Application of DERL3 as endoplasmic reticulum stress core gene in glioblastoma

Abstract

The invention discloses application of DERL3 as an endoplasmic reticulum stress core gene in glioblastoma, and belongs to the field of biological medicine. The invention discloses a mechanism of DERL3 as an endoplasmic reticulum stress (ER Stress) core effector in malignant progression of glioblastoma (GBM). By establishing an ER stress risk scoring system, it is found that DERL3 high expression is positively correlated with GBM poor prognosis. Functional experiments show that knock-down of DERL3 significantly inhibits proliferation, migration and invasion of tumor cells, and the mechanism is that ATF6 mediates ER stress to up-regulate DERL3, DERL3 is combined with and stabilizes HNRNPA2B1 protein, and then an NF-kappa B signal channel is activated. In-vivo experiments prove that the targeted DERL3 can inhibit the growth of intracranial tumors and prolong the lifetime. Based on this, the invention provides application of DERL3 as a prognostic marker and a drug target in GBM diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of DERL3 as a core gene for endoplasmic reticulum stress in glioblastoma. Background Technology

[0002] Gliomas are the most common primary brain tumors, associated with the highest rates of intracranial disability and mortality among central nervous system (CNS) malignancies. Glioblastoma multiforme (GBM), the most aggressive subtype of glioma, has a median survival of only 14.6 months even after multiple treatments, including surgical resection, standard radiotherapy, and chemotherapy. This poor prognosis stems from several characteristics of GBM: extensive invasiveness, migratory capacity, malignant proliferation, and significant microenvironmental adaptation. Recent studies have shown that various external signals or therapeutic interventions often induce a state of high stress in tumor cells. While this stress condition can trigger autophagy and apoptosis in tumor cells, thereby exerting anti-tumor effects, glioma cells, like many other malignancies, rapidly develop treatment resistance. This adaptive response primarily stems from their intrinsic plasticity and ability to translate stress signals into self-renewal drivers. Therefore, elucidating stress-related signaling targets and their regulatory mechanisms in gliomas is of significant scientific and clinical importance for understanding tumor cell adaptation and developing targeted therapies.

[0003] Mounting evidence suggests that endoplasmic reticulum (ER) stress activation is closely associated with the occurrence and development of malignant tumors. Multiple factors can trigger ER stress, including radiotherapy, chemotherapy, oxidative stress, nutritional deficiencies, and inflammatory responses. When ER stress is activated, tumor cells rapidly initiate adaptive protective mechanisms, particularly the unfolded protein response (UPR). Recent research indicates that the UPR can activate the HIF1α signaling pathway, a crucial regulator of tumor cell proliferation, invasion, migration, angiogenesis, and glycolytic metabolism. In triple-negative breast cancer, IRE1α promotes the formation of the XBP1-HIF1α transcriptional complex, which recruits RNA polymerase II to regulate HIF1α target genes, thereby promoting cancer cell proliferation and invasion. Furthermore, Jiang et al. discovered that IRE1α activation induces IκB phosphorylation and degradation through interaction with the tumor necrosis factor receptor eLF2α, thereby activating the NF-κB signaling pathway.

[0004] Current treatment strategies increasingly target endoplasmic reticulum (ER) stress or related pathways to influence tumor development. For example, ER stress-induced exosome miR-27a-3p promotes immune escape in breast cancer by regulating PD-L1 expression in macrophages. The ER stress mediator ERO1 triggers cancer metastasis under hypoxic conditions by activating angiogenesis. In lung adenocarcinoma, p62 promotes tumor development by mediating the upregulation of NRF2 and ATF6 through autophagy. However, how ER stress affects the pathogenesis of gliomas remains unclear. Therefore, a systematic analysis of ER stress status in gliomas and the identification of relevant molecular markers have significant scientific and clinical implications. Summary of the Invention

[0005] To address the problems existing in current technologies, this invention provides the application of DERL3 as a core gene for endoplasmic reticulum stress in glioblastoma. Through in vitro and in vivo experiments, this invention reveals that DERL3 acts as a feedback upregulated target molecule during endoplasmic reticulum stress, mediating the role of stress-induced effects in glioma cells. This demonstrates that DERL3 is a promising endoplasmic reticulum stress-related regulatory molecule with potential therapeutic value.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0007] This invention discloses the use of DERL3 inhibitors in the preparation of drugs for the prevention or treatment of glioblastoma.

[0008] Furthermore, the DELL3 inhibitor is a shRNA or siRNA that targets DELL3.

[0009] This invention also discloses the application of NF-κB inhibitors in the preparation of drugs for treating glioblastoma, wherein the glioblastoma is a high-expressing DERL3 tumor.

[0010] Furthermore, the NF-κB inhibitor is BAY 11-7082 or a pharmaceutically acceptable salt thereof.

[0011] The present invention also discloses the use of a compound that inhibits the interaction between the DERL3-HNRNPA2B1 protein in the preparation of an anti-glioblastoma drug.

[0012] This invention also discloses a pharmaceutical composition for treating glioblastoma, comprising: (a) DERL3 inhibitors or NF-κB inhibitors; and (b) Pharmaceutically acceptable carriers.

[0013] Furthermore, the DELL3 inhibitor is a shRNA or siRNA that targets DELL3.

[0014] The present invention also discloses the application of reagents for detecting DERL3 expression levels in the preparation of kits for the diagnosis, screening, and prognostic assessment of glioblastoma.

[0015] Furthermore, the glioblastoma diagnostic screening kit detects the expression level of the DERL3 gene in the sample, and the degree of DERL3 gene expression is positively correlated with the incidence of glioblastoma.

[0016] Furthermore, the glioblastoma prognostic assessment kit detects the expression level of the DERL3 gene in the sample, and the degree of intra-oncological expression of the DERL3 gene is negatively correlated with the prognostic survival of glioblastoma.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] This invention utilizes glioma transcriptome data to establish a personalized ER stress risk scoring system, demonstrating its association with poor prognosis, aggressive molecular subtypes, and pro-tumor biological functions. Through systematic screening, DERL3 was identified as a core effector gene mediating ER stress adaptation. Functional validation showed that DERL3 promotes glioma proliferation and invasion by binding to and stabilizing HNRNPA2B1, thereby activating the NF-κB signaling pathway. These experiments reveal the DERL3-HNRNPA2B1-NF-κB axis as a crucial mechanistic link between ER stress adaptation and glioma malignancy. Treatment strategies targeting this axis offer new avenues for overcoming treatment resistance, possess significant translational potential, contribute to improved glioma management, advance the understanding of stress response mechanisms in tumorigenesis, and emphasize the clinical significance of ER stress-related pathways in precision oncology.

[0019] Diagnostic value: DERL3 expression level can serve as a biomarker for prognostic assessment of GBM (especially applicable to wild-type / mesenchymal subtype of IDH).

[0020] Treatment target: Targeting the DERL3-HNRNPA2B1-NF-κB axis can overcome ER stress-induced treatment resistance.

[0021] Mechanism innovation: This study reveals for the first time the pathway mechanism by which DERL3 activates NF-κB through stabilizing HNRNPA2B1, providing a new target for intervention in GBM.

[0022] Translational potential: The NF-κB inhibitor (BAY 11-7082) has been proven to block the oncogenic effects of DERL3, supporting the strategy of repurposing existing drugs. Attached Figure Description

[0023] Figure 1DERL3-mediated endoplasmic reticulum stress drives malignant progression of gliomas. (A) Expression patterns and clinical significance of seven constitutive risk score genes in the CGGA database. (B) Survival analysis comparing high-ERS-risk and low-ERS-risk groups in three databases. (C) Immunohistochemical staining of DERL3 in glioma tissues (grades II-IV). (DE) Protein levels of DERL3 in different grades of gliomas (grades II-IV). (F) mRNA expression of DERL3 in grade II-IV gliomas.

[0024] Figure 2 .Correlation analysis of DELL3 with poor prognosis in GBM. (A)-(B) Analysis of 7 genes significantly associated with survival in the CGGA dataset. (C) Multivariate analysis of DELL3 as an independent prognostic biomarker. (D)-(F) ROC curves of the CGGA and TCGA datasets.

[0025] Figure 3 .DERL3 expression level and survival analysis. (AF) Elevated DERL3 expression level predicts poorer survival. (GL) DERL3 expression level versus MGMT and IDH status results.

[0026] Figure 4 Relationship between tumor grade and DERL3. (AC) DERL3 expression increases with increasing tumor grade. (DI) Expression analysis in stromal GBM and IDH wild-type tumors.

[0027] Figure 5 DELL3 knockdown suppressed the malignant phenotype in GBM. (A) Protein and mRNA expression of DELL3 in different cell lines. (B) Verification of DELL3 knockdown efficiency in U87 and LN229 cells. (C) Proliferation curves of U87 and LN229 cells after DELL3 knockdown. (D) Colony-forming ability of U87 and LN229 cells after DELL3 knockdown. (EF) Effects of DELL3 knockdown on invasion and migration of U87 and LN229 cells.

[0028] Figure 6 Effects of DERL3 overexpression on glioma cells. (AB) U87 and LN229 cells overexpressing DERL3. (CD) Effects of DERL3 overexpression on the migration and invasion of glioma cells. (E) Correlation between DERL3 and ATF6.

[0029] Figure 7Protein expression and malignant behavior of various molecules in GBM cells under endoplasmic reticulum stress and inhibition. (A) Protein expression of multiple related genes in U87 and LN229 cells under the action of different endoplasmic reticulum stress activators and inhibitors. (B) ATF6 can regulate DERL3 expression in U87 and LN229 cells. (C) ATF6 cannot be regulated by DERL3 in U87 and LN229 cells. (DE) Cell migration and invasion abilities were detected after DERL3 knockdown and administration of endoplasmic reticulum stress activators. (F) Cell proliferation ability was detected after DERL3 knockdown and administration of endoplasmic reticulum stress activators.

[0030] Figure 8 DELL3 promotes GBM cell invasion through the NF-κB signaling pathway. (A) KEGG pathway analysis was performed on differentially expressed genes related to DELL3 in the CGGA GBM database. (B) NF-κB pathway activity was analyzed by Western blot after knocking down DELL3 in LN229 and U87 cells. (C) NF-κB activation was verified by Western blot in U87 and LN229 cells after DELL3 overexpression and treatment with different concentrations of BAY (NF-κB inhibitor). (D) The effects of DELL3 overexpression ± BAY on glioma cell proliferation were investigated. (EF) The effects of DELL3 overexpression ± BAY on glioma cell migration and invasion were investigated.

[0031] Figure 9 DELL3 maintains its stability by binding to HNRNPA2B1. (A) CO-IP assays verified the binding relationship between DELL3 and HNRNPA2B1 in LN229 and U87 cell lines. (B) Western blot analysis showed that DELL3 regulates the expression of HNRNPA2B1 in U87 and LN229 cell lines. (C) Knockdown of DELL3 did not significantly affect the mRNA expression of HNRNPA2B1. (DF) After the addition of CHX, CHX+MG132, and CHX+CQ, the degradation rate and degradation curves of HNRNPA2B1 protein in the DELL3 knockdown group and the control group were plotted.

[0032] Figure 10Knockdown of HNRNPA2B1 and DERL3 inhibited the malignant progression of GBM. (A) HNRNPA2B1 regulates the NF-κB signaling pathway in U87 and LN299 cell lines. (B) Changes in cell migration and invasion after HNRNPA2B1 knockdown in U87 and LN299 cell lines. (CD) Cell proliferation before and after HNRNPA2B1 knockdown in U87 and LN299 cell lines. (E) The survival time of mice in the DERL3 knockdown group was significantly prolonged. (F) Immunohistochemical staining results of DERL3 and Ki67 in the DERL3 control and knockdown groups.

[0033] Figure 11 The relationship between DELL3 and HNRNPA2B1 in glioma progression. (A) In the U87 cell line, HNRNPA2B1 regulates the NF-κB signaling pathway. (B) In the LN299 cell line, HNRNPA2B1 regulates the NF-κB signaling pathway. (C) Changes in cell migration and invasion after HNRNPA2B1 knockdown in the U87 cell line. (D) Changes in cell migration and invasion after HNRNPA2B1 knockdown in the LN299 cell line. (E) Cell proliferation before and after HNRNPA2B1 knockdown in the U87 cell line. (F) Cell proliferation before and after HNRNPA2B1 knockdown in the LN299 cell line.

[0034] Figure 12 DERL3 knockdown in xenograft models. (A) H&E staining results of brain slices from nude mice implanted with glioma cells intracranially. (B) Tumor volume histogram. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0036] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0037] I. Materials and Methods.

[0038] 1. Clinical specimens.

[0039] This study has been approved by the Ethics Committee of the First Affiliated Hospital of China Medical University, and all human tissue samples were collected with the informed consent of the patients or their legal guardians.

[0040] During the Western blot analysis, 26 surgically resected glioma specimens were obtained between January 2018 and October 2020, including 4 non-tumor controls, 8 WHO grade II, 7 grade III, and 7 grade IV. The immunohistochemical (IHC) study used 61 specimens collected between February 2014 and October 2020, including 6 non-tumor controls, 10 grade II, 24 grade III, and 31 grade IV.

[0041] 2. Cell culture model.

[0042] Human glioma cell lines (U87, U251, LN229) were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Normal human astrocytes (NHA) were generously provided by Professor Jiang Tao of the Beijing Neurosurgical Institute. Primary glioblastoma stem cell-like cells (GSC21, GSC40) were established in our laboratory from fresh surgical specimens according to approved protocols.

[0043] 3. RNA isolation and qRT-PCR.

[0044] Total RNA was extracted using TRIzol reagent (Takara) according to the manufacturer's instructions. RNA purity and concentration were determined spectrophotometrically (NanoDrop). cDNA synthesis was performed using PrimeScript RT Master Mix (Takara). Quantitative PCR was performed using SYBR Green Master Mix (Takara) on a QuantStudio system under the following cycling conditions: 95°C pre-denaturation for 30 s, followed by 40 cycles, each consisting of 5 s at 95°C and 30 s at 60°C. Relative mRNA expression levels were determined by 2... -ΔΔCt The method was calculated using 18S rRNA as an internal reference.

[0045] .

[0046] 4. Protein blotting.

[0047] Protein lysates were quantified using the BCA method. In the Western blot assay, 20 μg of protein per band was separated by 10% SDS-PAGE and transferred to a PVDF membrane. The membrane was first blocked with 5% skim milk, then incubated overnight with primary antibody (4°C), followed by incubation with HRP-labeled secondary antibody for 1 h (room temperature). The signal was developed using ECL substrate and quantified using ImageJ software.

[0048] 5. Immunohistochemistry (IHC).

[0049] Formalin-fixed paraffin sections underwent antigen retrieval in citrate buffer (pH 6.0), endogenous peroxidase was blocked using 3% H2O2, and serum blocking was also performed. Primary antibody was incubated overnight at 4°C. Detection was performed using the biotin-streptavidin-HRP system with DAB chromogenic reagent. Sections were counterstained with hematoxylin, dehydrated, and then mounted.

[0050] 6. Endoplasmic reticulum stress activation and inhibition experiments.

[0051] U87 and LN229 cells were seeded at a density of 500,000 cells per well in six-well plates. When the cell density reached 70%, the experimental group was treated with 1 μM of the endoplasmic reticulum stress activator thiapsigargin, while the control group was treated with PBS. The expression of the target protein was measured after 48 h. In addition, the experimental group was treated with 2 mM of the endoplasmic reticulum stress inhibitor tauroursodeoxycholate sodium, while the control group was also treated with PBS. The expression of the target protein was measured again after 48 h.

[0052] 7. Cell proliferation assay.

[0053] Sixty hours after transfection, cells were seeded into 96-well plates at a density of 1000 cells per well, with a total volume of 100 μL per well. The plates were cultured at 37°C and 5% CO2 for 5 days. Simultaneously, on days 0, 1, 2, 3, 4, and 5, 20 μL of MTS solution (Promega) was added to each well, and the plates were incubated at 37°C for 3 hours. The optical density (OD) of each well was measured at 490 nm using a Molecular Devices (Spectramax M5) spectrophotometer to assess cell proliferation.

[0054] 8. Detection of cell invasion, migration and cloning capabilities.

[0055] To assess cell invasion and migration capabilities, 24-well plates and Transwell chambers were used. In the migration assay, 5 × 10⁶ cells were seeded per well. 4 100 cells per well; in the invasion test, 1 × 10⁶ cells per well. 5Cells were seeded in the upper chamber containing 0.2% fetal bovine serum (FBS) (the invasion chamber was coated with 80 μL of 1:8 diluted matrix gel). The lower chamber was filled with medium containing 20% ​​FBS. After 16 h and 24 h, cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet. Cell counts were performed from five random regions under a 100x microscope. In cell cloning experiments, 2000 cells were seeded into 6-well plates. Two weeks later, cells were fixed with methanol, stained with 0.1% crystal violet, and the number of visible colonies was counted. The results showed that the invasion and migration abilities of cells were enhanced in the overexpression group.

[0056] 9. Co-immunoprecipitation (Co-IP) experiment.

[0057] Take 1×10 7 Cells were lysed using IP-specific lysis buffer as instructed. After lysis, the cells were incubated at 4°C for 3 hours on a rotor, followed by centrifugation to collect the supernatant. A / G magnetic beads were added to the rotor, and the supernatant was collected again after incubation for 10 minutes to prevent non-specific protein binding. In a new EP tube, 30 μL of protein was added as the input group, and 300 μL of protein and the corresponding IP antibody were added as the IP group. IgG antibody was added as a control in another tube. The antigen-antibody complexes were incubated overnight at 4°C on a rotor. The next day, magnetic beads were added to the antigen-antibody complexes, mixed at room temperature, and incubated on a rotor for 120 minutes. The antigen-antibody complexes were collected using a magnetic rack, and 1× heat-loading buffer was added to denature the proteins before Western blotting.

[0058] .

[0059] 10. Protein stability test.

[0060] Forty-eight hours after cell transfection, cells were washed with PBS. To investigate the protein degradation rate under different treatment conditions, 20 mM cyclohexylimide (CHX) was added to each group to inhibit intracellular protein synthesis, and the expression of the target protein was detected at 0 h, 3 h, 6 h, and 9 h. To further explore the protein degradation pathway, 20 μM proteasome inhibitor MG132 or 20 μM lysosome inhibitor chloroquine (CQ) was added simultaneously, and the expression of the target protein was detected at 0 h, 3 h, 6 h, and 9 h.

[0061] 11. Brain transplantation in nude mice.

[0062] Using a stereotactic instrument, 5×10 5U87 cells (a specific tumor cell line) were injected into the substantia nigra region of the right brain of nude mice under different treatment conditions, with a vertical depth of 3 mm. When the tumor-bearing mice developed neurological symptoms, mice in different groups were simultaneously euthanized, and then the brain tissue was fixed and embedded for HE staining and immunohistochemical staining. The above experiments were repeated to study the differences in survival under different conditions.

[0063] 12. Bioinformatics analysis.

[0064] The clinical expression profiles and molecular data required for this study were obtained from three open and shareable data sources: The Cancer Genome Atlas (TCGA), the Glial Cell Gene Atlas (CGGA), and the GlioVis glioma data platform. The gene set associated with endoplasmic reticulum stress (ERS) was selected from the GSEA website (http: / / www.gseamsigdb.org / gsea / msigdb / search.jsp), including both activated and suppressed ERS stress genes (GO_REF: 0000058). Using CGGA RNA-seq, TCGA RNA-seq, CGGA microarray glioma expression profiles, and clinical data, univariate Cox regression analysis was employed to identify prognostic ERS stress-related genes. The ssGSEA algorithm was used to weight and filter the expression levels of relevant genes and clinical prognostic data in the database to calculate the ERS risk score for each sample. Clinical prognostic and molecular subtype data from the CGGA database were used to demonstrate the association between the ERS risk score and different molecular subtypes, grades, and prognoses.

[0065] 13. Statistical analysis.

[0066] Statistical data analysis and plotting were performed using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA) and R (version 3.6.1; R packages: pheatmap, corrplot). Univariate Cox regression analysis was performed using R; ROC curves were plotted using Medcalc 19.0.7 (www.medcalc.org); survival analysis was performed using the Kaplan-Meier method combined with the log-rank test; and relevant signaling pathways were identified through KEGG enrichment analysis. P < 0.05 was considered statistically significant, and differences between groups were determined using two-tailed t-tests, one-way ANOVA, and chi-square tests.

[0067] II. Results.

[0068] 1. DELL3 is a core gene associated with ER stress, which is associated with poor prognosis of GBM.

[0069] To identify prognostic genes associated with ER stress, we used univariate Cox regression analysis on 81 genes in the CGGA dataset. The results showed that 7 genes were significantly associated with survival (P<0.0001), among which PPP1R15A, FCGR2B, EDEM1, DERL3, and OS9 were risk factors (HR>1), while UBXN1 and UBQLN1 were protective factors (HR<1). The ER stress risk score derived from these genes was associated with aggressive glioma subtypes (e.g., stromal glioma, IDH wild-type) and higher WHO classification. Figure 1 A and Figure 2 AB). High-risk patients have lower survival rates ( Figure 1 B). Multivariate analysis further confirmed the status of DERL3 as an independent prognostic biomarker, and this was verified by ROC curves in the CGGA and TCGA datasets. Figure 2 CF). Elevated DERL3 expression levels predict poorer survival, especially in GBM ( Figure 3 AF), and the results were stratified according to MGMT and IDH states ( Figure 3 GL). DERL3 expression increases with increasing tumor grade ( Figure 4 AC was most highly expressed in stromal GBM and IDH wild-type tumors. Figure 4 DI). Histopathological results validated by immunohistochemistry further confirmed these findings, showing that DERL3 expression was gradually upregulated with increasing tumor grade. Figure 1 C). Furthermore, analysis of mRNA and protein levels in glioma tissue further confirmed that DERL3 expression increased with increasing malignancy. Figure 1 DF).

[0070] 2. DELL3 promotes the malignant progression of GBM cells.

[0071] To assess the expression of DERL3 in glioma cells, we analyzed its mRNA and protein levels in human glioma cell lines (U87, U251, LN229), primary glioma stem cell-like cells (GSC21, GSC40), and normal human astrocytes (NHA). Notably, DERL3 expression was significantly increased in glioma cell lines compared to normal human astrocytes, with high levels observed particularly in LN229 and U87 cells. Figure 5A). To investigate the functional role of DEL3 in glioma progression, we established stable DEL3 knockdown models in U87 and LN229 cells using a lentiviral shRNA construct. Quantitative PCR and Western blotting confirmed the effective silencing of DEL3 at both transcriptional and translational levels. Figure 5 B).

[0072] .

[0073] .

[0074] Functional tests showed that DERL3 knockdown significantly inhibited glioma cell proliferation. MTS experiments demonstrated that, compared to the control group, the survival rate of DERL3-depleted U87 and LN229 cells decreased significantly over time. Figure 5 C). Consistent with this, colony formation experiments also showed that inhibition of DERL3 led to a decrease in colony-forming ability. Figure 5 D). To assess the effect of DERL3 on glioma cell migration, we performed Transwell migration and Matrigel invasion assays. The results showed that DERL3 knockdown significantly reduced cell migration and invasion abilities, manifested as a weakened ability of cells to penetrate membrane pores and the Matrigel matrix. Figure 5 To further validate these findings, we generated DELL3-overexpressing U87 and LN229 cells via plasmid transfection. Figure 6 AB). Functional tests showed that, compared with the vector control group, overexpression of DERL3 significantly enhanced the migration and invasion abilities of glioma cells. Figure 6 (CD). In summary, these data indicate that DERL3 plays a crucial role in promoting the proliferation, migration, and invasion of glioma cells, highlighting its potential as a key driver of GBM malignancies.

[0075] 3. ATF6-mediated DERL3 regulation drives GBM malignancy by activating ER stress.

[0076] Our preliminary study found that DERL3 is a gene associated with endoplasmic reticulum stress response and contributes to the malignant development of gliomas. To explore its regulatory mechanism, we induced endoplasmic reticulum stress in U87 and LN229 cells using drug therapy. The results showed that treatment with the endoplasmic reticulum stress inducer thapsigargin significantly increased the expression of ATF6 and BiP, two classic endoplasmic reticulum stress markers, as well as DERL3. Figure 7 A). Conversely, the endoplasmic reticulum stress inhibitor sodium taurocholate (TUDCA) inhibited the levels of ATF6, BiP, and DERL3. Figure 7 A). To determine the specific endoplasmic reticulum stress pathway regulating DERL3 expression, we performed a comprehensive correlation analysis on the CGGA glioma dataset. Among the three major endoplasmic reticulum stress branches (PERK-eIF2α-ATF4, ATF6, and IRE1-XBP1 pathways), DERL3 showed the strongest positive correlation with ATF6. Figure 6 E). Subsequent experiments further confirmed this regulatory relationship: ATF6 knockdown significantly reduced DERL3 protein levels (E). Figure 7 B), while DERL3 silencing had no opposite effect on ATF6 expression (B). Figure 7 (C), which indicates that ATF6 regulates DERL3 in a unidirectional manner.

[0077] Functional characterization showed that endoplasmic reticulum stress activation significantly enhanced the proliferation, migration, and invasion capabilities of glioma cells. Figure 7 Importantly, this pro-tumor effect was significantly attenuated in DERL3 knockdown cells (DF). Figure 7 The results indicate that DERL3 mediates the malignant promotion of endoplasmic reticulum stress in gliomas. These findings establish the ATF6-DERL3 axis as an important mechanistic link between endoplasmic reticulum stress and glioblastoma progression, suggesting that targeting this pathway may be a novel strategy for the treatment of glioblastoma.

[0078] 4. DERL3 promotes the development of GBM malignancies by activating the NF-κB signaling pathway.

[0079] KEGG pathway enrichment analysis showed that, in the NF-κB signaling pathway, there was a significant enrichment of differentially expressed genes between the high-expression and low-expression groups of DERL3. Figure 8 A). Subsequent Western blot analysis showed that DERL3 knockdown led to a significant decrease in phosphorylated NF-κB protein levels, while total NF-κB expression levels remained unchanged. Figure 8 B). To further investigate the role of DERL3 in glioma progression via the NF-κB signaling pathway, we performed pharmacological inhibition experiments using the specific NF-κB inhibitor BAY 11-7082. Dose-response analysis in DERL3-overexpressing cells showed that 10 μmol of BAY 11-7082 effectively inhibited NF-κB protein expression (B). Figure 8 C). Notably, this treatment eliminated the pro-malignant effect induced by DERL3 overexpression, manifested as a significant reduction in glioma cell invasiveness (C). Figure 8 These findings collectively demonstrate that DERL3 promotes the malignant development of gliomas by activating the NF-κB signaling pathway.

[0080] 5. DELL3 interacts with HNRNPA2B1 and regulates its protein stability.

[0081] To further elucidate the molecular mechanism by which DERL3 promotes the malignant development of gliomas, we attempted to identify potential binding partners for DERL3. Through immunoprecipitation (IP) combined with mass spectrometry analysis, we discovered that HNRNPA2B1 may be an interacting protein with DERL3, a finding subsequently validated by co-immunoprecipitation (Co-IP) experiments. Figure 9 A). Interestingly, when DERL3 was knocked down, the HNRNPA2B1 protein level decreased significantly ( Figure 9 B), while its mRNA expression level remained unchanged (B). Figure 9 (C), indicating that the regulation of HNRNPA2B1 by DELL3 is at the posttranscriptional level.

[0082] To determine whether DERL3 stabilizes HNRNPA2B1 through direct binding, we performed a cyclohexylimide (CHX) tracking experiment to assess HNRNPA2B1 protein turnover. The results showed that DERL3 depletion significantly accelerated the degradation of HNRNPA2B1. Figure 9 D), which supports the role of DERL3 in maintaining the stability of HNRNPA2B1. Further investigation into the degradation mechanism revealed that the proteasome inhibitor MG132, rather than the lysosomal inhibitor chloroquine (CQ), could attenuate the degradation of HNRNPA2B1 (D). Figure 9 (EF), indicating that the turnover of HNRNPA2B1 is mainly mediated by the ubiquitin-proteasome pathway.

[0083] 6. DELL3 activates NF-κB signaling through an HNRNPA2B1-dependent mechanism.

[0084] To investigate the functional relationship between DELL3 and HNRNPA2B1 in NF-κB signaling pathway activation, we first established stable HNRNPA2B1 knockdown models in LN229 and U87 glioma cell lines. Western blot analysis showed that HNRNPA2B1 depletion significantly reduced the level of phosphorylated NF-κB (p-NF-κB) protein. Figure 10 A), and functional tests showed that the malignant phenotype was significantly impaired. MTS proliferation assays showed that, compared to the control group, the viability of HNRNPA2B1-deficient cells gradually decreased over time (A). Figure 10 B). Furthermore, Transwell migration and invasion assays showed that HNRNPA2B1 depleted cells exhibited significantly reduced migration and invasion capabilities. Figure 10 These results collectively indicate that knockdown of HNRNPA2B1 weakens the proliferation and metastasis of glioma cells.

[0085] To elucidate the hierarchical relationship between DERL3 and HNRNPA2B1 in glioma progression, we conducted rescue experiments by overexpressing DERL3 in HNRNPA2B1 knockdown cells and control cells. The results showed that DERL3 overexpression significantly enhanced the proliferation, migration, and invasion abilities of control cells; however, these oncogenic effects were completely absent in the context of HNRNPA2B1 deficiency. Figure 11 These results indicate that HNRNPA2B1, as a key downstream effector molecule, mediates DERL3-dependent NF-κB signaling activation, thereby promoting the malignant development of gliomas.

[0086] 7. DERL3 knockdown suppressed GBM progression in an intracranial xenograft model.

[0087] To verify the tumorigenic effect of DERL3 in vivo, we established an orthotopic xenograft model by intracranial implantation of U87 glioma cells into nude mice. Thirty-one days post-implantation, H&E staining of brain sections revealed a significant reduction in tumor volume compared to the control group (DELL3 knockdown group). Figure 12 AB). Importantly, Kaplan-Meier survival analysis showed that mice carrying DERL3-deficient tumors had significantly prolonged survival (AB). Figure 10 E). Immunohistochemical analysis confirmed the successful knockdown of DERL3. Figure 10 F), and it was found that the Ki67 proliferation index decreased simultaneously (F). Figure 10 The F-value indicates reduced tumor cell proliferation. These in vivo findings collectively demonstrate that DERL3 depletion effectively inhibits glioblastoma growth and improves host survival, further supporting its crucial role in glioma progression.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of DERL3 inhibitors in the preparation of drugs for the prevention or treatment of glioblastoma.

2. The application as described in claim 1, characterized in that, The DELL3 inhibitors mentioned above are shRNAs or siRNAs that target DELL3.

3. Application of NF-κB inhibitors in the preparation of drugs for treating glioblastoma, wherein the glioblastoma is characterized by high expression of DERL3.

4. The application as described in claim 3, characterized in that, The NF-κB inhibitor mentioned therein is BAY 11-7082 or a pharmaceutically acceptable salt thereof.

5. Application of a compound that inhibits the interaction between DERL3 and HNRNPA2B1 proteins in the preparation of an anti-glioma drug.

6. A pharmaceutical composition for treating glioblastoma, comprising: (a) DERL3 inhibitors or NF-κB inhibitors; and (b) Pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, The DELL3 inhibitors mentioned above are shRNAs or siRNAs that target DELL3.

8. Application of reagents for detecting DERL3 expression levels in the preparation of kits for the diagnosis, screening, and prognostic assessment of glioblastoma.

9. The application as described in claim 8, characterized in that, The aforementioned glioblastoma diagnostic screening kit detects the expression level of the DERL3 gene in samples, and the degree of DERL3 gene expression is positively correlated with the incidence of glioblastoma.

10. The application as described in claim 8, characterized in that, The aforementioned glioblastoma prognostic assessment kit detects the expression level of the DERL3 gene in the sample. The degree of intra-cancerous expression of the DERL3 gene is negatively correlated with the prognostic survival of glioblastoma.

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