Transcription factor SP1-Mediated Upregulation of B-Myb Promotes Glioma Aggression Through Transcriptomic Mechanism

Transcription factor SP1-Mediated Upregulation of B-Myb Promotes Glioma Aggression Through Transcriptomic Mechanism | 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 Transcription factor SP1-Mediated Upregulation of B-Myb Promotes Glioma Aggression Through Transcriptomic Mechanism Qiuming Pan, Hongrui Li, Chengyin Huang, Junxi Wang, Yudi Huang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7699276/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 Given the limited therapeutic options for gliomas, identifying molecules with therapeutic potential remains critical. This study aimed to investigate the functional significance and regulatory mechanisms of B-Myb in glioma progression. Methods B-Myb expression was assessed in glioma tissues through multimodal analyses (qRT-PCR, Western blotting, immunohistochemistry, and immunofluorescence). Prognostic relevance was evaluated in 325 glioma patients. Functional consequences of B-Myb modulation were examined via overexpression and knockdown approaches, with subsequent evaluation of proliferation, invasion, and DNA damage responses. Transcriptional regulation of B-Myb by SP1 was validated using chromatin immunoprecipitation (ChIP) and luciferase reporter assays. Results B-Myb was markedly upregulated in gliomas, with elevated expression correlating with advanced histopathological grades and reduced patient survival ( P < 0.01). Functionally, B-Myb overexpression promoted tumor cell proliferation and invasion while suppressing DNA damage responses, whereas its knockdown reversed these phenotypes. Mechanistically, SP1 activated B-Myb transcription by binding to its promoter, thereby driving glioma malignancy. Vorinostat, a histone deacetylase inhibitor, demonstrated potent antitumor effects by suppressing B-Myb expression in preclinical models. Conclusions Our findings establish B-Myb as a clinically relevant oncoprotein in gliomas, with its expression driven by SP1-mediated transcriptional activation. The therapeutic efficacy of Vorinostat via B-Myb targeting highlights its potential for clinical translation. These results position B-Myb as both a prognostic biomarker and a promising therapeutic target for glioma management. Gliomas B-Myb Transcription mechanism Biomarker Malignant progression Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Gliomas, identified as originating from glial stem cells, constitute one of the most common primary tumors in the central nervous system (CNS)(Ostrom, et al., 2014 ). Tumors of this type are typically notorious for a high incidence rate, aggressiveness, recurrence rate, and a generally unsatisfactory prognosis(Altieri, et al., 2014 ; Huang, Qiu, Mao, Hu, & Qu, 2022 ). According to the latest epidemiological data, gliomas show an annual incidence of five to six cases per 100,000 individuals in the United States, and account for up to 80% of all malignant intracranial tumors(Ostrom, et al., 2014 ; Tan, et al., 2020 ). Unfortunately, patients with gliomas have an average five-year survival rate of under 35% (Lapointe, Perry, & Butowski, 2018 ). Of all gliomas, glioblastoma (GBM) is the most aggressive and malignant subtype, accounting for 45% of all cases(Ostrom, et al., 2014 ). Despite the incorporation of surgical resection and temozolomide (TMZ) adjuvant therapy, the median overall survival (OS) for GBM patients is less than 15 months, with relative five-year survival rates remaining abysmal, at only 5%(Ostrom, et al., 2019 ). Thus, new treatment modalities are urgently needed for patients with gliomas. Targeted therapy primarily addresses gene mutant and abnormal signaling pathways underlying molecular events in tumors(Wang, et al., 2020 ), and has proven to be highly effective in treating different types of solid tumors(Cheng, Zhang, & Xu, 2021 ; Kung & Yu, 2023 ; Shariati & Meric-Bernstam, 2019 ). For instance, targeted medications such as gefitinib have been utilized to address EGFR -mutated lung cancer with an efficacy rate exceeding 70%(Cho, et al., 2022 ). In comparison to conventional chemotherapy, these targeted drugs offer markedly reduced adverse side effects and greater effectiveness. However, the clinical use of targeted therapies for gliomas remains highly constrained. Transcription factors (TFs) as novel targets for therapy and promoters of gliomas progression, which comprise around 10% of all genes in the genome and are ubiquitous throughout different organisms(Brivanlou & Darnell, 2002 ). Regulatory dysfunction of transcription presents a significant contributing factor in numerous diseases, warranting it as a feasible therapeutic target(Pink, et al., 2011 ; Poliseno, 2012 ). B-Myb belongs to the Myb transcription factor family that is highly conserved. Significantly, B-Myb directionally impacts the regulation of cell cycle and division behaviors, indicating a substantive and contributory role in the pathogenesis of cancers(Li & McDonnell, 2002 ; Oh & Reddy, 1998 ). To date, few studies have been reported which assess the relationship between B-Myb gene expression and clinicopathological features of gliomas, or its biological functionality in glioma cells. In this study, we performed a clinical analysis of 325 glioma patients in order to assess the relationship between B-Myb expression and pathological features and prognosis. Furthermore, we conducted in vitro evaluations to determine the effect of B-Myb expression on the malignancy of glioma cells. We then referenced the GDSC database to screen for potential drugs that target the B-Myb protein, thereby illuminating a potential path towards creating clinical treatment strategies for gliomas. Ultimately, we endeavored to shed light on the underlying mechanisms driving the upregulation of B-Myb expression in gliomas. Through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter experiments, we further demonstrate the binding region and interaction between them. Concisely put, B-Myb presents a prospective prognostic marker for glioma patients and therapeutic approaches focused on B-Myb may hold considerable promise in enhancing glioma treatment. Materials and methods Tissues and cell lines We collected glioma and adjacent tissues from patients who underwent glioma resections at Nanfang Glioma Center of Nanfang Hospital. All tissues were obtained with the consent of patients and were approved by the Medical Ethics Committee of Nanfang Hospital, ensuring ethical and legal compliance. The glioma cell lines under investigation, namely the U87-MG and LN229, were procured from the Precision Laboratory of Neurosurgery, Nanfang Hospital (Guangzhou, China). The culturing of all glioma cells was carried out in Dulbecco's Modified Eagle Medium (Cat.no. C3113-0500; Vivacell) supplemented with 10% fetal bovine serum (FBS; Cat.no. 04-001-1A; BI Biotech), and the cells were incubated at 37°C under CO 2 enriched conditions. Mycoplasma contamination checks were routinely conducted on all cells. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) The qRT–PCR assay was carried out using previously described methods(Garelli, et al., 2015 ). Briefly, total RNA was extracted with the TRIzol reagent (Cat.no. 15596-026; Transgene). The assay was conducted in a Lightcycler 96 (Roche), utilizing the FastStart Essential DNA Green Master dye and polymerase (Roche). Each reaction's final volume was 10 µl, comprising of 5 µl of dye and polymerase (master mix), 2 µl of a cDNA sample (diluted to approximately 1–10 ng/µl equivalence of RNA), and 3 µl of specific primer pairs. All data represent the average of three replicates. The primers used are listed in Table S1 . Western blot Total cell proteins were obtained using cell RIPA lysis buffer (Cat. no. P0013B; Beyotime) complemented with both protease and phosphatase inhibitors (Cat. no. P1264-100T; Solarbio). Equal quantities of total proteins were separated by a 10% SDS-Page gel electrophoresis and subsequently transferred to PVDF membranes. The PVDF membrane was blocked by a 5% BSA solution for 1 h before being incubated with the primary antibodies at 4℃ for an overnight period. The primary antibodies included anti-B-Myb (Cat.no. ab191064; abcam), anti-SP1 (Cat.no. ab227383; abcam), anti-FOXM1 (Cat.no. PA1429; abmart) and anti-GAPDH (Cat.no. # 5014; CST). Next day, the PVDF membranes underwent three separate washes of 10 minutes each using 1×TBST. Furthermore, the PVDF membranes were incubated with secondary antibodies for 50 min at room temperature. 1×TBST was used to wash the PVDF membrane three times. Afterward, the sample was subjected to dark surroundings, liquid removal by absorption, luminescence with Enhanced Chemiluminescent (Cat.no. P2100; NCM). Cell viability assay To determine cell viability, we utilized the Cell Counting Kit-8 (CCK-8) assay. This assay was performed utilizing a CCK-8 kit (Cat.no. C6005; New Cell & Molecular Biotech). Briefly, approximately 3,000 cells were seeded into each of the wells of 96-well plates (SORFA Life Science Research, Co., Ltd., Huzhou, China), with 5 replicate wells allocated for each group. These plates were incubated at 37℃ for a period. Subsequently, the medium from each well was replaced with 100 µL DMEM that contained 10% of CCK-8, and the cells were allowed to incubate for an additional 2 h. Finally, we determined the absorbance at 450 nm using a microplate reader (Thermo Fisher, MA, USA). Colony formation assay In the colony formation assay, approximately 500 cells were seeded into each well of 6-well plates (SORFA Life Science Research, Co., Ltd., Huzhou, China) and incubated for approximately three weeks. Throughout this duration, the cell culture medium was refreshed every three days. Following this, the colonies underwent two sequential washes with PBS before being fixed with 4% paraformaldehyde and stained with 0.4% crystal violet. After a final wash with PBS, the colonies were oven-dried and imaged using a camera. Immunohistochemistry (IHC) IHC assays were performed on glioma samples to analyze B-Myb and SP1 protein expression. In brief, the paraffin-embedded blocks were sectioned into 4-µm sections, deparaffinized, and subsequently rehydrated. To retrieve the antigens' activity, pressure cooking for 5 min in citrate buffer (pH = 6.0) was carried out, followed by blocking the endogenous peroxidase's activity using 0.3% H 2 O 2 . After blocking with 5% bovine serum albumin (BSA) for an hour, the sections were sequentially incubated with primary antibodies (anti-SP1 (Cat.no. ab227383; abcam), and anti-B-Myb (Cat.no. ab191064; abcam)) for overnight. Next day, the sections were incubated with horseradish peroxidase-linked secondary antibody (Cat.no. SAP-9100; Zhongshan Golden Bridge Biotechnology Co., Ltd). Color was developed with a DAB chromogenic solution. Nuclei were counterstained with Meyer's hematoxylin. Immunofluorescence In the case of immunofluorescence, 5x10³ cells were grown on 20 mm confocal petri dishes, and the cells were incubated for the indicated period at 37℃. Subsequently, the culture medium was removed, and cells underwent three washes with PBS. Additionally, cells were fixed for ten minutes with 4% paraformaldehyde, permeabilized using 0.5% Triton-X100 for 15 minutes, and blocked for one hour with 5% BSA. Following the removal of BSA, cells were incubated overnight with their respective primary antibody. Next day, the primary antibody was removed, and the cells were then washed with PBS. Subsequently, the cells were incubated in the dark with fluorophore-conjugated secondary antibodies (Alexa Fluor® 488) for 45 min. After incubation, the secondary antibody solution was removed, and the cells were washed thrice with PBS before staining the nuclei with DAPI (Cat.no. ab228549; abcam). The images were captured using a Carl-Zeiss confocal microscope. Tissue section immunofluorescence staining was conducted according to a previously published protocol(Qu, et al., 2023 ). The primary antibodies employed were anti-γ-H2AX (Cat.no. ab11174; abcam), anti-Ki-67 (Cat.no. #9449S; CST), and anti-B-Myb (Cat.no. ab191064; abcam). Transwell assays Transwell assays were performed in accordance with a previously described method(Mao, Huang, Hu, & Qu, 2022 ). Briefly, for cell invasion examinations, we precoated the transwell chambers (Cat.no. 140629; Thermo Fisher Scientific) with Extra Matrigel (Cat.no. BD354248; Becton-Dickinson). A total of 1×10 5 cells were loaded in the upper chamber, while DMEM containing 10% FBS was introduced to the lower chamber. After an incubation period of 24 h, migratory cells located on the lower membrane were fixed with a 4% paraformaldehyde solution for 30 min. After three washed with PBS, the cells were stained with a 0.4% crystal violet solution, then rinsed with PBS once again, and finally air-dried. Chromatin immunoprecipitation (ChIP) assay The ChIP assay was conducted following the manufacturer's guidelines for the ChIP assay kit (cat. no. P2078, Beyotime Institute of Biotechnology). Briefly, we initiated crosslinking by incubating cells in 1% formaldehyde and terminated this process using 1% glycin, before applying ultrasonication to fracture chromatin into fragments. Fragments were exposed to either anti-SP1 (Cat. no. ab227383; abcam) or normal rabbit IgG, with an incubation of 12 h at 4℃. Following this, the mixture was again incubated, this time with Protein A/G MagBeads (Thermo Fisher Scientific, Inc.) for a period of 2 h at 4°C, followed by washing with radio-immunoprecipitation assay (RIPA) buffer, lithium buffer, and Tris‐EDTA (TE) buffer. Following the cross-linking unfastening with the use of 0.2 µM NaCl at 65°C for 6 h before proceeding to ethanol precipitation of immunoprecipitated DNA. We then measured the fold enrichment relative to the IgG control via qPCR. Dual-luciferase reporter assays To clarify whether SP1 regulation of B-Myb expression involved modulation of its promoter activity, we performed a dual-luciferase reporter assay. We seeded cells at a density of 20,000 cells/well into a 12-well plate to initiate the assay. Prior to transfection, cells were cultured in 12-well culture plates until reaching a 60–80% confluency. A luciferase reporter plasmid was generated by embedding a 2 kb sequence of B-Myb promoter into the pGL3-basic vector (Promega Corporation, Madison, WI, USA). This vector expresses the firefly luciferase gene. Luciferase activity was measured as previously described(Gan & Zhang, 2009 ). Forty-eight hours after transfection, we utilized a Dual-Luciferase Activity Assay Kit (Genomeditech, Shanghai, China) to quantify luminescent values for Firefly and Renilla luciferase activities by following the manufacturer's protocol. All assays were conducted in triplicate. Molecular docking The B-Myb prediction structure was generated via state-of-the-art computational modelling utilizing Alphafold. The small molecule's protonation state was standardized to pH = 7.4, and Open Babel was employed to propagate the compound into a highly detailed 3D configuration. To prepare the receptor proteins and ligands, a series of techniques were executed using the expertly designed AutoDock tool (ADT3). Subsequent to generating the butt box using the accomplished AutoGrid program, a molecular docking simulation was executed via Autodock Vina (1.2.0). The optimal combination conformation was selected to effectively scrutinize the interaction, with due consideration to various factors. The subsequent protein-ligand interaction diagrams were meticulously produced with the aid of PyMOL. Statistical analysis For statistical analyses, we employed the use of GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA). Quantitative data between two groups were compared by performing a Student's t-test. Spearman's correlation coefficient was calculated to examine the association between the expression of two genes. We utilized the Kaplan-Meier analysis and log-rank test to examine overall survival and cumulative recurrence rate. Statistical significance was defined as P < 0.05. Results B-Myb is highly expression in glioma tissues To investigate the mRNA expression patterns of B-Myb in gliomas, we obtained the microarray datasets (GSE16011 and GSE12657) from the GEO database (www.ncbi.nlm.nih.gov/geo/). The data demonstrated a significant up-regulation of B-Myb mRNA in glioma tissues compared with normal brain tissues ( P <0.01 and P <0.05, Figure 1A). Further validation was conducted through qRT-PCR and Western blot experiments, and substantial increases in both mRNA and protein levels of B-Myb were observed in glioma tissues (Figure 1B-C). The immunohistochemical analysis also demonstrated a significant up-regulation of B-Myb protein expression in glioma tissues, and the levels increased with WHO grade (Figure 1D). Additionally, the immunofluorescence assay indicated that the expression of B-Myb was correlated with Ki-67 expression (Figure 1E). Moreover, we also assessed the differential expression of B-Myb between pan-cancer tissues and corresponding control tissues (Figure 1F), and our analysis revealed that 55.6% (10 out of 18) of cancer types exhibited abnormal B-Myb expression. These findings suggest that B-Myb expression is a probable indicator of tumorigenesis and progression, with wide-ranging potential for clinical applications. Correlation of B-Myb overexpression with adverse clinicopathologic features and an unfavorable prognosis in gliomas To gain further insight into the clinical implications of B-Myb , we collected clinical and mRNA-seq data of 325 patients diagnosed with gliomas. The clinicopathological details of the patients are outlined in Table S2. The median age of the cohort was 42 years, with an age range of 8 to 79 years. Out of the 325 patients, 203 (62.5%) were male and 122 (37.5%) were female (M: F=1.66:1). Of these cases, 103 (31.7%) were categorized as WHO grade II, 79 (24.3%) as WHO grade III, and 139 (42.8%) as WHO grade IV. Clinical follow-up records were available for these cases, ranging from 0.63 to 160.3 months. To investigate the relationship between the B-Myb mRNA levels and the clinicopathologic features of gliomas, we divided the patient cohort into two groups based on B-Myb levels using the median value: high-expression (n=163) and low-expression (n=162), as illustrated in Figure 1G. The results of the chi-square test revealed a notable correlation between B-Myb high-expression and advanced age ( P =0.006), higher WHO grade ( P <0.0001), malignant histopathology ( P <0.0001), IDH wildtype ( P <0.0001), 1p/19q non-codeletion ( P <0.0001), and chemotherapy ( P <0.0001) (Table 1). These findings point towards a potential association between the up-regulation of B-Myb and malignant progression in gliomas. We conducted an additional analysis to investigate the prognostic impact of B-Myb on patients with gliomas. The Kaplan-Meier curve revealed that B-Myb high-expression corresponded to a poor prognosis for patients (Figure 1H). The Spearman correlation analysis revealed a significant positive correlation between the expression of B-Myb and Ki-67 ( r =0.783, R 2 =0.614, P <0.0001, Figure 1I). We further performed both univariate and multivariate Cox regression analyses to identify potential prognostic biomarkers. Variables that displayed significant differences in the univariate Cox regression analysis were subjected to a stepwise multivariate Cox regression analysis. As shown in Figure 1J, the results indicated that B-Myb high-expression was an independent prognostic biomarker for glioma patients (HR=1.86, 95%CI=1.31-2.66, P <0.01). Overall, these findings suggest that B-Myb high-expression may potentially be accountable for malignant characteristics observed in gliomas. B-Myb promotes glioma cells proliferation, invasion, and suppresses DNA damage in vitro To identify the mechanistic function of B-Myb protein in glioma cells, we conducted loss- or gain-of-function experiments. The efficacy of B-Myb overexpression or knockdown was validated by Western blot (Figure 2A). The CCK-8 and clone formation were performed to determine the cell proliferation. The CCK-8 assay showed that overexpression of B-Myb promoted U87-MG and LN229 cells' proliferation in a time-dependent manner (Figure 2B). Conversely, knockdown of B-Myb demonstrated an opposite trend (Figure 2B). The clone formation assays further confirmed the effects of B-Myb on the proliferation of LN229 cells (Figure 2C). Next, to explore the potential role of B-Myb in the regulation of glioma cell invasion, we conducted transwell assays. The results showed that overexpression of B-Myb significantly enhanced the invasiveness of U87-MG and LN229 cells whereas knockdown of B-Myb displayed an inhibitory effect (Figure 2D). Together, these observations suggested that B-Myb overexpression might play a critical role in regulating malignant biological behavior of gliomas. To strengthen the evidence base, we performed an analysis to determine the relationship between B-Myb and gene sets related to proliferation and invasiveness using mRNA-sequencing data of glioma patients from TCGA. As shown in Figure 2E, the results confirmed our in vitro results. Interestingly, our analysis also revealed a significant correlation between the expression of B-Myb and the activation of DNA damage repair pathways (Figure 2E), suggesting that B-Myb knockdown might promote DNA damage. Subsequently, this result was verified by confocal laser assays. Indeed, the results demonstrated that knockdown of B-Myb significantly promoted the DNA damage of U87-MG and LN229 cells (Figure 2F), indicating that B-Myb may be a promising target for glioma treatment. The Genomics of Drug Sensitivity in Cancer (GDSC, https://www.cancerrxgene.org/) is currently recognized as the largest publicly available database of information on drug sensitivity and molecular markers of cancer cells. Using this database, our analysis revealed that Vorinostat exhibited the most significant inhibitory effect on B-Myb in glioma cells (Figure 2G). To evaluate the affinity of the candidate drugs for B-Myb, we performed molecular docking analysis. The interactions between the protein and molecular drug were comprehensively analyzed, with a high-resolution map available in Figure 2H. Functional residues were interrogated and systematically classified based on their contributions to the interactions. Multiple residue groups are utilized to foster interaction between the receptor protein and ligand, exemplified by a hydrophobic interaction involving TRP138 of B-Myb and the ligand. With these interaction forces, the binding energy of the protein-ligand complex was gauged to be -5.0 kcal/mol, attesting to a commendable degree of performance. Further CCK-8 assays confirmed a dose-dependent inhibitory effect of Vorinostat on U87 and LN229 cells (Figure 2I). The live/dead staining also showed that Vorinostat (3µM) could significantly induce glioma cells death (Figure 2J). Finally, transwell experiment also proved that Vorinostat could significantly inhibit the invasion ability of glioma cells (Figure 2K). In short, Vorinostat showed good antitumor efficacy for gliomas in vitro . SP1 regulates B-Myb expression by binding promoter sequences of target genes The underlying mechanisms of B-Myb up-regulation in gliomas remained poorly understood. To identify potential TFs, we employed the UCSC Genome Browser (https://genome-store.ucsc.edu/) and Animal TFDB3.0 databases (http://bioinfo.life.hust.edu.cn/AnimalTFDB/) to predict 27 and 31 TFs for B-Myb gene, respectively (Figure 3A). Upon further scrutiny, we discovered four shared TFs-SP1, ZNF384, KLF5, and ELF1-at the intersection (Figure 3A). To assess the relationship between these TFs and B-Myb expression, we analyzed the TCGA database and found that SP1 exhibited the strongest correlation coefficient ( r =0.415, P <0.0001). Additionally, utilizing the JASPAR database, we were able to make predictions regarding potential SP1 binding sites. In addition, the Spearman correlation analysis revealed that SP1 was associated with B-Myb mRNA levels ( r = 0.428, R 2 = 0.184, P < 0.0001; Figure 3B). Previous study had established that SP1 could bind to the promoter region of FOXM1, resulting in the positive regulation of FOXM1 transcription(Petrovic, Costa, Lau, Raychaudhuri, & Tyner, 2010). Xue Zhang et al. also reported that the AKT/FOXM1 axis is the upstream signal regulating B-Myb expression(Zhang, Lv, Huang, Zhang, & Zhou, 2017). Next, we also found that FOXM1 displayed a strong correlation with B-Myb expression in glioma tissues ( r = 0.855, R 2 = 0.732, P < 0.0001). To evaluate the potential roles of SP1 and FOXM1 genes in gliomas, we employed GEPIA (http://gepia.cancer-pku.cn/index.html) to compare their expression in glioma and normal brain tissues. Our results demonstrated a significant upregulation of both genes in glioma tissues (Figure 3C). Additionally, we performed immunohistochemical analysis and observed an elevation in SP1 protein expression in gliomas, with a positive correlation between SP1 expression and tumor grade (Figure 3D). Next, to explore the regulation effect of SP1 on B-Myb, we conducted SP1 overexpression and knockdown experiments (Figure 3E). The results of qRT-PCR and Western blot revealed that overexpression of SP1 in U87-MG and LN229 cells led to a significant increase in both mRNA and protein levels of the B-Myb gene (Figure 3G-F). Conversely, knockdown of SP1 resulted in a considerable reduction of B-Myb mRNA and protein expression. These findings indicate that SP1 may play a vital role in the regulation of B-Myb genes at the transcriptional level. To further confirm the direct involvement of SP1 in the B-Myb promoter, we conducted ChIP. As illustrated in Figure 3H, the ChIP assay demonstrated SP1's direct binding to the B-Myb promoter in vitro . To determine the biological significance of SP1 binding to the B-Myb gene promoter, we performed a luciferase reporter gene experiment. As shown in Figure 3I, the luciferase reporter gene experiment confirmed the transcriptional regulation of the B-Myb promoter by SP1. To search for the SP1 binding site on B-Myb , truncated B-Myb promoter reporter gene sequences with varying lengths were constructed, as shown in Figure 3I. Through relative luciferase activity analysis, the results showed that only a specific DNA sequence (50 and 500 bp upstream of the transcription initiation site) exhibited significantly higher B-Myb expression as compared to the control group (Figure 3I, P ˂ 0.001), indicating the significance of this region in SP1-mediated transcriptional regulation of B-Myb , which suggested that the binding site of SP1 on B-Myb was between -50 and -500 bp upstream of the transcription start site. Collectively, our results strongly suggest that B-Myb is a direct target of SP1. SP1 mediates proliferation, invasion and DNA damage of glioma cells through regulating target genes Next, we investigated the impact of SP1 on glioma cell proliferation using CCK-8 and plate clone formation assays. The results showed that overexpression of SP1 significantly enhanced glioma cell proliferation, while overexpression of SP1 coupled with B-Myb knockdown effectively reversed this effect (Figure 4A). Consistent with the CCK-8 assays, plate cloning experiments revealed that overexpression of SP1 led to a notable increase in the number of LN229 cell clones, whereas knockout of SP1 resulted in the opposite trend (Figure 4B). B-Myb was able to rescue the number of SP1-regulated cell clones (Figure 4B). The transwell assays revealed that overexpression of SP1 significantly enhanced the invasion capability of glioma cells (Figure 4C-D). Conversely, downregulation of SP1 led to a reduction in the invasion ability of glioma cells, and B-Myb was able to restore the invasion ability of glioma cells mediated by SP1. Furthermore, we sought to assess the impact of SP1 on DNA damage in glioma cells via laser confocal experiments. Our results indicated that knockdown of SP1 significantly promoted DNA damage in glioma cells (Figure 4E). Likewise, overexpression of B-Myb effectively rescued this phenotype (Figure 4E). These data suggest that SP1 might affect the progression of glioma cells through B-Myb expression. We proceeded to analyze mRNA-sequencing data from 325 glioma patients. Our analysis indicated a significant correlation between SP1 mRNA levels and advanced age, high WHO grade, and 1p/19q non-co-deletion, but not with recurrence, IDH wild-type, or MGMT promoter methylation (Figure 4F). Furthermore, the Spearman correlation analysis revealed that SP1 expression was associated with the expression of Ki-67 ( r =0.53, R 2 =0.28, P <0.0001), Vim ( r =0.38, R 2 =0.14, P <0.0001), and FOXM1 ( r =0.45, R 2 =0.21, P <0.0001), as shown in Figure 4. Kaplan-Meier survival curves revealed that patients exhibiting SP1 high-expression had worse prognosis (Figure 4H). Next, we included data from three patients, presented here with their MRI imaging, pathological indicators, B-Myb immunohistochemistry results, and prognostic data (Figure 4I). Tumor size, degree of peritumoral edema and the Ki-67 proliferation index demonstrated a positive correlation with B-Myb protein expression, indicating a poor prognosis for glioma patients. This degree of peritumoral edema is somewhat reflective of the overall invasion(Cheon, et al., 2017). Discussion At present, treatment options for gliomas are extremely limited and patients continue to face a poor overall prognosis(Ene & Holland, 2015 ). Therefore, there is a need for effective strategies to comprehensively address the disease and improve patients’ outcomes. Targeted therapy, as an effective strategy for the comprehensive treatment of tumor, may be a promising approach to alleviate symptoms and extend survival for individuals afflicted by gliomas(Mehta, Abi Nader, Waddington, & David, 2011). In our work, high expression of B-Myb is associated with malignant clinicopathological features and poor prognosis in gliomas from a clinical perspective. Functionally, B-Myb high-expression is involved in regulating DNA damage response in addition to proliferation and invasion of glioma cells. Mechanically, we discovered a new mechanism that SP1 interacts with the B-Myb promoter and facilitates B-Myb transcription in gliomas. Therapeutically, Vorinostat was found to inhibit tumor cell proliferation, invasion and induction of cell death by inhibiting B-Myb. TFs constitute critical proteins that regulate gene expression via initiation or inhibition of gene transcription, accomplished by effector binding to either enhancers or promoters of target genes(Debnath, Huirem, Dutta, & Palchaudhuri, 2022 ; Mao, et al., 2022 ). MYB TFs exhibit remarkable conservation across a wide range of species, spanning from plants to vertebrates, signifying that their functions represent fundamental mechanisms in the biology of both cells and organisms (Dubos, et al., 2010 ). The human MYB gene family comprises of three members: MYB, MYBL1 and MYBL2. These genes encode for MYB, MYBL1, and MYBL2 TFs, which are alternatively referred to as c-MYB, A-MYB, and B-MYB, correspondingly. In MCF-7 breast cancer cells, c-MYB has been observed to bind to over 10,000 promoters, thereby being identified as a crucial stimulant of downstream targets. These targets encompass genes that are associated with cancer metastasis and progression, such as BCL2, BCLXL, JUN, CXCR4, MYC, NANOG, KLF4, and COX-2(Quintana, Liu, O'Rourke, & Ness, 2011 ). B-Myb and C-Myb belong to the same family and exhibit functional similarities. The MYBL2 gene was initially discovered as cellular homologs of the v-myb oncogene, which is recognized for triggering leukemia in chickens(Oh & Reddy, 1999 ). The expression of MYBL2 in proliferative cells is critical for governing cellular proliferation and differentiation, while also playing a pivotal role in directing the progression of the cell cycle (Fischer, Quaas, Steiner, & Engeland, 2016 ). B-Myb exerts profound influences on cell behavior, as its plethora of protein partners and target genes permit it to govern multiple crucial cellular processes, including proliferation, senescence, apoptosis, and mitosis(Musa, Aynaud, Mirabeau, Delattre, & Grünewald, 2017 ). Considering its wide-ranging functions and correlation with human cancer, it may be viable to utilize B-Myb as a significant diagnostic, prognostic and therapeutic instrument. A study conducted by Xiaoyan Fan and colleagues revealed that overexpression of B-Myb in colorectal cancer cells accelerates cell proliferation, cell cycle progression, and cell motility (Fan, et al., 2021 ). Furthermore, in vivo investigation utilizing orthotopic nude mouse models demonstrated that B-Myb overexpression promotes tumor growth. The functional roles of B-Myb in the above are similar to our findings in gliomas. It is unclear why B-Myb expression was increased in our study. Our investigation on gliomas has revealed a novel mechanism through which SP1 facilitates oncogenic transcription of B-Myb. Traditionally, the transcription factor SP1 was regarded as a fundamental transcriptional regulator and was assigned a secondary role in the regulation of what are known as housekeeping genes (Beishline & Azizkhan-Clifford, 2015 ; Chiefari, et al., 2002 ). The overexpression of SP1 is frequently observed in several types of cancers, and it is positively correlated with a poor prognosis. In a publication authored by Fuzhen Qi et al., it was observed that SP1 mediated upregulation of ASAP2-AS1 functions as a crucial oncogenic driver in the development and progression of gastric cancer, by impeding P21 and E-cadherin (Qi, et al., 2017 ). Vladimir Petrovic et al. reported that the transcription factor SP1 binds to the promoter region of FOXM1 and subsequently augments FOXM1 transcription (Petrovic, et al., 2010 ). The results of our experimentation further validated the affirmative regulation of the transcription factor SP1 on the production of FOXM1 protein. Xue Zhang et al. reported that MYBL2 is a key downstream factor of Akt/FOXM1 signaling to promote progression of gliomas (Zhang, et al., 2017 ). This implies that SP1 may operate diverse pathways to modulate B-Myb expression in glioma cells. Consequently, B-Myb emerges as a noteworthy downstream target implicated in the onset and evolution of gliomas, while potentially displaying minimal adverse effects. Collectively, our study has established that B-Myb expression is upregulated in glioma tissues, and its overexpression is strongly associated with malignant pathological characteristics and unfavorable prognosis in glioma patients. Moreover, our investigations have demonstrated that B-Myb overexpression significantly promotes glioma cell proliferation and invasion, while also suppressing DNA damage response. Mechanistically, SP1 may exert its pro-tumorigenic effects by directly binding to the B-Myb promoter, thereby activating its transcription and facilitating gliomas development. Therapeutically, Vorinostat was found to exhibit significant anti-tumor efficacy for gliomas. In light of our new insights, B-Myb may hold promise as both a novel prognostic biomarker and promising therapeutic target for gliomas. Declarations Author contribution Qiuming Pan and Hongrui Li: Conceptualization, Methodology, Investigation, Formal analysis, Writing - Original Draft; Junxi Wang, Yudi Huang and Huafu Wang: Data Curation, Visualization, Writing - Review & Editing; Guozhong Yi and Zhiyong Li: Investigation, Resources, Software; Rongyang Xu and Ye Zhu: Methodology, Validation, In vitro experiments; Luyao Wang and Yuou Qin: Bioinformatics analysis, Statistical validation, Clinical sample collection; Guanglong Huang and Songtao Qi: Funding acquisition, Project administration, Data Interpretation; Shanqiang Qu: Conceptualization, Supervision, Writing - Review & Editing. All authors have reviewed and approved the final manuscript. Funding This work was supported by President Foundation of Nanfang Hospital, Southern Medical University (2022A018), Funding by Science and Technology Projects in Guangzhou (2024A04J5111), GuangDong Basic and Applied Basic Research Foundation (2023A15111129) and the Basic Public Welfare Research Program of Zhejiang Province (LGF22H160052). Ethical approval statement All tissues were obtained with the consent of patients and were approved by the Medical Ethics Committee of Nanfang Hospital, ensuring ethical and legal compliance. Human Ethics and Consent to Participate declarations: not applicable. Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. Disclosure statement The authors declare no conflict of interests. References Altieri, R., Agnoletti, A., Quattrucci, F., Garbossa, D., Calamo Specchia, F.M., Bozzaro, M., et al. (2014). Molecular biology of gliomas: present and future challenges. 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Adv Sci (Weinh), 7 (24), 2003036, doi:10.1002/advs.202003036. Wang, L., Qin, W., Huo, Y.J., Li, X., Shi, Q., Rasko, J.E.J., et al. (2020). Advances in targeted therapy for malignant lymphoma. Signal Transduct Target Ther, 5 (1), 15, doi:10.1038/s41392-020-0113-2. Zhang, X., Lv, Q.L., Huang, Y.T., Zhang, L.H., & Zhou, H.H. (2017). Akt/FoxM1 signaling pathway-mediated upregulation of MYBL2 promotes progression of human glioma. J Exp Clin Cancer Res, 36 (1), 105, doi:10.1186/s13046-017-0573-6. Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.docx Table S1.List of primers used in this study. Sequences of forward and reverse primers used for the amplification of GAPDH and B-Myb transcripts by reverse transcription-PCR. Table S2.Clinicopathological characteristics of patients. The table summarizes the distribution of key demographics, tumor pathology, molecular markers, and treatment history for all patients included in the analysis. NA, not appliable; A, astrocytoma; AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; GBM, glioblastoma; O, oligodendroglioma. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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09:42:17","extension":"xml","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":131851,"visible":true,"origin":"","legend":"","description":"","filename":"aa37549b81cb43ff8015ecfd8efd063c1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/e7d29e987875731f3ede85f2.xml"},{"id":93027684,"identity":"af382ce0-9869-433b-8bc3-391c6b7b6928","added_by":"auto","created_at":"2025-10-08 09:42:17","extension":"html","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":144300,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/9f0f72fc179db091ec5be911.html"},{"id":93026756,"identity":"eadce54b-1196-43d6-b301-f4107c2f0dac","added_by":"auto","created_at":"2025-10-08 09:34:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2909751,"visible":true,"origin":"","legend":"\u003cp\u003eUpregulation of B-Myb is observed in glioma and has been significantly correlated with unfavorable prognostic outcomes in patients.\u003c/p\u003e\n\u003cp\u003e(A) The expression profile of B-Myb exhibits significant differences between glioma and control brain tissues, as evidenced by analysis of the GEO datasets (GSE16011 and GSE12657). (B-C) The differential expression of B-Myb was verified by qRT-PCR and Western blotting analysis of paired glioma samples. (D) Immunohistochemical expression of B-Myb in glioma tissues. Scale bar, 50µm. (E)Immunofluorescence for Ki-67 and B-Myb expression in paired glioma tissues. Scale bar, 50µm. (F) The expression profile of B-Myb between tumor and control tissues from a pan-cancer perspective. (G) The 325 glioma patients were divided into two groups on the basis of high (n=163) and low (n=162) B-Myb expression values. (H) Results of B-Myb in the prognosis of gliomas in Kaplan-Meier survival curves. (I) Spearman correlation analysis between B-Myb and Ki-67 (MKI67) mRNA levels in 325 glioma patients. (J) Investigating the potential prognostic markers, a univariate and multivariate Cox regression analysis was carried out in a cohort of 325 glioma patients.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/5d77ba9d66d717dc73fdf5de.png"},{"id":93026761,"identity":"d28e4eda-bf48-40d9-bfc6-f76e8f0fc734","added_by":"auto","created_at":"2025-10-08 09:34:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6081358,"visible":true,"origin":"","legend":"\u003cp\u003eB-Myb protein modulates the proliferation, invasion, and DNA damage response of glioma cells \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Western blot validated the B-Myb overexpression and interference efficiency. (B) The impact of B-Myb overexpression or knockdown on cell proliferation was assessed using the CCK-8 assay at 0, 12, 24, 48 and 72 h, respectively. (C) Plate clone formation assay was conducted to investigate the effects of B-Myb on the clonogenic ability of LN229 cells. (D) Transwell assays were performed to examine the effects of B-Myb on the invasion capacity of U87-MG and LN229 cells. Scale bar, 50µm. (E) The heatmaps was employed to represent the associations between B-Myb mRNA levels and diverse gene-sets in the TCGA glioma dataset. (F）Confocal microscopy was utilized to observe changes in DNA damage response signaling subsequent to B-Myb knockdown. (G）Drug sensitivity analysis of ARPs in GBM was conducted using the GDSC drug sensitivity database. The Spearman correlation coefficient was employed to determine the relationship between gene expression and small molecule drug response. A positive correlation signifies that high gene expression correlates with drug resistance, whereas a negative correlation reflects the opposite. (H) Molecular docking between Vorinostat and core targets protein. The B-Myb protein is represented as a slate cartoon model, Vorinostat is shown as a cyan stick, and their binding sites are shown as magentas stick structures. Nonpolar hydrogen atoms are omitted. The hydrogen bond, ionic interactions, and hydrophobic interactions are depicted as yellow, magentas and green dashed lines, respectively. Source data are provided as Source Data file. (I-J) The in vitro cytotoxicity of Vorinostat against U87-MG and LN229 cells was assessed using the CCK-8 assay and Live/Dead cell staining technique. Scale bar, 50µm. (K) The impact of Vorinostat on invasion suppressing activity against U87-MG and LN229 cells was evaluated using the Transwell assay. Scale bar, 50µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/b8244637f851e0a2665d22c3.png"},{"id":93027680,"identity":"53aec34b-e7be-4f2a-a40f-361466fb615c","added_by":"auto","created_at":"2025-10-08 09:42:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3068747,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eB-Myb\u003c/em\u003e is a direct transcriptional target of SP1.\u003c/p\u003e\n\u003cp\u003e(A)The identification of potential transcription factors for the \u003cem\u003eB-Myb\u003c/em\u003e promoter was performed using the UCSC and AnimalTFDB 3.0 databases. Three predicted binding sites were identified using the JASPAR database. (B) The expression correlation was assessed in a cohort of 325 glioma patients using the Spearman correlation analysis. (C) The correlation between B-Myb expression and the expression of SP1 and FOXM1 in glioma was assessed using the GEPIA database. (D) Immunohistochemical expression of SP1 in glioma tissues. Scale bar, 50µm. (E) Western blot validated the SP1 overexpression and interference efficiency. (F) The impact of SP1 on the expression of B-Myb and FOXM1 proteins in glioma cells was assessed using Western blot analysis in vitro. (G) The regulatory influence of SP1 on B-Myb in glioma cells was assessed using qRT-PCR. (H) Left: Sketch view of ChIP assay. Right: The determination of SP1 binding on B-Myb promoter using ChIP assay with anti-SP1 antibody, followed by qRT-PCR analysis. (I) Up: Schematic of luciferase reporter constructs. Bottom: The luciferase reporter assay in glioma cells. The promoter was truncated and luciferase reporter gene experiment was conducted\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/e6a3e5a60eaf8112b1a6b9e7.png"},{"id":93026766,"identity":"9e52fb3f-9ea9-45aa-9d06-f8a1cfe195a0","added_by":"auto","created_at":"2025-10-08 09:34:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6720807,"visible":true,"origin":"","legend":"\u003cp\u003eB-Myb is indispensable for SP1 regulation of proliferation, invasion and DNA damage response in vitro.\u003c/p\u003e\n\u003cp\u003e(A)The CCK-8 assay showed that SP1 promoted the proliferation of glioma cells, which could be rescue by B-Myb knockdown \u003cem\u003ein vitro\u003c/em\u003e.(B) The plate clone formation assay showed that SP1 promoted the proliferation of glioma cells, which could be rescued by B-Myb knockdown \u003cem\u003ein vitro\u003c/em\u003e. (C-D) Transwell assay showed that the invasion ability of glioma cells was influenced by SP1, which could be rescued by B-Myb knockdown. Scale bar, 50µm.\u003c/p\u003e\n\u003cp\u003e(E) Knockdown of SP1 promotes DNA damage response, which could be rescued by B-Myb overexpression in U87-MG and LN229 cell lines. Scale bar, 10µm. (F) The association between SP1 expression and clinicopathologic features in 325 glioma patients. (G) Spearman correlation analysis between SP1 expression and Ki-67, Vim, FOXM1 expression in 325 glioma patients. (H) The impact of SP1 expression on prognosis of glioma patients. (I) We elucidate a comprehensive set of multimodal data comprising MRI imaging, pathological information, Ki-67 proliferation index, B-Myb protein expression data through immunohistochemical analysis, and crucial prognostic data of glioma patients. Scale bar, 100µm.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/b7b35dcc9824a8450c684dc3.png"},{"id":109356689,"identity":"3772dc66-0893-46c4-ac5f-41e7bef89628","added_by":"auto","created_at":"2026-05-16 05:55:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18029118,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/fe2910ba-4d3e-4ada-a264-35470e852f3c.pdf"},{"id":93026757,"identity":"92f4f7f5-7503-4fac-9341-012eb5e55389","added_by":"auto","created_at":"2025-10-08 09:34:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22057,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003eList of primers used in this study.\u003c/p\u003e\n\u003cp\u003eSequences of forward and reverse primers used for the amplification of GAPDH and B-Myb transcripts by reverse transcription-PCR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003eClinicopathological characteristics of patients.\u003c/p\u003e\n\u003cp\u003eThe table summarizes the distribution of key demographics, tumor pathology, molecular markers, and treatment history for all patients included in the analysis. NA, not appliable; A, astrocytoma; AA, anaplastic astrocytoma; AO, anaplastic oligodendroglioma; GBM, glioblastoma; O, oligodendroglioma.\u003c/p\u003e","description":"","filename":"Supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-7699276/v1/ebf254a2a806ddc4907a7bff.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcription factor SP1-Mediated Upregulation of B-Myb Promotes Glioma Aggression Through Transcriptomic Mechanism","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGliomas, identified as originating from glial stem cells, constitute one of the most common primary tumors in the central nervous system (CNS)(Ostrom, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Tumors of this type are typically notorious for a high incidence rate, aggressiveness, recurrence rate, and a generally unsatisfactory prognosis(Altieri, et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Huang, Qiu, Mao, Hu, \u0026amp; Qu, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). According to the latest epidemiological data, gliomas show an annual incidence of five to six cases per 100,000 individuals in the United States, and account for up to 80% of all malignant intracranial tumors(Ostrom, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tan, et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Unfortunately, patients with gliomas have an average five-year survival rate of under 35% (Lapointe, Perry, \u0026amp; Butowski, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Of all gliomas, glioblastoma (GBM) is the most aggressive and malignant subtype, accounting for 45% of all cases(Ostrom, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite the incorporation of surgical resection and temozolomide (TMZ) adjuvant therapy, the median overall survival (OS) for GBM patients is less than 15 months, with relative five-year survival rates remaining abysmal, at only 5%(Ostrom, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thus, new treatment modalities are urgently needed for patients with gliomas.\u003c/p\u003e\u003cp\u003eTargeted therapy primarily addresses gene mutant and abnormal signaling pathways underlying molecular events in tumors(Wang, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and has proven to be highly effective in treating different types of solid tumors(Cheng, Zhang, \u0026amp; Xu, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kung \u0026amp; Yu, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shariati \u0026amp; Meric-Bernstam, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For instance, targeted medications such as gefitinib have been utilized to address \u003cem\u003eEGFR\u003c/em\u003e-mutated lung cancer with an efficacy rate exceeding 70%(Cho, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In comparison to conventional chemotherapy, these targeted drugs offer markedly reduced adverse side effects and greater effectiveness. However, the clinical use of targeted therapies for gliomas remains highly constrained. Transcription factors (TFs) as novel targets for therapy and promoters of gliomas progression, which comprise around 10% of all genes in the genome and are ubiquitous throughout different organisms(Brivanlou \u0026amp; Darnell, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Regulatory dysfunction of transcription presents a significant contributing factor in numerous diseases, warranting it as a feasible therapeutic target(Pink, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Poliseno, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). B-Myb belongs to the Myb transcription factor family that is highly conserved. Significantly, B-Myb directionally impacts the regulation of cell cycle and division behaviors, indicating a substantive and contributory role in the pathogenesis of cancers(Li \u0026amp; McDonnell, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Oh \u0026amp; Reddy, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). To date, few studies have been reported which assess the relationship between \u003cem\u003eB-Myb\u003c/em\u003e gene expression and clinicopathological features of gliomas, or its biological functionality in glioma cells.\u003c/p\u003e\u003cp\u003eIn this study, we performed a clinical analysis of 325 glioma patients in order to assess the relationship between \u003cem\u003eB-Myb\u003c/em\u003e expression and pathological features and prognosis. Furthermore, we conducted \u003cem\u003ein vitro\u003c/em\u003e evaluations to determine the effect of B-Myb expression on the malignancy of glioma cells. We then referenced the GDSC database to screen for potential drugs that target the B-Myb protein, thereby illuminating a potential path towards creating clinical treatment strategies for gliomas. Ultimately, we endeavored to shed light on the underlying mechanisms driving the upregulation of B-Myb expression in gliomas. Through chromatin immunoprecipitation (ChIP) and dual-luciferase reporter experiments, we further demonstrate the binding region and interaction between them. Concisely put, \u003cem\u003eB-Myb\u003c/em\u003e presents a prospective prognostic marker for glioma patients and therapeutic approaches focused on B-Myb may hold considerable promise in enhancing glioma treatment.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eTissues and cell lines\u003c/h2\u003e\u003cp\u003eWe collected glioma and adjacent tissues from patients who underwent glioma resections at Nanfang Glioma Center of Nanfang Hospital. All tissues were obtained with the consent of patients and were approved by the Medical Ethics Committee of Nanfang Hospital, ensuring ethical and legal compliance. The glioma cell lines under investigation, namely the U87-MG and LN229, were procured from the Precision Laboratory of Neurosurgery, Nanfang Hospital (Guangzhou, China). The culturing of all glioma cells was carried out in Dulbecco's Modified Eagle Medium (Cat.no. C3113-0500; Vivacell) supplemented with 10% fetal bovine serum (FBS; Cat.no. 04-001-1A; BI Biotech), and the cells were incubated at 37\u0026deg;C under CO\u003csub\u003e2\u003c/sub\u003e enriched conditions. Mycoplasma contamination checks were routinely conducted on all cells.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eQuantitative reverse-transcription polymerase chain reaction (qRT-PCR)\u003c/h3\u003e\n\u003cp\u003eThe qRT\u0026ndash;PCR assay was carried out using previously described methods(Garelli, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Briefly, total RNA was extracted with the TRIzol reagent (Cat.no. 15596-026; Transgene). The assay was conducted in a Lightcycler 96 (Roche), utilizing the FastStart Essential DNA Green Master dye and polymerase (Roche). Each reaction's final volume was 10 \u0026micro;l, comprising of 5 \u0026micro;l of dye and polymerase (master mix), 2 \u0026micro;l of a cDNA sample (diluted to approximately 1\u0026ndash;10 ng/\u0026micro;l equivalence of RNA), and 3 \u0026micro;l of specific primer pairs. All data represent the average of three replicates. The primers used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eWestern blot\u003c/h3\u003e\n\u003cp\u003eTotal cell proteins were obtained using cell RIPA lysis buffer (Cat. no. P0013B; Beyotime) complemented with both protease and phosphatase inhibitors (Cat. no. P1264-100T; Solarbio). Equal quantities of total proteins were separated by a 10% SDS-Page gel electrophoresis and subsequently transferred to PVDF membranes. The PVDF membrane was blocked by a 5% BSA solution for 1 h before being incubated with the primary antibodies at 4℃ for an overnight period. The primary antibodies included anti-B-Myb (Cat.no. ab191064; abcam), anti-SP1 (Cat.no. ab227383; abcam), anti-FOXM1 (Cat.no. PA1429; abmart) and anti-GAPDH (Cat.no. # 5014; CST). Next day, the PVDF membranes underwent three separate washes of 10 minutes each using 1\u0026times;TBST. Furthermore, the PVDF membranes were incubated with secondary antibodies for 50 min at room temperature. 1\u0026times;TBST was used to wash the PVDF membrane three times. Afterward, the sample was subjected to dark surroundings, liquid removal by absorption, luminescence with Enhanced Chemiluminescent (Cat.no. P2100; NCM).\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eTo determine cell viability, we utilized the Cell Counting Kit-8 (CCK-8) assay. This assay was performed utilizing a CCK-8 kit (Cat.no. C6005; New Cell \u0026amp; Molecular Biotech). Briefly, approximately 3,000 cells were seeded into each of the wells of 96-well plates (SORFA Life Science Research, Co., Ltd., Huzhou, China), with 5 replicate wells allocated for each group. These plates were incubated at 37℃ for a period. Subsequently, the medium from each well was replaced with 100 \u0026micro;L DMEM that contained 10% of CCK-8, and the cells were allowed to incubate for an additional 2 h. Finally, we determined the absorbance at 450 nm using a microplate reader (Thermo Fisher, MA, USA).\u003c/p\u003e\n\u003ch3\u003eColony formation assay\u003c/h3\u003e\n\u003cp\u003eIn the colony formation assay, approximately 500 cells were seeded into each well of 6-well plates (SORFA Life Science Research, Co., Ltd., Huzhou, China) and incubated for approximately three weeks. Throughout this duration, the cell culture medium was refreshed every three days. Following this, the colonies underwent two sequential washes with PBS before being fixed with 4% paraformaldehyde and stained with 0.4% crystal violet. After a final wash with PBS, the colonies were oven-dried and imaged using a camera.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eIHC assays were performed on glioma samples to analyze B-Myb and SP1 protein expression. In brief, the paraffin-embedded blocks were sectioned into 4-\u0026micro;m sections, deparaffinized, and subsequently rehydrated. To retrieve the antigens' activity, pressure cooking for 5 min in citrate buffer (pH\u0026thinsp;=\u0026thinsp;6.0) was carried out, followed by blocking the endogenous peroxidase's activity using 0.3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. After blocking with 5% bovine serum albumin (BSA) for an hour, the sections were sequentially incubated with primary antibodies (anti-SP1 (Cat.no. ab227383; abcam), and anti-B-Myb (Cat.no. ab191064; abcam)) for overnight. Next day, the sections were incubated with horseradish peroxidase-linked secondary antibody (Cat.no. SAP-9100; Zhongshan Golden Bridge Biotechnology Co., Ltd). Color was developed with a DAB chromogenic solution. Nuclei were counterstained with Meyer's hematoxylin.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eIn the case of immunofluorescence, 5x10\u0026sup3; cells were grown on 20 mm confocal petri dishes, and the cells were incubated for the indicated period at 37℃. Subsequently, the culture medium was removed, and cells underwent three washes with PBS. Additionally, cells were fixed for ten minutes with 4% paraformaldehyde, permeabilized using 0.5% Triton-X100 for 15 minutes, and blocked for one hour with 5% BSA. Following the removal of BSA, cells were incubated overnight with their respective primary antibody. Next day, the primary antibody was removed, and the cells were then washed with PBS. Subsequently, the cells were incubated in the dark with fluorophore-conjugated secondary antibodies (Alexa Fluor\u0026reg; 488) for 45 min. After incubation, the secondary antibody solution was removed, and the cells were washed thrice with PBS before staining the nuclei with DAPI (Cat.no. ab228549; abcam). The images were captured using a Carl-Zeiss confocal microscope. Tissue section immunofluorescence staining was conducted according to a previously published protocol(Qu, et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The primary antibodies employed were anti-γ-H2AX (Cat.no. ab11174; abcam), anti-Ki-67 (Cat.no. #9449S; CST), and anti-B-Myb (Cat.no. ab191064; abcam).\u003c/p\u003e\n\u003ch3\u003eTranswell assays\u003c/h3\u003e\n\u003cp\u003eTranswell assays were performed in accordance with a previously described method(Mao, Huang, Hu, \u0026amp; Qu, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Briefly, for cell invasion examinations, we precoated the transwell chambers (Cat.no. 140629; Thermo Fisher Scientific) with Extra Matrigel (Cat.no. BD354248; Becton-Dickinson). A total of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were loaded in the upper chamber, while DMEM containing 10% FBS was introduced to the lower chamber. After an incubation period of 24 h, migratory cells located on the lower membrane were fixed with a 4% paraformaldehyde solution for 30 min. After three washed with PBS, the cells were stained with a 0.4% crystal violet solution, then rinsed with PBS once again, and finally air-dried.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eChromatin immunoprecipitation (ChIP) assay\u003c/h2\u003e\u003cp\u003e The ChIP assay was conducted following the manufacturer's guidelines for the ChIP assay kit (cat. no. P2078, Beyotime Institute of Biotechnology). Briefly, we initiated crosslinking by incubating cells in 1% formaldehyde and terminated this process using 1% glycin, before applying ultrasonication to fracture chromatin into fragments. Fragments were exposed to either anti-SP1 (Cat. no. ab227383; abcam) or normal rabbit IgG, with an incubation of 12 h at 4℃. Following this, the mixture was again incubated, this time with Protein A/G MagBeads (Thermo Fisher Scientific, Inc.) for a period of 2 h at 4\u0026deg;C, followed by washing with radio-immunoprecipitation assay (RIPA) buffer, lithium buffer, and Tris‐EDTA (TE) buffer. Following the cross-linking unfastening with the use of 0.2 \u0026micro;M NaCl at 65\u0026deg;C for 6 h before proceeding to ethanol precipitation of immunoprecipitated DNA. We then measured the fold enrichment relative to the IgG control via qPCR.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eDual-luciferase reporter assays\u003c/h2\u003e\u003cp\u003eTo clarify whether SP1 regulation of B-Myb expression involved modulation of its promoter activity, we performed a dual-luciferase reporter assay. We seeded cells at a density of 20,000 cells/well into a 12-well plate to initiate the assay. Prior to transfection, cells were cultured in 12-well culture plates until reaching a 60\u0026ndash;80% confluency. A luciferase reporter plasmid was generated by embedding a 2 kb sequence of \u003cem\u003eB-Myb\u003c/em\u003e promoter into the pGL3-basic vector (Promega Corporation, Madison, WI, USA). This vector expresses the firefly luciferase gene. Luciferase activity was measured as previously described(Gan \u0026amp; Zhang, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Forty-eight hours after transfection, we utilized a Dual-Luciferase Activity Assay Kit (Genomeditech, Shanghai, China) to quantify luminescent values for Firefly and Renilla luciferase activities by following the manufacturer's protocol. All assays were conducted in triplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eMolecular docking\u003c/h2\u003e\u003cp\u003eThe B-Myb prediction structure was generated via state-of-the-art computational modelling utilizing Alphafold. The small molecule's protonation state was standardized to pH\u0026thinsp;=\u0026thinsp;7.4, and Open Babel was employed to propagate the compound into a highly detailed 3D configuration. To prepare the receptor proteins and ligands, a series of techniques were executed using the expertly designed AutoDock tool (ADT3). Subsequent to generating the butt box using the accomplished AutoGrid program, a molecular docking simulation was executed via Autodock Vina (1.2.0). The optimal combination conformation was selected to effectively scrutinize the interaction, with due consideration to various factors. The subsequent protein-ligand interaction diagrams were meticulously produced with the aid of PyMOL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eFor statistical analyses, we employed the use of GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA). Quantitative data between two groups were compared by performing a Student's t-test. Spearman's correlation coefficient was calculated to examine the association between the expression of two genes. We utilized the Kaplan-Meier analysis and log-rank test to examine overall survival and cumulative recurrence rate. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eB-Myb is highly expression in glioma tissues\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the mRNA expression patterns of \u003cem\u003eB-Myb\u003c/em\u003e in gliomas, we obtained the microarray datasets (GSE16011 and GSE12657) from the GEO database (www.ncbi.nlm.nih.gov/geo/). The data demonstrated a significant up-regulation of \u003cem\u003eB-Myb\u003c/em\u003e mRNA in glioma tissues compared with normal brain tissues (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01 and \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, Figure 1A). Further validation was conducted through qRT-PCR and Western blot experiments, and substantial increases in both mRNA and protein levels of B-Myb were observed in glioma tissues (Figure 1B-C). The immunohistochemical analysis also demonstrated a significant up-regulation of B-Myb protein expression in glioma tissues, and the levels increased with WHO grade (Figure 1D). Additionally, the immunofluorescence assay indicated that the expression of B-Myb was correlated with Ki-67 expression (Figure 1E). Moreover, we also assessed the differential expression of \u003cem\u003eB-Myb\u0026nbsp;\u003c/em\u003ebetween pan-cancer tissues and corresponding control tissues (Figure 1F), and our analysis revealed that 55.6% (10 out of 18) of cancer types exhibited abnormal \u003cem\u003eB-Myb\u0026nbsp;\u003c/em\u003eexpression. These findings suggest that \u003cem\u003eB-Myb\u003c/em\u003e expression is a probable indicator of tumorigenesis and progression, with wide-ranging potential for clinical applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCorrelation of B-Myb overexpression with adverse clinicopathologic features and an unfavorable prognosis in gliomas\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain further insight into the clinical implications of \u003cem\u003eB-Myb\u003c/em\u003e, we collected clinical and mRNA-seq data of 325 patients diagnosed with gliomas. The clinicopathological details of the patients are outlined in Table S2. The median age of the cohort was 42 years, with an age range of 8 to 79 years. Out of the 325 patients, 203 (62.5%) were male and 122 (37.5%) were female (M: F=1.66:1). Of these cases, 103 (31.7%) were categorized as WHO grade II, 79 (24.3%) as WHO grade III, and 139 (42.8%) as WHO grade IV. Clinical follow-up records were available for these cases, ranging from 0.63 to 160.3 months.\u003c/p\u003e\n\u003cp\u003eTo investigate the relationship between the \u003cem\u003eB-Myb\u0026nbsp;\u003c/em\u003emRNA levels and the clinicopathologic features of gliomas, we divided the patient cohort into two groups based on\u003cem\u003e\u0026nbsp;B-Myb\u003c/em\u003e levels using the median value: high-expression (n=163) and low-expression (n=162), as illustrated in Figure 1G. The results of the chi-square test revealed a notable correlation between \u003cem\u003eB-Myb\u003c/em\u003e high-expression and advanced age (\u003cem\u003eP\u003c/em\u003e=0.006), higher WHO grade (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001), malignant histopathology (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001), IDH wildtype (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001), 1p/19q non-codeletion (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001), and chemotherapy (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001) (Table 1). These findings point towards a potential association between the up-regulation of \u003cem\u003eB-Myb\u003c/em\u003e and malignant progression in gliomas. We conducted an additional analysis to investigate the prognostic impact of \u003cem\u003eB-Myb\u003c/em\u003e on patients with gliomas. The Kaplan-Meier curve revealed that \u003cem\u003eB-Myb\u003c/em\u003e high-expression corresponded to a poor prognosis for patients (Figure 1H). The Spearman correlation analysis revealed a significant positive correlation between the expression of \u003cem\u003eB-Myb\u003c/em\u003e and Ki-67 (\u003cem\u003er\u003c/em\u003e=0.783, R\u003csup\u003e2\u003c/sup\u003e=0.614, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001, Figure 1I). We further performed both univariate and multivariate Cox regression analyses to identify potential prognostic biomarkers. Variables that displayed significant differences in the univariate Cox regression analysis were subjected to a stepwise multivariate Cox regression analysis. As shown in Figure 1J, the results indicated that \u003cem\u003eB-Myb\u003c/em\u003e high-expression was an independent prognostic biomarker for glioma patients (HR=1.86, 95%CI=1.31-2.66, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Overall, these findings suggest that \u003cem\u003eB-Myb\u003c/em\u003e high-expression may potentially be accountable for malignant characteristics observed in gliomas.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eB-Myb promotes glioma cells proliferation, invasion, and suppresses DNA damage in vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the mechanistic function of B-Myb protein in glioma cells, we conducted loss- or gain-of-function experiments. The efficacy of B-Myb overexpression or knockdown was validated by Western blot (Figure 2A). The CCK-8 and clone formation were performed to determine the cell proliferation. The CCK-8 assay showed that overexpression of B-Myb promoted U87-MG and LN229 cells' proliferation in a time-dependent manner (Figure 2B). Conversely, knockdown of B-Myb demonstrated an opposite trend (Figure 2B). The clone formation assays further confirmed the effects of B-Myb on the proliferation of LN229 cells (Figure 2C). Next, to explore the potential role of B-Myb in the regulation of glioma cell invasion, we conducted transwell assays. The results showed that overexpression of B-Myb significantly enhanced the invasiveness of U87-MG and LN229 cells whereas knockdown of B-Myb displayed an inhibitory effect (Figure 2D). Together, these observations suggested that B-Myb overexpression might play a critical role in regulating malignant biological behavior of gliomas.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo strengthen the evidence base, we performed an analysis to determine the relationship between B-Myb and gene sets related to proliferation and invasiveness using mRNA-sequencing data of glioma patients from TCGA. As shown in Figure 2E, the results confirmed our \u003cem\u003ein vitro\u003c/em\u003e results. Interestingly, our analysis also revealed a significant correlation between the expression of B-Myb and the activation of DNA damage repair pathways (Figure 2E), suggesting that B-Myb knockdown might promote DNA damage. Subsequently, this result was verified by confocal laser assays. Indeed, the results demonstrated that knockdown of B-Myb significantly promoted the DNA damage of U87-MG and LN229 cells (Figure 2F), indicating that B-Myb may be a promising target for glioma treatment.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe Genomics of Drug Sensitivity in Cancer (GDSC, https://www.cancerrxgene.org/) is currently recognized as the largest publicly available database of information on drug sensitivity and molecular markers of cancer cells. Using this database, our analysis revealed that Vorinostat exhibited the most significant inhibitory effect on B-Myb in glioma cells (Figure 2G). To evaluate the affinity of the candidate drugs for B-Myb, we performed molecular docking analysis. The interactions between the protein and molecular drug were comprehensively analyzed, with a high-resolution map available in Figure 2H. Functional residues were interrogated and systematically classified based on their contributions to the interactions. Multiple residue groups are utilized to foster interaction between the receptor protein and ligand, exemplified by a hydrophobic interaction involving TRP138 of B-Myb and the ligand. With these interaction forces, the binding energy of the protein-ligand complex was gauged to be -5.0 kcal/mol, attesting to a commendable degree of performance.\u003c/p\u003e\n\u003cp\u003eFurther CCK-8 assays confirmed a dose-dependent inhibitory effect of Vorinostat on U87 and LN229 cells (Figure 2I). The live/dead staining also showed that Vorinostat (3µM) could significantly induce glioma cells death (Figure 2J). Finally, transwell experiment also proved that Vorinostat could significantly inhibit the invasion ability of glioma cells (Figure 2K). In short, Vorinostat showed good antitumor efficacy for gliomas \u003cem\u003ein vitro\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSP1 regulates B-Myb expression by binding promoter sequences of target genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe underlying mechanisms of B-Myb up-regulation in gliomas remained poorly understood. To identify potential TFs, we employed the UCSC Genome Browser (https://genome-store.ucsc.edu/) and Animal TFDB3.0 databases (http://bioinfo.life.hust.edu.cn/AnimalTFDB/) to predict 27 and 31 TFs for \u003cem\u003eB-Myb\u003c/em\u003e gene, respectively (Figure 3A). Upon further scrutiny, we discovered four shared TFs-SP1, ZNF384, KLF5, and ELF1-at the intersection (Figure 3A). To assess the relationship between these TFs and \u003cem\u003eB-Myb\u003c/em\u003e expression, we analyzed the TCGA database and found that SP1 exhibited the strongest correlation coefficient (\u003cem\u003er\u003c/em\u003e=0.415, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001). Additionally, utilizing the JASPAR database, we were able to make predictions regarding potential SP1 binding sites.\u003c/p\u003e\n\u003cp\u003eIn addition, the Spearman correlation analysis revealed that SP1 was associated with B-Myb mRNA levels (\u003cem\u003er\u003c/em\u003e = 0.428, R\u003csup\u003e2\u003c/sup\u003e = 0.184, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001; Figure 3B). Previous study had established that SP1 could bind to the promoter region of FOXM1, resulting in the positive regulation of FOXM1 transcription(Petrovic, Costa, Lau, Raychaudhuri, \u0026amp; Tyner, 2010). Xue Zhang et al. also reported that the AKT/FOXM1 axis is the upstream signal regulating B-Myb expression(Zhang, Lv, Huang, Zhang, \u0026amp; Zhou, 2017). Next, we also found that FOXM1 displayed a strong correlation with B-Myb expression in glioma tissues (\u003cem\u003er\u003c/em\u003e = 0.855, R\u003csup\u003e2\u003c/sup\u003e = 0.732, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.0001). To evaluate the potential roles of SP1 and FOXM1 genes in gliomas, we employed GEPIA (http://gepia.cancer-pku.cn/index.html) to compare their expression in glioma and normal brain tissues. Our results demonstrated a significant upregulation of both genes in glioma tissues (Figure 3C). Additionally, we performed immunohistochemical analysis and observed an elevation in SP1 protein expression in gliomas, with a positive correlation between SP1 expression and tumor grade (Figure 3D).\u003c/p\u003e\n\u003cp\u003eNext, to explore the regulation effect of SP1 on B-Myb, we conducted SP1 overexpression and knockdown experiments (Figure 3E). The results of qRT-PCR and Western blot revealed that overexpression of SP1 in U87-MG and LN229 cells led to a significant increase in both mRNA and protein levels of the\u003cem\u003e\u0026nbsp;B-Myb\u003c/em\u003e gene (Figure 3G-F). Conversely, knockdown of SP1 resulted in a considerable reduction of \u003cem\u003eB-Myb\u003c/em\u003e mRNA and protein expression. These findings indicate that SP1 may play a vital role in the regulation of \u003cem\u003eB-Myb\u003c/em\u003e genes at the transcriptional level.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further confirm the direct involvement of SP1 in the \u003cem\u003eB-Myb\u003c/em\u003e promoter, we conducted ChIP. As illustrated in Figure 3H, the ChIP assay demonstrated SP1's direct binding to the B-Myb promoter \u003cem\u003ein vitro\u003c/em\u003e. To determine the biological significance of SP1 binding to the\u003cem\u003e\u0026nbsp;B-Myb\u0026nbsp;\u003c/em\u003egene promoter, we performed a luciferase reporter gene experiment. As shown in Figure 3I, the luciferase reporter gene experiment confirmed the transcriptional regulation of the \u003cem\u003eB-Myb\u003c/em\u003e promoter by SP1. To search for the SP1 binding site on\u003cem\u003e\u0026nbsp;B-Myb\u003c/em\u003e, truncated \u003cem\u003eB-Myb\u003c/em\u003e promoter reporter gene sequences with varying lengths were constructed, as shown in Figure 3I. Through relative luciferase activity analysis, the results showed that only a specific DNA sequence (50 and 500 bp upstream of the transcription initiation site) exhibited significantly higher \u003cem\u003eB-Myb\u0026nbsp;\u003c/em\u003eexpression as compared to the control group (Figure 3I, \u003cem\u003eP\u003c/em\u003e˂ 0.001), indicating the significance of this region in SP1-mediated transcriptional regulation of \u003cem\u003eB-Myb\u003c/em\u003e, which suggested that the binding site of SP1 on \u003cem\u003eB-Myb\u003c/em\u003e was between -50 and -500 bp upstream of the transcription start site. Collectively, our results strongly suggest that \u003cem\u003eB-Myb\u003c/em\u003e is a direct target of SP1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSP1 mediates proliferation, invasion and DNA damage of glioma cells through regulating target genes\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we investigated the impact of SP1 on glioma cell proliferation using CCK-8 and plate clone formation assays. The results showed that overexpression of SP1 significantly enhanced glioma cell proliferation, while overexpression of SP1 coupled with B-Myb knockdown effectively reversed this effect (Figure 4A). Consistent with the CCK-8 assays, plate cloning experiments revealed that overexpression of SP1 led to a notable increase in the number of LN229 cell clones, whereas knockout of SP1 resulted in the opposite trend (Figure 4B). B-Myb was able to rescue the number of SP1-regulated cell clones (Figure 4B). The transwell assays revealed that overexpression of SP1 significantly enhanced the invasion capability of glioma cells (Figure 4C-D). Conversely, downregulation of SP1 led to a reduction in the invasion ability of glioma cells, and B-Myb was able to restore the invasion ability of glioma cells mediated by SP1. Furthermore, we sought to assess the impact of SP1 on DNA damage in glioma cells via laser confocal experiments. Our results indicated that knockdown of SP1 significantly promoted DNA damage in glioma cells (Figure 4E). Likewise, overexpression of B-Myb effectively rescued this phenotype (Figure 4E). These data suggest that SP1 might affect the progression of glioma cells through B-Myb expression.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe proceeded to analyze mRNA-sequencing data from 325 glioma patients. Our analysis indicated a significant correlation between \u003cem\u003eSP1\u0026nbsp;\u003c/em\u003emRNA levels and advanced age, high WHO grade, and 1p/19q non-co-deletion, but not with recurrence, IDH wild-type, or MGMT promoter methylation (Figure 4F). Furthermore, the Spearman correlation analysis revealed that \u003cem\u003eSP1\u0026nbsp;\u003c/em\u003eexpression was associated with the expression of Ki-67 (\u003cem\u003er\u003c/em\u003e=0.53, R\u003csup\u003e2\u003c/sup\u003e=0.28, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001), \u003cem\u003eVim\u0026nbsp;\u003c/em\u003e(\u003cem\u003er\u003c/em\u003e=0.38, R\u003csup\u003e2\u003c/sup\u003e=0.14, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001), and \u003cem\u003eFOXM1\u003c/em\u003e (\u003cem\u003er\u003c/em\u003e=0.45, R\u003csup\u003e2\u003c/sup\u003e=0.21, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.0001), as shown in Figure 4. Kaplan-Meier survival curves revealed that patients exhibiting SP1 high-expression had worse prognosis (Figure 4H). Next, we included data from three patients, presented here with their MRI imaging, pathological indicators, B-Myb immunohistochemistry results, and prognostic data (Figure 4I). Tumor size, degree of peritumoral edema and the Ki-67 proliferation index demonstrated a positive correlation with B-Myb protein expression, indicating a poor prognosis for glioma patients. This degree of peritumoral edema is somewhat reflective of the overall invasion(Cheon, et al., 2017).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAt present, treatment options for gliomas are extremely limited and patients continue to face a poor overall prognosis(Ene \u0026amp; Holland, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Therefore, there is a need for effective strategies to comprehensively address the disease and improve patients\u0026rsquo; outcomes. Targeted therapy, as an effective strategy for the comprehensive treatment of tumor, may be a promising approach to alleviate symptoms and extend survival for individuals afflicted by gliomas(Mehta, Abi Nader, Waddington, \u0026amp; David, 2011). In our work, high expression of \u003cem\u003eB-Myb\u003c/em\u003e is associated with malignant clinicopathological features and poor prognosis in gliomas from a clinical perspective. Functionally, B-Myb high-expression is involved in regulating DNA damage response in addition to proliferation and invasion of glioma cells. Mechanically, we discovered a new mechanism that SP1 interacts with the \u003cem\u003eB-Myb\u003c/em\u003e promoter and facilitates \u003cem\u003eB-Myb\u003c/em\u003e transcription in gliomas. Therapeutically, Vorinostat was found to inhibit tumor cell proliferation, invasion and induction of cell death by inhibiting B-Myb.\u003c/p\u003e\u003cp\u003eTFs constitute critical proteins that regulate gene expression via initiation or inhibition of gene transcription, accomplished by effector binding to either enhancers or promoters of target genes(Debnath, Huirem, Dutta, \u0026amp; Palchaudhuri, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Mao, et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). MYB TFs exhibit remarkable conservation across a wide range of species, spanning from plants to vertebrates, signifying that their functions represent fundamental mechanisms in the biology of both cells and organisms (Dubos, et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The human MYB gene family comprises of three members: MYB, MYBL1 and MYBL2. These genes encode for MYB, MYBL1, and MYBL2 TFs, which are alternatively referred to as c-MYB, A-MYB, and B-MYB, correspondingly. In MCF-7 breast cancer cells, c-MYB has been observed to bind to over 10,000 promoters, thereby being identified as a crucial stimulant of downstream targets. These targets encompass genes that are associated with cancer metastasis and progression, such as BCL2, BCLXL, JUN, CXCR4, MYC, NANOG, KLF4, and COX-2(Quintana, Liu, O'Rourke, \u0026amp; Ness, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). B-Myb and C-Myb belong to the same family and exhibit functional similarities. The \u003cem\u003eMYBL2\u003c/em\u003e gene was initially discovered as cellular homologs of the v-myb oncogene, which is recognized for triggering leukemia in chickens(Oh \u0026amp; Reddy, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The expression of MYBL2 in proliferative cells is critical for governing cellular proliferation and differentiation, while also playing a pivotal role in directing the progression of the cell cycle (Fischer, Quaas, Steiner, \u0026amp; Engeland, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). B-Myb exerts profound influences on cell behavior, as its plethora of protein partners and target genes permit it to govern multiple crucial cellular processes, including proliferation, senescence, apoptosis, and mitosis(Musa, Aynaud, Mirabeau, Delattre, \u0026amp; Gr\u0026uuml;newald, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Considering its wide-ranging functions and correlation with human cancer, it may be viable to utilize B-Myb as a significant diagnostic, prognostic and therapeutic instrument. A study conducted by Xiaoyan Fan and colleagues revealed that overexpression of B-Myb in colorectal cancer cells accelerates cell proliferation, cell cycle progression, and cell motility (Fan, et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Furthermore, \u003cem\u003ein vivo\u003c/em\u003e investigation utilizing orthotopic nude mouse models demonstrated that B-Myb overexpression promotes tumor growth. The functional roles of B-Myb in the above are similar to our findings in gliomas. It is unclear why B-Myb expression was increased in our study.\u003c/p\u003e\u003cp\u003eOur investigation on gliomas has revealed a novel mechanism through which SP1 facilitates oncogenic transcription of B-Myb. Traditionally, the transcription factor SP1 was regarded as a fundamental transcriptional regulator and was assigned a secondary role in the regulation of what are known as housekeeping genes (Beishline \u0026amp; Azizkhan-Clifford, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chiefari, et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The overexpression of SP1 is frequently observed in several types of cancers, and it is positively correlated with a poor prognosis. In a publication authored by Fuzhen Qi et al., it was observed that SP1 mediated upregulation of ASAP2-AS1 functions as a crucial oncogenic driver in the development and progression of gastric cancer, by impeding P21 and E-cadherin (Qi, et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Vladimir Petrovic et al. reported that the transcription factor SP1 binds to the promoter region of \u003cem\u003eFOXM1\u003c/em\u003e and subsequently augments \u003cem\u003eFOXM1\u003c/em\u003e transcription (Petrovic, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The results of our experimentation further validated the affirmative regulation of the transcription factor SP1 on the production of FOXM1 protein. Xue Zhang et al. reported that MYBL2 is a key downstream factor of Akt/FOXM1 signaling to promote progression of gliomas (Zhang, et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This implies that SP1 may operate diverse pathways to modulate B-Myb expression in glioma cells. Consequently, B-Myb emerges as a noteworthy downstream target implicated in the onset and evolution of gliomas, while potentially displaying minimal adverse effects.\u003c/p\u003e\u003cp\u003eCollectively, our study has established that B-Myb expression is upregulated in glioma tissues, and its overexpression is strongly associated with malignant pathological characteristics and unfavorable prognosis in glioma patients. Moreover, our investigations have demonstrated that B-Myb overexpression significantly promotes glioma cell proliferation and invasion, while also suppressing DNA damage response. Mechanistically, SP1 may exert its pro-tumorigenic effects by directly binding to the B-Myb promoter, thereby activating its transcription and facilitating gliomas development. Therapeutically, Vorinostat was found to exhibit significant anti-tumor efficacy for gliomas. In light of our new insights, B-Myb may hold promise as both a novel prognostic biomarker and promising therapeutic target for gliomas.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQiuming Pan and Hongrui Li: Conceptualization, Methodology, Investigation, Formal analysis, Writing - Original Draft; Junxi Wang, Yudi Huang and Huafu Wang: Data Curation, Visualization, Writing - Review \u0026amp; Editing; Guozhong Yi and Zhiyong Li: Investigation, Resources, Software; Rongyang Xu and Ye Zhu: Methodology, Validation, In vitro experiments; Luyao Wang and Yuou Qin: Bioinformatics analysis, Statistical validation, Clinical sample collection; Guanglong Huang and Songtao Qi: Funding acquisition, Project administration, Data Interpretation; Shanqiang Qu: Conceptualization, Supervision, Writing - Review \u0026amp; Editing. All authors have reviewed and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by President Foundation of Nanfang Hospital, Southern Medical University (2022A018), Funding by Science and Technology Projects in Guangzhou (2024A04J5111), GuangDong Basic and Applied Basic Research Foundation (2023A15111129) and the Basic Public Welfare Research Program of Zhejiang Province (LGF22H160052).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll tissues were obtained with the consent of patients and were approved by the Medical Ethics Committee of Nanfang Hospital, ensuring ethical and legal compliance. Human Ethics and Consent to Participate declarations: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAltieri, R., Agnoletti, A., Quattrucci, F., Garbossa, D., Calamo Specchia, F.M., Bozzaro, M., et al. (2014). Molecular biology of gliomas: present and future challenges. \u003cem\u003eTransl Med UniSa, 10\u003c/em\u003e, 29-37.\u003c/li\u003e\n\u003cli\u003eBeishline, K., \u0026amp; Azizkhan-Clifford, J. (2015). 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Akt/FoxM1 signaling pathway-mediated upregulation of MYBL2 promotes progression of human glioma. \u003cem\u003eJ Exp Clin Cancer Res, 36\u003c/em\u003e(1), 105, doi:10.1186/s13046-017-0573-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Gliomas, B-Myb, Transcription mechanism, Biomarker, Malignant progression","lastPublishedDoi":"10.21203/rs.3.rs-7699276/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7699276/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the limited therapeutic options for gliomas, identifying molecules with therapeutic potential remains critical. This study aimed to investigate the functional significance and regulatory mechanisms of B-Myb in glioma progression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB-Myb expression was assessed in glioma tissues through multimodal analyses (qRT-PCR, Western blotting, immunohistochemistry, and immunofluorescence). Prognostic relevance was evaluated in 325 glioma patients. Functional consequences of B-Myb modulation were examined via overexpression and knockdown approaches, with subsequent evaluation of proliferation, invasion, and DNA damage responses. Transcriptional regulation of B-Myb by SP1 was validated using chromatin immunoprecipitation (ChIP) and luciferase reporter assays.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB-Myb was markedly upregulated in gliomas, with elevated expression correlating with advanced histopathological grades and reduced patient survival (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Functionally, B-Myb overexpression promoted tumor cell proliferation and invasion while suppressing DNA damage responses, whereas its knockdown reversed these phenotypes. Mechanistically, SP1 activated B-Myb transcription by binding to its promoter, thereby driving glioma malignancy. Vorinostat, a histone deacetylase inhibitor, demonstrated potent antitumor effects by suppressing B-Myb expression in preclinical models.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur findings establish B-Myb as a clinically relevant oncoprotein in gliomas, with its expression driven by SP1-mediated transcriptional activation. The therapeutic efficacy of Vorinostat via B-Myb targeting highlights its potential for clinical translation. These results position B-Myb as both a prognostic biomarker and a promising therapeutic target for glioma management.\u003c/p\u003e","manuscriptTitle":"Transcription factor SP1-Mediated Upregulation of B-Myb Promotes Glioma Aggression Through Transcriptomic Mechanism","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 09:34:12","doi":"10.21203/rs.3.rs-7699276/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":"03b2f71d-7480-4ad4-b4c9-472da1792a45","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-05-16T05:55:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-08 09:34:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7699276","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7699276","identity":"rs-7699276","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}