minus; mice to assess the contribution of endogenous MG53 in the tumor microenvironment, together with a subcutaneous xenograft model in nude mice to evaluate the cell-autonomous effects of MG53 modulation in tumor cells.\u003c/p\u003e \u003cp\u003eIn the orthotopic model, in vivo bioluminescence imaging revealed a progressive increase in tumor burden following tumor cell inoculation in mg53\u0026minus;/\u0026minus; mice, with significantly higher signal intensity compared to wild-type (WT) controls by day 21 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, c). Consistent with these imaging data, gross examination demonstrated markedly larger tumors in mg53\u0026minus;/\u0026minus;mice, accompanied by a significant increase in tumor wet weight relative to WT mice (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, d). These findings indicate that loss of endogenous MG53 creates a permissive environment for bladder tumor growth, supporting a tumor-suppressive role for MG53 in vivo.\u003c/p\u003e \u003cp\u003eTo further dissect tumor cell-intrinsic effects, we evaluated MG53 gain- and loss-of-function in a nude mouse subcutaneous xenograft model. Tumor growth kinetics showed that xenografts derived from MG53-overexpressing T24 cells exhibited significantly reduced growth compared with vector controls, with a marked difference observed by day 22 post-implantation (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In contrast, tumors derived from MG53-silenced cells grew more rapidly and reached significantly larger sizes than those in the knockdown control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee, f). Consistent with these observations, endpoint tumor weights were significantly decreased in the MG53 overexpression group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and increased in the MG53 knockdown group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) relative to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eg).\u003c/p\u003e \u003cp\u003eTogether, these complementary in vivo approaches demonstrate that MG53 suppresses bladder cancer growth through both tumor cell-intrinsic and microenvironment-dependent mechanisms. These findings provide in vivo validation of MG53 as a tumor suppressor and reinforce the MG53\u0026ndash;AMPKα axis as a key regulatory pathway governing bladder cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we identify MG53/TRIM72 as a previously unrecognized tumor suppressor in bladder cancer and define a mechanistic link between MG53-mediated ubiquitination and metabolic reprogramming. We demonstrate that MG53 is significantly downregulated at both the mRNA and protein levels in human bladder cancer tissues, and that its loss is associated with increased expression of AMPKα, enhanced proliferative signaling, and aggressive tumor behavior. These findings are consistent with prior reports in pancreatic, lung, and breast cancers, supporting the concept that MG53 functions as a broadly acting tumor suppressor across multiple malignancies [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Importantly, our combined in vitro and in vivo data establish that restoration of MG53 suppresses proliferation, invasion, and tumor growth, whereas its depletion promotes malignant progression. In the context of high-grade invasive bladder cancer, characterized by high recurrence rates, metastatic potential, and limited targeted treatment options, these findings highlight MG53 as a candidate biomarker and therapeutic target with potential clinical relevance [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMetabolic reprogramming is a defining feature of bladder cancer, with tumor cells exhibiting a strong dependence on aerobic glycolysis to sustain rapid growth and survival under stress conditions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While current metabolic interventions have largely focused on targeting individual glycolytic enzymes such as GLUT1, these approaches are often limited by metabolic plasticity and compensatory pathway activation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Our study provides an alternative perspective by identifying MG53 as an upstream regulator of tumor metabolism. Specifically, we show that MG53 overexpression reduces glucose uptake and lactate production, thereby attenuating glycolytic flux and suppressing the Warburg phenotype. These findings suggest that targeting regulatory nodes that coordinate metabolic pathways, rather than individual enzymes, may represent a more effective strategy to disrupt tumor metabolism.\u003c/p\u003e \u003cp\u003eA key mechanistic insight from our study is the identification of AMPKα as a direct downstream target of MG53. AMPKα serves as a central sensor of cellular energy status, but its role in cancer is highly context-dependent. In bladder cancer, accumulating evidence indicates that AMPKα is upregulated and contributes to tumor proliferation, migration, and therapeutic resistance [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Consistent with these observations, we found that AMPKα expression is elevated in bladder cancer tissues and inversely correlated with MG53 levels. Mechanistically, MG53 directly interacts with AMPKα and promotes its ubiquitination and proteasomal degradation, thereby limiting its abundance and downstream signaling. As a member of the TRIM family of E3 ubiquitin ligases [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], MG53 appears to exert its tumor-suppressive effects, at least in part, by regulating protein turnover of key metabolic and signaling mediators. Notably, previous work by Jiang et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] demonstrated that MG53 mediates AMPKα ubiquitination under high-glucose conditions in metabolic disease models. Our findings extend this mechanism to bladder cancer and suggest that MG53-dependent regulation of AMPKα represents a conserved pathway that integrates metabolic control with disease-specific signaling contexts. Together, these data establish the MG53\u0026ndash;AMPKα axis as a critical regulatory node linking ubiquitin-mediated proteostasis to metabolic reprogramming in cancer.\u003c/p\u003e \u003cp\u003eThe in vivo studies further strengthen the biological and translational significance of our findings. Using both an orthotopic bladder cancer model in mg53\u0026minus;/\u0026minus; mice and a xenograft model with MG53-modulated tumor cells, we demonstrate that MG53 deficiency accelerates tumor growth, whereas MG53 overexpression suppresses tumor progression. The use of complementary models allows us to distinguish tumor cell-intrinsic effects from microenvironmental contributions of MG53. The observation that systemic loss of MG53 enhances tumor growth suggests that MG53 may also influence the tumor microenvironment, potentially through modulation of metabolic stress responses, inflammation, or stromal interactions. These results provide strong preclinical evidence supporting MG53 as a tumor suppressor and reinforce the therapeutic relevance of targeting the MG53\u0026ndash;AMPKα pathway.\u003c/p\u003e \u003cp\u003eCollectively, our findings support a model in which MG53 restrains bladder cancer progression by promoting ubiquitin-mediated degradation of AMPKα, thereby suppressing glycolytic metabolism and limiting the metabolic flexibility required for tumor growth. This work expands the functional repertoire of MG53 beyond its established roles in membrane repair and metabolic homeostasis, positioning it as a key regulator of cancer cell metabolism and signaling. From a translational perspective, strategies aimed at restoring MG53 expression or activity, such as gene delivery, recombinant protein administration, or small-molecule modulators, may represent promising approaches for bladder cancer therapy.\u003c/p\u003e \u003cp\u003eDespite these advances, several limitations should be considered. First, although both T24 and J82 cell lines were introduced, most functional studies were performed in T24 cells; validation across additional bladder cancer models will be important to confirm the generalizability of our findings. Second, the specific ubiquitination sites and chain topology (e.g., K48- versus K63-linked ubiquitination) of AMPKα targeted by MG53 remain undefined and warrant further investigation using site-directed mutagenesis, linkage-specific antibodies, and proteomic approaches. Third, the potential roles of MG53-regulated cell death, including pyroptosis and ferroptosis, remain to be explored. Fourth, the clinical cohort analyzed in this study was limited in size and derived from a single center; larger, multicenter studies will be required to establish the prognostic and diagnostic value of MG53 in bladder cancer. Finally, the present work is preclinical, and further studies are needed to evaluate the feasibility, safety, and efficacy of MG53-based therapeutic strategies in translational and clinical settings.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eIn summary, our study identifies MG53 as a critical tumor suppressor in bladder cancer and reveals a novel MG53\u0026ndash;AMPKα signaling axis that links ubiquitin-mediated protein degradation to metabolic reprogramming. These findings provide new mechanistic insights into bladder cancer biology and lay a foundation for developing MG53-targeted therapeutic strategies.\u003c/p\u003e "},{"header":"Declarations","content":"\u003ch2\u003eCRediT Author Contribution Statement\u003c/h2\u003e\n\u003cp\u003eXiangfei He: Conceptualization, Supervision, Funding acquisition, Writing – original draft. He Zhang: Methodology, Software, Investigation, Data curation, Visualization, Writing – original draft. Yanping Zhang: Validation. Junwei Wu: Formal analysis. Tianen Wang: Resources. Yunlong Liu: Resources. Ke Jin: Validation. Weixiang Tang: Validation. Qinghua Ye: Validation. Qingwei Wang: Conceptualization, Writing – review and editing, Project administration, Supervision.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Competing Interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eDeclaration of Generative AI and AI-Assisted Technologies in the Manuscript Preparation Process\u003c/h2\u003e\n\u003cp\u003e(I used the SCISPACE AI tool to format the layout of the paper.)\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis research was funded by the Medical Science and Technology Research Program of Henan Province, Joint Project (grant number: LHGJ20220398). The funding source had no role in the study design, data collection, analysis, interpretation, or decision to submit the article for publication.\u003c/p\u003e\n\u003ch2\u003eEthics Approval and Consent to Participate\u003c/h2\u003e\n\u003cp\u003eThis study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Ethics Committee of the First Affiliated Hospital of Zhengzhou University (Approval No.: 2022-KY-1274; approval date: 09-15-2022). Animal experiments were approved by the Institutional Animal Care and Use Committee of Zhengzhou University (Approval No.: 2022-KY-1274; approval date: 09-15-2022). Written informed consent was obtained from all human subjects involved in the study.\u003c/p\u003e\n\u003ch2\u003eData Availability Statement\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed in this study are included in this article. Further inquiries regarding the data supporting the findings of this study can be directed to the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eThe authors thank the staff of the Department of Pathology at the First Affiliated Hospital of Zhengzhou University for their assistance with tissue processing and histological analyses.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegel RL, Kratzer TB, Wagle NS, Sung H, Jemal A. Cancer statistics, 2026. CA Cancer J Clin. 2026;76:e70043. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3322/caac.21820\u003c/span\u003e\u003cspan address=\"10.3322/caac.21820\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVolanis D, Kadiyska T, Galanis A, Delakas D, Logotheti S, Zoumpourlis V. 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Nature. 2013;494:375\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nature11834\u003c/span\u003e\u003cspan address=\"10.1038/nature11834\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"bladder cancer, MG53/TRIM72, AMPKα, E3 ubiquitin ligase, Warburg effect, tumor suppressor","lastPublishedDoi":"10.21203/rs.3.rs-9590464/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9590464/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eMG53/TRIM72 is a member of the tripartite motif family of E3 ubiquitin ligases that regulates metabolic homeostasis and tumor-associated signaling; however, its role in bladder cancer remains undefined.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe investigated the expression, function, and molecular mechanism of MG53 in bladder cancer. MG53 expression was analyzed in bladder cancer tissues and cell lines. Functional assays including proliferation, invasion, and glycolytic metabolism were performed. The interaction between MG53 and AMPKα was examined using co-immunoprecipitation and ubiquitination assays. In vivo tumor growth was assessed in xenograft models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMG53 was significantly downregulated in bladder cancer tissues compared to adjacent normal tissues, and its expression was inversely correlated with AMPKα levels. Overexpression of MG53 suppressed cell proliferation, invasion, and glycolytic metabolism in bladder cancer cells. Mechanistically, MG53 promoted the ubiquitination and proteasomal degradation of AMPKα. In vivo experiments demonstrated that MG53 overexpression inhibited tumor growth in xenograft models.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur findings reveal that MG53 functions as a tumor suppressor in bladder cancer by promoting AMPKα degradation through its E3 ubiquitin ligase activity, thereby attenuating the Warburg effect. MG53 represents a potential therapeutic target for bladder cancer treatment.\u003c/p\u003e","manuscriptTitle":"MG53 Suppresses the Progression of Bladder Cancer Through E3 Ubiquitin Ligase-Mediated Degradation of AMPKα","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 13:41:12","doi":"10.21203/rs.3.rs-9590464/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-12T16:43:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-10T11:18:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"139012410008388700323675141207370283214","date":"2026-05-08T16:56:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"197081150857350157715762952774309930654","date":"2026-05-08T06:05:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2724064312216103264643211472892414365","date":"2026-05-08T02:55:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"64737304351597974473176635320268296788","date":"2026-05-08T00:51:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"319589386420782601572151256168678406663","date":"2026-05-08T00:16:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"83596496258658896092676406605296651966","date":"2026-05-07T23:35:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-07T22:55:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-05T14:33:56+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-04T14:26:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2026-05-02T03:59:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cc295851-c0c2-4f36-8d7f-b09040cbbc60","owner":[],"postedDate":"May 18th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-12T16:43:38+00:00","index":21,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-10T11:18:30+00:00","index":20,"fulltext":""},{"type":"reviewerAgreed","content":"139012410008388700323675141207370283214","date":"2026-05-08T16:56:10+00:00","index":19,"fulltext":""},{"type":"reviewerAgreed","content":"197081150857350157715762952774309930654","date":"2026-05-08T06:05:04+00:00","index":18,"fulltext":""},{"type":"reviewerAgreed","content":"2724064312216103264643211472892414365","date":"2026-05-08T02:55:48+00:00","index":17,"fulltext":""},{"type":"reviewerAgreed","content":"64737304351597974473176635320268296788","date":"2026-05-08T00:51:26+00:00","index":15,"fulltext":""},{"type":"reviewerAgreed","content":"319589386420782601572151256168678406663","date":"2026-05-08T00:16:32+00:00","index":14,"fulltext":""},{"type":"reviewerAgreed","content":"83596496258658896092676406605296651966","date":"2026-05-07T23:35:18+00:00","index":13,"fulltext":""},{"type":"reviewersInvited","content":"9","date":"2026-05-07T22:55:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-05T14:33:56+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-18T13:41:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-18 13:41:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9590464","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9590464","identity":"rs-9590464","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}