A new target: AlkBH2 promotes bladder cancer by upregulation of inflammation

A new target: AlkBH2 promotes bladder cancer by upregulation of inflammation | 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 A new target: AlkBH2 promotes bladder cancer by upregulation of inflammation Zhangjie Yang, Jinhu Ma, Ziyang Qiang, Wenhao Xie, Liang Jiao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7087922/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background : A close relationship exists between inflammation and cancer. Recent studies have highlighted inflammation as a significant contributor to the progression of bladder cancer. However, the role of alkyladenine DNA glycosylase homolog 2 (ALKBH2), an enzyme involved in DNA repair and a member of the ALKB family, in the context of bladder cancer inflammation remains largely unexplored. Methods : We evaluated ALKBH2 expression in bladder cancer tissues and adjacent normal tissues using hematoxylin and eosin (H&E) staining and immunohistochemistry. The clinical significance of ALKBH2 expression was further assessed through quantitative real-time polymerase chain reaction (Q-PCR) and Western blotting. To explore the functional implications of ALKBH2, we generated stable cell lines with overexpression and knockdown of ALKBH2. Functional assays, including Cell Counting Kit-8 (CCK-8), colony formation, Transwell migration and invasion, and wound healing assays, were conducted to assess the impact of ALKBH2 on cell proliferation, migration, and invasion. Additionally, the influence of ALKBH2 on inflammation in bladder cancer cells was investigated. Results : Our findings demonstrate that ALKBH2 promotes the proliferation, colony formation, migration, and invasion of bladder cancer cells. Mechanistically, ALKBH2 activates the nuclear factor-kappa B (NF-κB) signaling pathway, which in turn drives the progression of bladder cancer. Conclusion : These results suggest that ALKBH2 plays a critical oncogenic role in bladder cancer by modulating inflammation through the activation of the NF-κB pathway and the suppression of the NRF2/HO-1 signaling pathway. These findings highlight the potential of ALKBH2 as a therapeutic target for bladder cancer treatment. AlkBH2 bladder cancer inflammation NF-κB NRF2/HO-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction According to the World Health Organization (WHO) report from 2024, bladder cancer ranks as the 9th most common cancer globally, accounting for over 220,000 annual deaths [1]. As a malignancy of the bladder mucosa, bladder cancer is the most prevalent urinary system tumor and is among the top ten most common cancers in China [2]. It is characterized by a higher incidence in men in developed regions, and the number of new cases is increasing annually worldwide [3]. Once muscular invasion occurs, bladder cancer tends to metastasize readily, leading to a poor prognosis [4]. These findings underscore the persistent global challenge posed by bladder cancer and highlight the urgent need for additional research and preventive strategies. Consequently, identifying novel mechanisms and therapeutic targets within the intricate regulatory network of bladder cancer is of paramount importance. Previous studies have emphasized the role of chronic inflammation in various malignancies, including bladder cancer [5]. However, the relationship between inflammatory factors and the risk of bladder cancer remains incompletely understood. Observational research has yielded inconsistent results, with some studies reporting a positive correlation between elevated levels of pro-inflammatory cytokines, such as IL-1 and TNF-α, and an increased risk of bladder cancer, while others have failed to establish a significant link [6]. The pre-surgical inflammatory environment in bladder cancer patients is closely associated with post-surgical prognosis [7]. Numerous studies on the inflammatory characteristics of bladder cancer have shown that it is intrinsically linked to inflammation [8]. The tumor microenvironment consists of various stromal tissues, inflammatory cells, and inflammatory mediators, and inflammation can initiate tumorigenesis, with tumor sites eliciting an inflammatory response that accelerates tumor progression [9]. The AlkB family of dioxygenases, including AlkBH2, plays a crucial role in DNA repair [10]. Early studies in Escherichia coli demonstrated that AlkB mutants exhibited hypersensitivity to alkylating agents [11]. Humans possess nine AlkB family isoenzymes (AlkBH1-8 and FTO) [12]. AlkBH2 is a key dioxygenase responsible for repairing alkylation damage in genomic DNA, particularly in ribosomal DNA genes and double-stranded DNA [13]. Its clinical significance in human cancers is increasingly recognized [14]. While AlkBH2 has been shown to modulate chemotherapy sensitivity in non-small cell lung cancer and influence the progression of digestive system tumors (e.g., rectal and gastric cancer), its specific role and underlying mechanisms in bladder cancer remain largely unexplored [15]. To date, only one study by Fujii et al. has reported on the relationship between AlkBH2 and bladder cancer, although the underlying mechanisms remain unclear [16]. Therefore, in this study, we investigated the effects of AlkBH2 on bladder cancer cell proliferation, colony formation, migration, and invasion. Our findings indicate that AlkBH2 promotes bladder cancer cell progression by upregulating inflammation, suggesting a novel mechanism by which AlkBH2 influences bladder cancer development and offering potential therapeutic strategies. Materials and Methods Patients and tissue samples A total of 58 paired tumor and adjacent non-tumor tissue samples were obtained from patients with bladder cancer who underwent transurethral bladder tumor resection or radical cystectomy at the Affiliated Hospital of Qinghai University. All surgical specimens were histopathologically confirmed to be urothelial carcinoma by an experienced pathologist. The study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University (Study Identifier: P-SL-2023-463). Written informed consent was obtained from all participants prior to their inclusion in the study. Histological analysis Bladder tissue samples were fixed in 4% paraformaldehyde for 12 hours and sectioned at a thickness of 5 μm. Hematoxylin and eosin (H&E) staining was performed as follows: slides were deparaffinized, stained with eosin for 5 seconds, and then immersed in hematoxylin for 1 minute. High-resolution images were captured using a Leica DM500 microscope (Leica Microsystems, Solms, Germany). Immunofluorescence (IF) Following deparaffinization, tissue sections were permeabilized with 0.5% Triton X-100 for 20 minutes and blocked with goat serum for 1 hour at room temperature. Sections were then incubated with primary antibodies (AlkBH2, NF-κB, 1:200) overnight at 4°C, followed by incubation with fluorescein- or rhodamine-conjugated goat anti-mouse/rabbit IgG secondary antibodies for 1 hour. Nuclei were counterstained with DAPI for 5 minutes before examination under a microscope (TissueGnostics, Austria). Cell culture and transfection Human umbilical vein endothelial cells (HUVEC), transitional cell carcinoma (TCC) cells, and T24 cells (obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were authenticated by short tandem repeat (STR) profiling. These cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin, under conditions of 95% humidity and 5% CO2 at 37°C. For hypoxia experiments, cells were cultured in an environment with 1% O2. Lentiviral particles expressing AlkBH2 overexpression or shRNA constructs (GenePharma, China) were used to generate stable cell lines. The target sequences are provided in Table S1. Cells (3-5×10 5 ) were seeded in 6-well plates to achieve 60-70% confluency and transduced with lentivirus at a multiplicity of infection (MOI) of 10 (virus titer: 2×10 8 TU/mL). After 6 hours, the medium was replaced with fresh DMEM. Transfection efficiency was evaluated 48 hours post-transduction by fluorescence microscopy, quantifying the percentage of green fluorescent protein (GFP)-positive cells. Cell viability (CCK-8) assay Cells were seeded in 96-well plates at a density of 10,000 cells per well. Following treatment, 10 μL of CCK-8 solution (Meilunbio, Dalian, China) was added to each well containing 100 μL of medium. Absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). Each experiment was conducted in triplicate and repeated independently three times to ensure reproducibility. Cell proliferation The proportion of cells in different phases of the cell cycle was determined by flow cytometry. Cells were fixed in 70% ethanol, stained with propidium iodide (PI) and RNase A, and analyzed to determine the distribution of cells in G0/G1, S, and G2/M phases based on DNA content using a cell cycle kit (Beyotime, Shanghai, China). Migration and invasion assays For the cell migration assay, 3×10 4 cells were seeded in the upper chamber of a Transwell plate (Corning, USA), while 500 μL of culture medium supplemented with 20% FBS was added to the lower chamber as a chemoattractant. For the invasion assay, the Transwell chambers were pre-coated with Matrigel (BD, USA) before seeding the cells. Cells were incubated at 37°C for 24 or 48 hours. Cells in the upper chamber were removed, and those that had migrated or invaded were fixed with 4% paraformaldehyde, stained with crystal violet, and quantified using ImageJ software (NIH, USA). Colony Formation Assay Cells were seeded in 6-well plates at a density of 2×10 3 cells per well and incubated for one week. Visible colonies were fixed with 95% ethanol for 30 minutes, stained with 1% crystal violet for 15 minutes, and then photographed and counted. Wound-healing Assay Cells (9×10 5 ) were plated in 6-well plates and allowed to reach 100% confluence. A linear wound was created by scratching the monolayer with a 10 μL pipette tip. Cells were cultured at 37°C in 5% CO2, and images were captured after 36 hours to assess the rate of wound closure. Vascularization experiments HUVEC cells were subjected to starvation by replacing the complete culture medium with DMEM containing 0.2% FBS for 24 hours. Matrix gel (Corning, USA) was thawed on ice, mixed thoroughly by inversion, and 20 μL was added to each well of a 24-well plate. The plate was incubated at 37°C for 30 minutes to allow the basement membrane to solidify. The cells were resuspended in culture medium supplemented with 10% FBS (including conditioned medium from three groups of bladder cancer cells) to achieve a single-cell suspension at a concentration of 5×10 4 cells/mL. The 24-well plate was then incubated at 37°C with 5% CO2 and 90% humidity. Images were captured at 8 hours to observe tube formation. ELISA assays For each experimental group, six samples were independently prepared for protein extraction and concentration determination. Microplates were coated with capture antibody (5 µg/mL in PBS, 100 µL per well) and incubated overnight at 4°C. After washing three times with PBS containing 0.05% Tween-20, the plates were blocked with 200 µL of 5% BSA in PBS for 2 hours at room temperature. Standards and samples, diluted in 1% BSA/PBS, were added to the wells (100 µL per well) and incubated for 2 hours at room temperature. Following another wash, biotinylated detection antibody (X µg/mL in 1% BSA/PBS, 100 µL per well) was added and incubated for 1 hour at room temperature, followed by Streptavidin-HRP (1:5000 dilution, 100 µL per well) for 30 minutes at room temperature. After the final wash, TMB substrate (100 µL per well) was added and allowed to develop in the dark at room temperature for 15 minutes. The reaction was stopped with 1M H₂SO₄ (50 µL per well), and absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). Detailed information on the specific ELISA kits used is provided in Table S1 Q-PCR Total RNA was isolated from cell samples using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from the extracted RNA using the Goldenstar RT6 cDNA Synthesis Kit (Tsingke Biotechnology). Quantitative real-time PCR (qPCR) was performed using the Master Q-PCR Mix (Tsingke Biotechnology) on a CFX96 Touch Q-PCR Detection System (Bio-Rad). Primers were synthesized by Tsingke Biotechnology, and GAPDH was used as an internal control to normalize gene expression levels. Relative gene expression was calculated using the 2−ΔΔCT method. Primer sequences are listed in Table S2 Western Blot Tissues or cells were homogenized in protein lysis buffer using ultrasonic equipment (KeyGEN, Jiangsu, China) and incubated on ice for 10 minutes. The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. They were then incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000 dilution) for 1 hour at room temperature. Immunoreactive bands were visualized using chemiluminescent reagents (West Chester, OH, USA). Details regarding the primary antibodies used are provided in Table S3. Statistical Analysis SPSS v22.0 software was used for statistical analysis. The data were quantified as the mean ± SD. One-way analysis of variance (ANOVA) was applied in more than three groups. A t-test was used to compare the mean values of the two groups of samples. The rates were analyzed using the rank test, and p < 0.05 indicated statistical significance. Results High expression of AlkBH2 in bladder cancer Although AlkBH3 is well-documented for its high expression in urological tumors, its homolog, AlkBH2, has garnered less attention [17]. To address this gap, we measured the expression levels of AlkBH2 in bladder cancer tissues relative to para-carcinoma tissues. A total of 58 bladder cancer cases from the Affiliated Hospital of Qinghai University were confirmed by HE staining. Histopathological examination revealed that bladder cancer tissues exhibited disorganized cellular architecture, loss of polarity, and disruption of the stratified arrangement from the basal to the superficial layers. Cells displayed marked pleomorphism, characterized by variations in size and shape, enlarged nuclei, an increased nuclear-to-cytoplasmic ratio, anisokaryosis (nuclear size variation), and irregular nuclear contours. Pathological mitotic figures were also observed (Fig. 1A). IF analysis demonstrated that AlkBH2 was significantly upregulated in bladder cancer tissues (Fig. 1B, C). Furthermore, both Q-PCR and Western blot analyses confirmed that the mRNA and protein levels of AlkBH2 were significantly higher in bladder cancer tissues compared to normal tissues. These findings suggest that high AlkBH2 expression is prevalent in bladder cancer patients and may play a crucial role in the malignant progression of the disease. Models of overexpression and silencing in bladder cancer To elucidate the functional role of AlkBH2 in bladder cancer, we utilized T24 and TCC bladder cancer cell lines. Lentiviral vectors were employed to establish stable models of AlkBH2 overexpression and silencing in these cell lines. Immunofluorescence microscopy revealed that the transfection efficiency in both T24 and TCC cells exceeded 85% (Fig. 2A, B, C). To validate the success of these models, Q-PCR and Western blot analyses were performed to quantify the expression of AlkBH2 RNA and protein in the control, overexpression, and silencing groups. Compared to the control, AlkBH2 expression was significantly upregulated in the overexpression group, while it was markedly downregulated in the silencing group (Fig. 2D-I). These results confirm the successful establishment of stable models for further exploration of AlkBH2's effects. AlkBH2 enhances the proliferation of bladder cancer Building upon the successful establishment of these models, we investigated the impact of AlkBH2 on tumor proliferation in bladder cancer. CCK-8 assays demonstrated that AlkBH2 overexpression significantly increased cell viability, whereas AlkBH2 knockdown had the opposite effect in both T24 and TCC cells (Fig. 3A). Additionally, colony formation assays showed that AlkBH2 overexpression promoted colony formation, while AlkBH2 knockdown inhibited it (Fig. 3B, C). Flow cytometry analysis of the cell cycle distribution revealed that AlkBH2 overexpression increased the proportion of cells in the S and M phases, indicative of enhanced cell division, while AlkBH2 knockdown caused a significant G1 phase arrest in both T24 and TCC cells (Fig. 3D-F). Collectively, these findings indicate that AlkBH2 promotes the growth of bladder cancer cells, while its knockdown inhibits proliferation. AlkBH2 promotes migration and invasion in bladder cancer Despite the impact of AlkBH2 on tumor proliferation, the influence of AlkBH2 on other aspects of tumor heterogeneity warrants further examination. Wound-healing assays demonstrated that overexpression of AlkBH2 significantly increased the migratory capacity of bladder cancer cells, whereas knockdown of AlkBH2 markedly inhibited cell migration compared to the control group (Fig. 4A, B). To elucidate the role of AlkBH2 in angiogenesis, we performed tube formation assays, which revealed that AlkBH2 overexpression substantially accelerated the formation of capillary-like structures by endothelial cells, while AlkBH2 knockdown suppressed this process (Fig. 4C, D). Transwell assays further confirmed that overexpression of AlkBH2 enhanced the invasive potential of bladder cancer cells (Fig. 4E, F). Collectively, these findings indicate that AlkBH2 plays a crucial role in promoting proliferation, migration, and invasion in bladder cancer, suggesting its potential as a key driver in tumor progression. AlkBH2 downregulation inhibits inflammation in bladder cancer While the mechanisms underlying the role of AlkBH2 in bladder cancer development remain unclear, our hypothesis posits that AlkBH2 may modulate tumor-associated inflammation. The absence of tumor suppressor factors can inhibit DNA repair mechanisms and accelerate DNA damage, leading to an inflammatory response [18]. Given that tumor-related inflammation is a fundamental characteristic of cancer, influencing various stages of tumor progression [19], we investigated the levels of inflammatory cytokines in T24 and TCC cells. Enzyme-linked immunosorbent assays (ELISAs) showed that overexpression of AlkBH2 upregulated proinflammatory factors such as IL-1β, TNF-α, IL-12, and IL-17, indicating that AlkBH2 activates inflammation to promote cancer development, malignancy, invasion, and metastasis (Fig. 5A-D). Conversely, knockdown of AlkBH2 upregulated anti-inflammatory factors such as IL-10, IL-4, TGF-β, and IL-38, thereby suppressing tumor growth (Fig. 5E-H). These results suggest that AlkBH2 contributes to bladder cancer progression by modulating the inflammatory microenvironment. AlkbH2 upregulates inflammation in bladder cancer via activation of NF-κB and suppression of NRF2/HO-1 To further explore the mechanisms by which AlkBH2 influences inflammation in bladder cancer, we focused on the NRF2/HO-1 signaling pathway, which plays a pivotal role in anti-inflammatory and antioxidant responses [20]. Overexpression of AlkBH2 downregulated the expression of NRF2 and HO-1, while knockdown of AlkBH2 restored the activity of the NRF2/HO-1 pathway (Fig. 6A-D). Additionally, we examined the role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), a key transcription factor involved in regulating inflammation [21], oxidative stress [22], immune responses [23], and tumorigenesis [24]. In unstimulated cells, NF-κB dimers are sequestered in the cytoplasm by inhibitory proteins (IκBα, IκBβ, IκBε) [25]. Upon activation by various stimuli, IκB proteins are phosphorylated, leading to their degradation and the subsequent nuclear translocation of NF-κB [26]. Our results showed that overexpression of AlkBH2 significantly increased the phosphorylation and nuclear translocation of NF-κB, while knockdown of AlkBH2 suppressed these processes (Fig. 6E-H). Immunofluorescence staining confirmed that overexpression of AlkBH2 enhanced NF-κB nuclear localization, thereby activating its downstream inflammatory responses and promoting bladder cancer development (Fig. 6A-D). In summary, AlkBH2 facilitates bladder cancer progression by upregulating inflammation through the activation of NF-κB and the suppression of the NRF2/HO-1 pathway. Discussion Bladder cancer ranks as the third leading cause of tumor-related mortality among urological neoplasms, characterized by a high incidence and mortality rate [27]. Current treatment modalities for bladder cancer, including surgical resection, intravesical instillation therapy, and systemic chemotherapy, often face limitations due to the disease's late presentation [28]. Symptoms of bladder cancer typically remain subtle until the onset of hematuria, which significantly hinders early detection and timely intervention [29]. Consequently, there is an urgent need to identify novel therapeutic targets to improve patient outcomes. AlkBH2, a prominent member of the dioxygenase family, plays a crucial role in repairing alkylation damage within genomic DNA, particularly in ribosomal DNA genes [30]. The AlkBH protein family exhibits elevated expression levels across various human malignancies and has been implicated in tumor progression and development [31]. Among the human homologs, only AlkBH2 and AlkBH3 have demonstrated repair activities analogous to the bacterial AlkB enzyme from Escherichia coli [32]. AlkBH2 functions as a housekeeping enzyme, safeguarding the mammalian genome from 1-methyladenine (1-meA) damage by facilitating the repair of double-stranded DNA (dsDNA) [33]. Although the role of AlkBH3 in tumorigenesis has been extensively investigated, relatively little is known about AlkBH2. This study aims to elucidate the role of AlkBH2 in bladder cancer, with our findings indicating that silencing AlkBH2 effectively inhibits tumor progression by suppressing cellular proliferation, migration, and invasion. These results suggest that AlkBH2 exerts a significant influence on bladder cancer development and represents a potential therapeutic target. The relationship between inflammation and cancer has been recognized since the 19th century, with inflammation identified as a potential etiological factor [34]. Epidemiological data reveal that approximately 25% of global cancer cases are attributed to infectious diseases and chronic inflammation [35]. Emerging evidence underscores the pivotal roles of local immune responses and systemic inflammation in tumor progression and patient survival [36]. Chronic inflammation arises when the body fails to resolve acute inflammatory responses, leading to persistent inflammation that can facilitate the development of various malignancies, including colorectal, gastric, mucosa-associated lymphoid tissue (MALT), lung, bladder, and liver cancers [37]. During tumor evolution, both endogenous and exogenous sources of inflammation and stimuli are induced [38]. The deficiency of tumor suppressor factors impairs DNA repair mechanisms, exacerbating DNA damage and triggering an inflammatory cascade [39]. Tumor-associated inflammation is now regarded as a hallmark of cancer, influencing multiple stages of tumor initiation, progression, malignancy, invasion, and metastasis [40]. Inflammatory processes involve intricate interactions among immune cells, inflammatory cells, chemokines, cytokines, and pro-inflammatory mediators [41]. Inflammatory cytokines are key mediators linking inflammation and cancer, exerting their effects through multiple pathways, such as direct modulation of tumor cells [42], interaction with the chemokine network, induction of epithelial-mesenchymal transition (EMT) [43], and promotion of metastasis [44]. Dysregulated inflammation within the tumor microenvironment can directly stimulate malignant cells via cytokine-induced transformation or indirectly promote tumor growth by inducing growth factors, angiogenesis, and tissue remodeling [45]. This study provides evidence that AlkBH2 contributes to the progression of bladder cancer cells by modulating inflammatory responses. To validate this hypothesis, we measured the levels of inflammatory factors in bladder cancer. Our results showed that the expression levels of pro-inflammatory cytokines, including IL-1β, TNF-α, IL-12, and IL-17, were significantly upregulated in cells overexpressing AlkBH2, indicating that AlkBH2 activates inflammation in bladder cancer. Conversely, the levels of anti-inflammatory cytokines, such as IL-10, IL-4, TGF-β, and IL-38, were downregulated. These findings suggest that one of the mechanisms by which AlkBH2 promotes bladder cancer progression involves the activation of inflammation, potentially increasing the heterogeneity of bladder cancer. During the oxidation-reduction processes within organisms, NRF2 functions as a critical and sensitive transcription factor. Upon activation, NRF2 effectively mitigates oxidative stress, enhances cell survival, and maintains the redox balance of cells [46]. HO-1, a member of the heme oxygenase family, plays a pivotal role in anti-inflammatory [47], anti-oxidative [48], and anti-apoptotic processes [49]. Given the significant contributions of the NRF2/HO-1 signaling pathway to anti-inflammatory, anti-oxidative, anti-apoptotic, and angiogenic activities in various biological processes, it has emerged as a vital signaling pathway in inflammation responses [50]. Research indicates that the NRF2/HO-1 pathway exerts protective effects in multiple organs under diverse stress conditions. Numerous studies have highlighted the role of the NRF2/HO-1 pathway in cancer, including colorectal cancer [51], hepatocellular carcinoma [52], lung cancer [53], and ovarian cancer [54]. However, AlkBH2 has been shown to suppress the NRF2/HO-1 pathway in bladder cancer, suggesting that AlkBH2 not only upregulates inflammation but also promotes angiogenesis, an area that warrants further exploration. To elucidate the mechanisms by which AlkBH2 promotes inflammation in bladder cancer, we focused on nuclear factor kappa B (NF-κB), a family of transcription factors involved in regulating a broad spectrum of biological responses [55]. NF-κB is well-known for its role in immune responses and inflammation, but recent evidence supports its significant involvement in oncogenesis. NF-κB is a key transcriptional regulatory factor that specifically binds to κB sequences in the promoters or enhancers of various genes, thereby promoting gene transcription [56]. The NF-κB family comprises five members: RelA (P65), RelB, c-Rel, P50, and P52, which can form homodimers or heterodimers [57]. In unstimulated cells, most NF-κB dimers remain inactive by binding to one of the three IκB family members (IκBα, IκBβ, IκBε) in the cytoplasm [58]. Upon stimulation by various signaling pathways, IκBs undergo phosphorylation and degradation, leading to NF-κB activation and translocation to the nucleus, where it binds to its target DNA sequences to induce gene transcription [59]. Extensive research has demonstrated that NF-κB exhibits broad transcriptional activity and plays a crucial role in multiple stages of tumor initiation and progression [60]. Specifically, NF-κB is highly relevant to bladder cancer, and AlkBH2 has been shown to upregulate NF-κB expression. Our findings indicate that overexpression of AlkBH2 increases NF-κB phosphorylation. Additionally, AlkBH2 promotes NF-κB nuclear translocation and binding to target DNA sequences, contributing to the development of bladder cancer. Since NF-κB activation involves a multi-step signaling pathway, various compounds may target different points within this process. For instance, some anti-inflammatory drugs inhibit NF-κB by interfering with IKK activity, thereby exerting anti-inflammatory effects and influencing tumor development [61]. These drugs can break down the histological barriers of tumor cell invasion, enhance the metastatic potential of tumor cells, and participate in tumor invasion and metastasis [62]. Consequently, several studies have explored the identification of potential NF-κB inhibitors as therapeutic agents for cancer [63]. In summary, our study investigated the effects of AlkBH2 on bladder cancer cell proliferation, colony formation, migration, and invasion. We found that AlkBH2 promotes bladder cancer progression by upregulating inflammation through increased NF-κB phosphorylation and nuclear translocation. This suggests a novel mechanism by which AlkBH2 influences bladder cancer development and offers potential therapeutic strategies. Declarations Acknowledgments Thank you to the Center laboratory of the Affiliated Hospital of Qinghai University for offering a plate to complete this experiment. Funding This work was supported by the Qinghai Key Construction Project of Specialized Departments of Qinghai University Affiliated Hospital (Qinghai Weijianwei [2023]133). The funding body played no role in the design of the study and collection, analysis, and interpretation of data, and in writing the manuscript. Availability of data and materials The RNA sequencing data generated in the present study may be found in the NCBI Gene Expression Omnibus repository. The data generated in the present study may be requested from the corresponding author. Authors ’ contributions Zhangjie Yang and Guojun Chen designed and revised the review. Zhangjie Yang drafted the manuscript. Guojun Chen helped to revise the manuscript. Ziyang Qiang, Wenhao Xie and Liang Jiao helped to revise the figures. All authors read and approved the final manuscript. Ethics approval and consent to participate The present study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University (Study identifier P-SL-2023-463). Informed consent was obtained from all the patients by the ethical principles of the Declaration of Helsinki. Patient consent for publication Not applicable. Competing interests The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 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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-7087922","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483583680,"identity":"9ec4de64-0a1c-4e82-9170-83f67b918cca","order_by":0,"name":"Zhangjie Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIie3RvQrCMBDA8ROhOASzSUSwPsKVLg59mBTBqUrBFxAcXBQcK76Ej3AaqEvFVdBBEJwcMio4+DGK2Lg55Dffn5A7AMv6S8XVVWNQ53xApC9GiSNrIm771SQNF9ORUcIagulliBT5quQYBHyYAQokiZRpBQxcXqHvicg6JGPcd6tqPFdxE7zpTOY8Q11JAk+9Mq3nKmEgcZeTuJuz12eowj5FB8UcgwS3kQ/PZEIRmCXe9tQuCnwtGR9LFvl/qW9aaUHfXqc8an0JXF7LSd6J38Yty7Ksz+5NEE7GWlmqKgAAAABJRU5ErkJggg==","orcid":"","institution":"Qinghai University","correspondingAuthor":true,"prefix":"","firstName":"Zhangjie","middleName":"","lastName":"Yang","suffix":""},{"id":483583681,"identity":"b3ef4bd7-4d00-4beb-a19a-9c1af630695c","order_by":1,"name":"Jinhu Ma","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Jinhu","middleName":"","lastName":"Ma","suffix":""},{"id":483583682,"identity":"e9b6bc08-5600-400e-8da1-8fafd906a47e","order_by":2,"name":"Ziyang Qiang","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Ziyang","middleName":"","lastName":"Qiang","suffix":""},{"id":483583683,"identity":"af0ff1d0-b4f4-4489-bb51-a53a32cb6a39","order_by":3,"name":"Wenhao Xie","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Wenhao","middleName":"","lastName":"Xie","suffix":""},{"id":483583684,"identity":"db47b861-897e-40af-84a6-ebf72d050e25","order_by":4,"name":"Liang Jiao","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Jiao","suffix":""},{"id":483583685,"identity":"ef4c7021-5c7a-4f54-9fc9-1c84278faf08","order_by":5,"name":"Guojun Chen","email":"","orcid":"","institution":"Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Guojun","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-07-10 01:38:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7087922/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7087922/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86889088,"identity":"b04fbd9f-1fe1-4f1c-b060-2915c4b0b421","added_by":"auto","created_at":"2025-07-16 19:07:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":5508787,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of AlkBH2 in bladder cancer. (A) HE stained images of bladder cancer and adjacent normal tissues from patients. Scale bar, 50 μm. (B) Representative immunohistochemical images showing AlkBH2 expression in a bladder cancer tissue microarray. Scale bar, 50 μm. (C) Quantitative analysis of relative fluorescence intensity of AlkBH2, normalized to the hypoxic group (n=6). (D) Representative Western blot image of AlkBH2 in bladder cancer tissue. (E) Analysis of AlkBH2 mRNA expression levels in normal and bladder cancer tissues (n=6). (F) Analysis of AlkBH2 protein expression levels in normal and bladder cancer tissues (n=6). Data are shown as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/3e7e718f7de46d6ad6d55662.png"},{"id":86889441,"identity":"f31a6c31-3422-4fc2-8838-b3a3ab8e7d81","added_by":"auto","created_at":"2025-07-16 19:15:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2590917,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression and knockdown of AlkBH2 in bladder cancer cell lines. (A) Immunofluorescence microscopy images of T24 and TCC cells transfected with lentiviruses. Scale bar, 50 μm. (B, C) Transfection efficiency was quantified in T24 and TCC cells, respectively (n=3). (D) Representative Western blot image of AlkBH2 in T24 cells. (E, F) Analysis of AlkBH2 mRNA and protein expression levels in T24 cells, compared to the control group (n=3). (G) Representative Western blot image of AlkBH2 in TCC cells. (H, I) Analysis of AlkBH2 mRNA and protein expression levels in TCC cells, compared to the control group (n=3). Data are shown as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/e457da577407b373f7d474fc.png"},{"id":86889089,"identity":"d3baa9cc-4d95-4cbe-a87d-4df57c5a1654","added_by":"auto","created_at":"2025-07-16 19:07:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1711696,"visible":true,"origin":"","legend":"\u003cp\u003eAlkBH2 promotes the proliferation of bladder cancer cells. (A) CCK-8 assay to quantify cell proliferation (n=6). (B) Colony formation assay to assess cell proliferation in bladder cancer cells. (C) Quantification of colony numbers at low magnification (n=3). (D) Flow cytometric analysis of the cell cycle in T24 and TCC cells. (E, F) Analysis of the distribution of T24 and TCC cells in the G1, S, and G2 phases, respectively (n=3). Data are shown as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/6b3e94243c22a7a27ae3275c.png"},{"id":86889093,"identity":"82bc3d4f-91c1-47c8-b5bc-d41f568d3e79","added_by":"auto","created_at":"2025-07-16 19:07:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":7512325,"visible":true,"origin":"","legend":"\u003cp\u003eAlkBH2 enhances the migration and invasion in bladder cancer. (A) Wound healing assay to evaluate the migratory potential of cells. Scale bar, 50 μm. (B) Quantification of wound closure rates, compared to the control group (n=3). (C) Tube formation assay to assess angiogenic potential under immunofluorescence microscopy. Scale bar, 10 μm. (D) Quantification of total tube length (n=3). (E) Transwell assay to evaluate cell invasion. Scale bar, 50 μm. (F) Quantification of invading cells (n=3). Data are shown as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/f9f442b1a5288e539bd3c864.png"},{"id":86889092,"identity":"ec4f0f79-ca9b-4539-8703-8ac4fd0f26a7","added_by":"auto","created_at":"2025-07-16 19:07:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":344653,"visible":true,"origin":"","legend":"\u003cp\u003eAlkBH2 upregulates inflammatory factors in bladder cancer. (A-D) ELISA to measure pro-inflammatory cytokine levels in T24 and TCC cells (n=6). (E-H) ELISA to measure anti-inflammatory cytokine levels in T24 and TCC cells (n=6). Data are shown as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/6532e5cc43fa6bcf69847355.png"},{"id":86889091,"identity":"8d67c6df-9e0d-4d13-94be-59507ec51e31","added_by":"auto","created_at":"2025-07-16 19:07:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":959587,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism of AlkbH2-mediated inflammation upregulation. (A, B) Representative Western blot images of NRF2 and HO-1 expression in T24 and TCC cells. (C, D) Densitometric analysis of NRF2 and HO-1 expression levels in the three groups, compared to the control (n=3). (E, F) Representative Western blot images of P-P65 and P65 expression in T24 and TCC cells. (G, H) Densitometric analysis of P-P65 and P65 expression levels in the three groups, compared to the control (n=3). Data are shown as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. *** \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/ec25310a1f8db7052dd5b108.png"},{"id":86889094,"identity":"858e2c3e-e21d-4261-8179-49d440572eee","added_by":"auto","created_at":"2025-07-16 19:07:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":3280539,"visible":true,"origin":"","legend":"\u003cp\u003eAlkbH2 upregulates inflammation in bladder cancer via nuclear translocation of NF-κB. (A, B) Representative immunohistochemical images of NF-κB signaling pathway expression in bladder cancer tissue microarrays from T24 and TCC cells. (C, D) Quantification of relative fluorescence intensity of NF-κB, compared to the control (n=3). Data are shown as mean ± SD. Statistical significance was assessed using a two-tailed unpaired Student’s t-test. *** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001, ** \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, * \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/e7755abe9b304b7323ce8303.png"},{"id":93360069,"identity":"c1e515a8-4088-4512-bdf3-ca56c5819dd5","added_by":"auto","created_at":"2025-10-13 03:02:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22053769,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/8497f6ae-8c12-4db6-8efb-ff9c332f2785.pdf"},{"id":86889087,"identity":"94286578-791f-428e-a751-83edf839be5f","added_by":"auto","created_at":"2025-07-16 19:07:30","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18919,"visible":true,"origin":"","legend":"","description":"","filename":"TableS.docx","url":"https://assets-eu.researchsquare.com/files/rs-7087922/v1/6fc45a417f109106511b2e18.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"A new target: AlkBH2 promotes bladder cancer by upregulation of inflammation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAccording to the World Health Organization (WHO) report from 2024, bladder cancer ranks as the 9th most common cancer globally, accounting for over 220,000 annual deaths [1]. As a malignancy of the bladder mucosa, bladder cancer is the most prevalent urinary system tumor and is among the top ten most common cancers in China [2]. It is characterized by a higher incidence in men in developed regions, and the number of new cases is increasing annually worldwide [3]. Once muscular invasion occurs, bladder cancer tends to metastasize readily, leading to a poor prognosis [4]. These findings underscore the persistent global challenge posed by bladder cancer and highlight the urgent need for additional research and preventive strategies. Consequently, identifying novel mechanisms and therapeutic targets within the intricate regulatory network of bladder cancer is of paramount importance.\u003c/p\u003e\n\u003cp\u003ePrevious studies have emphasized the role of chronic inflammation in various malignancies, including bladder cancer [5]. However, the relationship between inflammatory factors and the risk of bladder cancer remains incompletely understood. Observational research has yielded inconsistent results, with some studies reporting a positive correlation between elevated levels of pro-inflammatory cytokines, such as IL-1 and TNF-\u0026alpha;, and an increased risk of bladder cancer, while others have failed to establish a significant link [6]. The pre-surgical inflammatory environment in bladder cancer patients is closely associated with post-surgical prognosis [7]. Numerous studies on the inflammatory characteristics of bladder cancer have shown that it is intrinsically linked to inflammation [8]. The tumor microenvironment consists of various stromal tissues, inflammatory cells, and inflammatory mediators, and inflammation can initiate tumorigenesis, with tumor sites eliciting an inflammatory response that accelerates tumor progression [9].\u003c/p\u003e\n\u003cp\u003eThe AlkB family of dioxygenases, including AlkBH2, plays a crucial role in DNA repair [10]. Early studies in \u003cem\u003eEscherichia coli\u003c/em\u003e demonstrated that AlkB mutants exhibited hypersensitivity to alkylating agents [11]. Humans possess nine AlkB family isoenzymes (AlkBH1-8 and FTO) [12]. AlkBH2 is a key dioxygenase responsible for repairing alkylation damage in genomic DNA, particularly in ribosomal DNA genes and double-stranded DNA [13]. Its clinical significance in human cancers is increasingly recognized [14]. While AlkBH2 has been shown to modulate chemotherapy sensitivity in non-small cell lung cancer and influence the progression of digestive system tumors (e.g., rectal and gastric cancer), its specific role and underlying mechanisms in bladder cancer remain largely unexplored [15]. To date, only one study by Fujii et al. has reported on the relationship between AlkBH2 and bladder cancer, although the underlying mechanisms remain unclear [16].\u003c/p\u003e\n\u003cp\u003eTherefore, in this study, we investigated the effects of AlkBH2 on bladder cancer cell proliferation, colony formation, migration, and invasion. Our findings indicate that AlkBH2 promotes bladder cancer cell progression by upregulating inflammation, suggesting a novel mechanism by which AlkBH2 influences bladder cancer development and offering potential therapeutic strategies.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003ePatients and tissue samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of 58 paired tumor and adjacent non-tumor tissue samples were obtained from patients with bladder cancer who underwent transurethral bladder tumor resection or radical cystectomy at the Affiliated Hospital of Qinghai University. All surgical specimens were histopathologically confirmed to be urothelial carcinoma by an experienced pathologist. The study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University (Study Identifier: P-SL-2023-463). Written informed consent was obtained from all participants prior to their inclusion in the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBladder tissue samples were fixed in 4% paraformaldehyde for 12 hours and sectioned at a thickness of 5 \u0026mu;m. Hematoxylin and eosin (H\u0026amp;E) staining was performed as follows: slides were deparaffinized, stained with eosin for 5 seconds, and then immersed in hematoxylin for 1 minute. High-resolution images were captured using a Leica DM500 microscope (Leica Microsystems, Solms, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence (IF) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing deparaffinization, tissue sections were permeabilized with 0.5% Triton X-100 for 20 minutes and blocked with goat serum for 1 hour at room temperature. Sections were then incubated with primary antibodies (AlkBH2, NF-\u0026kappa;B, 1:200) overnight at 4\u0026deg;C, followed by incubation with fluorescein- or rhodamine-conjugated goat anti-mouse/rabbit IgG secondary antibodies for 1 hour. Nuclei were counterstained with DAPI for 5 minutes before examination under a microscope (TissueGnostics, Austria).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman umbilical vein endothelial cells (HUVEC), transitional cell carcinoma (TCC) cells, and T24 cells (obtained from the Cell Bank of the Chinese Academy of Sciences, Shanghai, China) were authenticated by short tandem repeat (STR) profiling. These cell lines were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin, under conditions of 95% humidity and 5% CO2 at 37\u0026deg;C. For hypoxia experiments, cells were cultured in an environment with 1% O2. Lentiviral particles expressing AlkBH2 overexpression or shRNA constructs (GenePharma, China) were used to generate stable cell lines. The target sequences are provided in Table S1. Cells (3-5\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were seeded in 6-well plates to achieve 60-70% confluency and transduced with lentivirus at a multiplicity of infection (MOI) of 10 (virus titer: 2\u0026times;10\u003csup\u003e8\u003c/sup\u003e TU/mL). After 6 hours, the medium was replaced with fresh DMEM. Transfection efficiency was evaluated 48 hours post-transduction by fluorescence microscopy, quantifying the percentage of green fluorescent protein (GFP)-positive cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell viability (CCK-8) assay \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 96-well plates at a density of 10,000 cells per well. Following treatment, 10 \u0026mu;L of CCK-8 solution (Meilunbio, Dalian, China) was added to each well containing 100 \u0026mu;L of medium. Absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). Each experiment was conducted in triplicate and repeated independently three times to ensure reproducibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell proliferation \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe proportion of cells in different phases of the cell cycle was determined by flow cytometry. Cells were fixed in 70% ethanol, stained with propidium iodide (PI) and RNase A, and analyzed to determine the distribution of cells in G0/G1, S, and G2/M phases based on DNA content using a cell cycle kit (Beyotime, Shanghai, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMigration and invasion assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the cell migration assay, 3\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were seeded in the upper chamber of a Transwell plate (Corning, USA), while 500 \u0026mu;L of culture medium supplemented with 20% FBS was added to the lower chamber as a chemoattractant. For the invasion assay, the Transwell chambers were pre-coated with Matrigel (BD, USA) before seeding the cells. Cells were incubated at 37\u0026deg;C for 24 or 48 hours. Cells in the upper chamber were removed, and those that had migrated or invaded were fixed with 4% paraformaldehyde, stained with crystal violet, and quantified using ImageJ software (NIH, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eColony Formation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 6-well plates at a density of 2\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per well and incubated for one week. Visible colonies were fixed with 95% ethanol for 30 minutes, stained with 1% crystal violet for 15 minutes, and then photographed and counted.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound-healing Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells (9\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were plated in 6-well plates and allowed to reach 100% confluence. A linear wound was created by scratching the monolayer with a 10 \u0026mu;L pipette tip. Cells were cultured at 37\u0026deg;C in 5% CO2, and images were captured after 36 hours to assess the rate of wound closure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVascularization experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHUVEC cells were subjected to starvation by replacing the complete culture medium with DMEM containing 0.2% FBS for 24 hours. Matrix gel (Corning, USA) was thawed on ice, mixed thoroughly by inversion, and 20 \u0026mu;L was added to each well of a 24-well plate. The plate was incubated at 37\u0026deg;C for 30 minutes to allow the basement membrane to solidify. The cells were resuspended in culture medium supplemented with 10% FBS (including conditioned medium from three groups of bladder cancer cells) to achieve a single-cell suspension at a concentration of 5\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/mL. The 24-well plate was then incubated at 37\u0026deg;C with 5% CO2 and 90% humidity. Images were captured at 8 hours to observe tube formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor each experimental group, six samples were independently prepared for protein extraction and concentration determination. Microplates were coated with capture antibody (5 \u0026micro;g/mL in PBS, 100 \u0026micro;L per well) and incubated overnight at 4\u0026deg;C. After washing three times with PBS containing 0.05% Tween-20, the plates were blocked with 200 \u0026micro;L of 5% BSA in PBS for 2 hours at room temperature. Standards and samples, diluted in 1% BSA/PBS, were added to the wells (100 \u0026micro;L per well) and incubated for 2 hours at room temperature. Following another wash, biotinylated detection antibody (X \u0026micro;g/mL in 1% BSA/PBS, 100 \u0026micro;L per well) was added and incubated for 1 hour at room temperature, followed by Streptavidin-HRP (1:5000 dilution, 100 \u0026micro;L per well) for 30 minutes at room temperature. After the final wash, TMB substrate (100 \u0026micro;L per well) was added and allowed to develop in the dark at room temperature for 15 minutes. The reaction was stopped with 1M H₂SO₄ (50 \u0026micro;L per well), and absorbance was measured at 450 nm using a microplate reader (Thermo Scientific, Waltham, MA, USA). Detailed information on the specific ELISA kits used is provided in Table S1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQ-PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was isolated from cell samples using the GeneJET RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized from the extracted RNA using the Goldenstar RT6 cDNA Synthesis Kit (Tsingke Biotechnology). Quantitative real-time PCR (qPCR) was performed using the Master Q-PCR Mix (Tsingke Biotechnology) on a CFX96 Touch Q-PCR Detection System (Bio-Rad). Primers were synthesized by Tsingke Biotechnology, and GAPDH was used as an internal control to normalize gene expression levels. Relative gene expression was calculated using the 2\u0026minus;\u0026Delta;\u0026Delta;CT method. Primer sequences are listed in Table S2\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern Blot \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues or cells were homogenized in protein lysis buffer using ultrasonic equipment (KeyGEN, Jiangsu, China) and incubated on ice for 10 minutes. The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. They were then incubated with primary antibodies overnight at 4\u0026deg;C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000 dilution) for 1 hour at room temperature. Immunoreactive bands were visualized using chemiluminescent reagents (West Chester, OH, USA). Details regarding the primary antibodies used are provided in Table S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS v22.0 software was used for statistical analysis. The data were quantified as the mean \u0026plusmn; SD. One-way analysis of variance (ANOVA) was applied in more than three groups. A t-test was used to compare the mean values of the two groups of samples. The rates were analyzed using the rank test, and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 indicated statistical significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eHigh expression of AlkBH2 in bladder cancer \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAlthough AlkBH3 is well-documented for its high expression in urological tumors, its homolog, AlkBH2, has garnered less attention [17]. To address this gap, we measured the expression levels of AlkBH2 in bladder cancer tissues relative to para-carcinoma tissues. A total of 58 bladder cancer cases from the Affiliated Hospital of Qinghai University were confirmed by HE staining. Histopathological examination revealed that bladder cancer tissues exhibited disorganized cellular architecture, loss of polarity, and disruption of the stratified arrangement from the basal to the superficial layers. Cells displayed marked pleomorphism, characterized by variations in size and shape, enlarged nuclei, an increased nuclear-to-cytoplasmic ratio, anisokaryosis (nuclear size variation), and irregular nuclear contours. Pathological mitotic figures were also observed (Fig. 1A). IF analysis demonstrated that AlkBH2 was significantly upregulated in bladder cancer tissues (Fig. 1B, C). Furthermore, both Q-PCR and Western blot analyses confirmed that the mRNA and protein levels of AlkBH2 were significantly higher in bladder cancer tissues compared to normal tissues. These findings suggest that high AlkBH2 expression is prevalent in bladder cancer patients and may play a crucial role in the malignant progression of the disease.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eModels of overexpression and silencing in bladder cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the functional role of AlkBH2 in bladder cancer, we utilized T24 and TCC bladder cancer cell lines. Lentiviral vectors were employed to establish stable models of AlkBH2 overexpression and silencing in these cell lines. Immunofluorescence microscopy revealed that the transfection efficiency in both T24 and TCC cells exceeded 85% (Fig. 2A, B, C). To validate the success of these models, Q-PCR and Western blot analyses were performed to quantify the expression of AlkBH2 RNA and protein in the control, overexpression, and silencing groups. Compared to the control, AlkBH2 expression was significantly upregulated in the overexpression group, while it was markedly downregulated in the silencing group (Fig. 2D-I). These results confirm the successful establishment of stable models for further exploration of AlkBH2's effects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlkBH2 enhances the proliferation of bladder cancer \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBuilding upon the successful establishment of these models, we investigated the impact of AlkBH2 on tumor proliferation in bladder cancer. CCK-8 assays demonstrated that AlkBH2 overexpression significantly increased cell viability, whereas AlkBH2 knockdown had the opposite effect in both T24 and TCC cells (Fig. 3A). Additionally, colony formation assays showed that AlkBH2 overexpression promoted colony formation, while AlkBH2 knockdown inhibited it (Fig. 3B, C). Flow cytometry analysis of the cell cycle distribution revealed that AlkBH2 overexpression increased the proportion of cells in the S and M phases, indicative of enhanced cell division, while AlkBH2 knockdown caused a significant G1 phase arrest in both T24 and TCC cells (Fig. 3D-F). Collectively, these findings indicate that AlkBH2 promotes the growth of bladder cancer cells, while its knockdown inhibits proliferation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlkBH2 promotes\u003c/strong\u003e\u003cstrong\u003e migration and invasion in bladder cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDespite the impact of AlkBH2 on tumor proliferation, the influence of AlkBH2 on other aspects of tumor heterogeneity warrants further examination. Wound-healing assays demonstrated that overexpression of AlkBH2 significantly increased the migratory capacity of bladder cancer cells, whereas knockdown of AlkBH2 markedly inhibited cell migration compared to the control group (Fig. 4A, B). To elucidate the role of AlkBH2 in angiogenesis, we performed tube formation assays, which revealed that AlkBH2 overexpression substantially accelerated the formation of capillary-like structures by endothelial cells, while AlkBH2 knockdown suppressed this process (Fig. 4C, D). Transwell assays further confirmed that overexpression of AlkBH2 enhanced the invasive potential of bladder cancer cells (Fig. 4E, F). Collectively, these findings indicate that AlkBH2 plays a crucial role in promoting proliferation, migration, and invasion in bladder cancer, suggesting its potential as a key driver in tumor progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlkBH2 downregulation inhibits inflammation in bladder cancer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhile the mechanisms underlying the role of AlkBH2 in bladder cancer development remain unclear, our hypothesis posits that AlkBH2 may modulate tumor-associated inflammation. The absence of tumor suppressor factors can inhibit DNA repair mechanisms and accelerate DNA damage, leading to an inflammatory response [18]. Given that tumor-related inflammation is a fundamental characteristic of cancer, influencing various stages of tumor progression [19], we investigated the levels of inflammatory cytokines in T24 and TCC cells. Enzyme-linked immunosorbent assays (ELISAs) showed that overexpression of AlkBH2 upregulated proinflammatory factors such as IL-1\u0026beta;, TNF-\u0026alpha;, IL-12, and IL-17, indicating that AlkBH2 activates inflammation to promote cancer development, malignancy, invasion, and metastasis (Fig. 5A-D). Conversely, knockdown of AlkBH2 upregulated anti-inflammatory factors such as IL-10, IL-4, TGF-\u0026beta;, and IL-38, thereby suppressing tumor growth (Fig. 5E-H). These results suggest that AlkBH2 contributes to bladder cancer progression by modulating the inflammatory microenvironment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlkbH2 upregulates inflammation in bladder cancer via activation of NF-\u0026kappa;B and suppression of NRF2/HO-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore the mechanisms by which AlkBH2 influences inflammation in bladder cancer, we focused on the NRF2/HO-1 signaling pathway, which plays a pivotal role in anti-inflammatory and antioxidant responses [20]. Overexpression of AlkBH2 downregulated the expression of NRF2 and HO-1, while knockdown of AlkBH2 restored the activity of the NRF2/HO-1 pathway (Fig. 6A-D). Additionally, we examined the role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-\u0026kappa;B), a key transcription factor involved in regulating inflammation [21], oxidative stress [22], immune responses [23], and tumorigenesis [24]. In unstimulated cells, NF-\u0026kappa;B dimers are sequestered in the cytoplasm by inhibitory proteins (I\u0026kappa;B\u0026alpha;, I\u0026kappa;B\u0026beta;, I\u0026kappa;B\u0026epsilon;) [25]. Upon activation by various stimuli, I\u0026kappa;B proteins are phosphorylated, leading to their degradation and the subsequent nuclear translocation of NF-\u0026kappa;B [26]. Our results showed that overexpression of AlkBH2 significantly increased the phosphorylation and nuclear translocation of NF-\u0026kappa;B, while knockdown of AlkBH2 suppressed these processes (Fig. 6E-H). Immunofluorescence staining confirmed that overexpression of AlkBH2 enhanced NF-\u0026kappa;B nuclear localization, thereby activating its downstream inflammatory responses and promoting bladder cancer development (Fig. 6A-D). In summary, AlkBH2 facilitates bladder cancer progression by upregulating inflammation through the activation of NF-\u0026kappa;B and the suppression of the NRF2/HO-1 pathway.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBladder cancer ranks as the third leading cause of tumor-related mortality among urological neoplasms, characterized by a high incidence and mortality rate [27]. Current treatment modalities for bladder cancer, including surgical resection, intravesical instillation therapy, and systemic chemotherapy, often face limitations due to the disease's late presentation [28]. Symptoms of bladder cancer typically remain subtle until the onset of hematuria, which significantly hinders early detection and timely intervention [29]. Consequently, there is an urgent need to identify novel therapeutic targets to improve patient outcomes.\u003c/p\u003e\n\u003cp\u003eAlkBH2, a prominent member of the dioxygenase family, plays a crucial role in repairing alkylation damage within genomic DNA, particularly in ribosomal DNA genes [30]. The AlkBH protein family exhibits elevated expression levels across various human malignancies and has been implicated in tumor progression and development [31]. Among the human homologs, only AlkBH2 and AlkBH3 have demonstrated repair activities analogous to the bacterial AlkB enzyme from \u003cem\u003eEscherichia coli\u003c/em\u003e [32]. AlkBH2 functions as a housekeeping enzyme, safeguarding the mammalian genome from 1-methyladenine (1-meA) damage by facilitating the repair of double-stranded DNA (dsDNA) [33]. Although the role of AlkBH3 in tumorigenesis has been extensively investigated, relatively little is known about AlkBH2. This study aims to elucidate the role of AlkBH2 in bladder cancer, with our findings indicating that silencing AlkBH2 effectively inhibits tumor progression by suppressing cellular proliferation, migration, and invasion. These results suggest that AlkBH2 exerts a significant influence on bladder cancer development and represents a potential therapeutic target.\u003c/p\u003e\n\u003cp\u003eThe relationship between inflammation and cancer has been recognized since the 19th century, with inflammation identified as a potential etiological factor [34]. Epidemiological data reveal that approximately 25% of global cancer cases are attributed to infectious diseases and chronic inflammation [35]. Emerging evidence underscores the pivotal roles of local immune responses and systemic inflammation in tumor progression and patient survival [36]. Chronic inflammation arises when the body fails to resolve acute inflammatory responses, leading to persistent inflammation that can facilitate the development of various malignancies, including colorectal, gastric, mucosa-associated lymphoid tissue (MALT), lung, bladder, and liver cancers [37]. During tumor evolution, both endogenous and exogenous sources of inflammation and stimuli are induced [38]. The deficiency of tumor suppressor factors impairs DNA repair mechanisms, exacerbating DNA damage and triggering an inflammatory cascade [39]. Tumor-associated inflammation is now regarded as a hallmark of cancer, influencing multiple stages of tumor initiation, progression, malignancy, invasion, and metastasis [40]. Inflammatory processes involve intricate interactions among immune cells, inflammatory cells, chemokines, cytokines, and pro-inflammatory mediators [41]. Inflammatory cytokines are key mediators linking inflammation and cancer, exerting their effects through multiple pathways, such as direct modulation of tumor cells [42], interaction with the chemokine network, induction of epithelial-mesenchymal transition (EMT) [43], and promotion of metastasis [44]. Dysregulated inflammation within the tumor microenvironment can directly stimulate malignant cells via cytokine-induced transformation or indirectly promote tumor growth by inducing growth factors, angiogenesis, and tissue remodeling [45].\u003c/p\u003e\n\u003cp\u003eThis study provides evidence that AlkBH2 contributes to the progression of bladder cancer cells by modulating inflammatory responses. To validate this hypothesis, we measured the levels of inflammatory factors in bladder cancer. Our results showed that the expression levels of pro-inflammatory cytokines, including IL-1\u0026beta;, TNF-\u0026alpha;, IL-12, and IL-17, were significantly upregulated in cells overexpressing AlkBH2, indicating that AlkBH2 activates inflammation in bladder cancer. Conversely, the levels of anti-inflammatory cytokines, such as IL-10, IL-4, TGF-\u0026beta;, and IL-38, were downregulated. These findings suggest that one of the mechanisms by which AlkBH2 promotes bladder cancer progression involves the activation of inflammation, potentially increasing the heterogeneity of bladder cancer.\u003c/p\u003e\n\u003cp\u003eDuring the oxidation-reduction processes within organisms, NRF2 functions as a critical and sensitive transcription factor. Upon activation, NRF2 effectively mitigates oxidative stress, enhances cell survival, and maintains the redox balance of cells [46]. HO-1, a member of the heme oxygenase family, plays a pivotal role in anti-inflammatory [47], anti-oxidative [48], and anti-apoptotic processes [49]. Given the significant contributions of the NRF2/HO-1 signaling pathway to anti-inflammatory, anti-oxidative, anti-apoptotic, and angiogenic activities in various biological processes, it has emerged as a vital signaling pathway in inflammation responses [50]. Research indicates that the NRF2/HO-1 pathway exerts protective effects in multiple organs under diverse stress conditions. Numerous studies have highlighted the role of the NRF2/HO-1 pathway in cancer, including colorectal cancer [51], hepatocellular carcinoma [52], lung cancer [53], and ovarian cancer [54]. However, AlkBH2 has been shown to suppress the NRF2/HO-1 pathway in bladder cancer, suggesting that AlkBH2 not only upregulates inflammation but also promotes angiogenesis, an area that warrants further exploration.\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanisms by which AlkBH2 promotes inflammation in bladder cancer, we focused on nuclear factor kappa B (NF-\u0026kappa;B), a family of transcription factors involved in regulating a broad spectrum of biological responses [55]. NF-\u0026kappa;B is well-known for its role in immune responses and inflammation, but recent evidence supports its significant involvement in oncogenesis. NF-\u0026kappa;B is a key transcriptional regulatory factor that specifically binds to \u0026kappa;B sequences in the promoters or enhancers of various genes, thereby promoting gene transcription [56]. The NF-\u0026kappa;B family comprises five members: RelA (P65), RelB, c-Rel, P50, and P52, which can form homodimers or heterodimers [57]. In unstimulated cells, most NF-\u0026kappa;B dimers remain inactive by binding to one of the three I\u0026kappa;B family members (I\u0026kappa;B\u0026alpha;, I\u0026kappa;B\u0026beta;, I\u0026kappa;B\u0026epsilon;) in the cytoplasm [58]. Upon stimulation by various signaling pathways, I\u0026kappa;Bs undergo phosphorylation and degradation, leading to NF-\u0026kappa;B activation and translocation to the nucleus, where it binds to its target DNA sequences to induce gene transcription [59]. Extensive research has demonstrated that NF-\u0026kappa;B exhibits broad transcriptional activity and plays a crucial role in multiple stages of tumor initiation and progression [60]. Specifically, NF-\u0026kappa;B is highly relevant to bladder cancer, and AlkBH2 has been shown to upregulate NF-\u0026kappa;B expression. Our findings indicate that overexpression of AlkBH2 increases NF-\u0026kappa;B phosphorylation. Additionally, AlkBH2 promotes NF-\u0026kappa;B nuclear translocation and binding to target DNA sequences, contributing to the development of bladder cancer. Since NF-\u0026kappa;B activation involves a multi-step signaling pathway, various compounds may target different points within this process. For instance, some anti-inflammatory drugs inhibit NF-\u0026kappa;B by interfering with IKK activity, thereby exerting anti-inflammatory effects and influencing tumor development [61]. These drugs can break down the histological barriers of tumor cell invasion, enhance the metastatic potential of tumor cells, and participate in tumor invasion and metastasis [62]. Consequently, several studies have explored the identification of potential NF-\u0026kappa;B inhibitors as therapeutic agents for cancer [63].\u003c/p\u003e\n\u003cp\u003eIn summary, our study investigated the effects of AlkBH2 on bladder cancer cell proliferation, colony formation, migration, and invasion. We found that AlkBH2 promotes bladder cancer progression by upregulating inflammation through increased NF-\u0026kappa;B phosphorylation and nuclear translocation. This suggests a novel mechanism by which AlkBH2 influences bladder cancer development and offers potential therapeutic strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThank you to the Center laboratory of the Affiliated Hospital of Qinghai University for offering a plate to complete this experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Qinghai Key Construction Project of Specialized Departments of Qinghai University Affiliated Hospital (Qinghai Weijianwei [2023]133). The funding body played no role in the design of the study and collection, analysis, and interpretation of data, and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RNA sequencing data generated in the present study may be found in the NCBI Gene Expression Omnibus repository. The data generated in the present study may be requested from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZhangjie Yang and Guojun Chen designed and revised the review. Zhangjie Yang drafted the manuscript. Guojun Chen helped to revise the manuscript. Ziyang Qiang, Wenhao Xie and Liang Jiao helped to revise the figures. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study was approved by the Ethics Committee of the Affiliated Hospital of Qinghai University (Study identifier P-SL-2023-463). Informed consent was obtained from all the patients by the ethical principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eChen M, Li C, Zhang J, Cui X, Tian W, Liao P, Wang Q, Sun J, Luo L, Wu H et al. Cancer and Atrial Fibrillation Comorbidities Among 25 Million Citizens in Shanghai, China: Medical Insurance Database Study. JMIR Public Health Surveill. 2023; 9:e40149.https://doi.org/10.2196/40149.\u003c/li\u003e\n \u003cli\u003eLi D, Zuo M, Hu X. 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Life Sci. 2024; 351:122840.https://doi.org/10.1016/j.lfs.2024.122840.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"AlkBH2, bladder cancer, inflammation, NF-κB, NRF2/HO-1","lastPublishedDoi":"10.21203/rs.3.rs-7087922/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7087922/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e: A close relationship exists between inflammation and cancer. Recent studies have highlighted inflammation as a significant contributor to the progression of bladder cancer. However, the role of alkyladenine DNA glycosylase homolog 2 (ALKBH2), an enzyme involved in DNA repair and a member of the ALKB family, in the context of bladder cancer inflammation remains largely unexplored.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: We evaluated ALKBH2 expression in bladder cancer tissues and adjacent normal tissues using hematoxylin and eosin (H\u0026amp;E) staining and immunohistochemistry. The clinical significance of ALKBH2 expression was further assessed through quantitative real-time polymerase chain reaction (Q-PCR) and Western blotting. To explore the functional implications of ALKBH2, we generated stable cell lines with overexpression and knockdown of ALKBH2. Functional assays, including Cell Counting Kit-8 (CCK-8), colony formation, Transwell migration and invasion, and wound healing assays, were conducted to assess the impact of ALKBH2 on cell proliferation, migration, and invasion. Additionally, the influence of ALKBH2 on inflammation in bladder cancer cells was investigated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Our findings demonstrate that ALKBH2 promotes the proliferation, colony formation, migration, and invasion of bladder cancer cells. Mechanistically, ALKBH2 activates the nuclear factor-kappa B (NF-κB) signaling pathway, which in turn drives the progression of bladder cancer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: These results suggest that ALKBH2 plays a critical oncogenic role in bladder cancer by modulating inflammation through the activation of the NF-κB pathway and the suppression of the NRF2/HO-1 signaling pathway. These findings highlight the potential of ALKBH2 as a therapeutic target for bladder cancer treatment.\u003c/p\u003e","manuscriptTitle":"A new target: AlkBH2 promotes bladder cancer by upregulation of inflammation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-16 19:07:25","doi":"10.21203/rs.3.rs-7087922/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"51abe6e3-50c2-4b48-af8f-279e4f90cca1","owner":[],"postedDate":"July 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-13T02:53:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-16 19:07:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7087922","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7087922","identity":"rs-7087922","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}