Application of succinylation modification of PGAM1 protein at k225-k228 site in diagnosis, treatment or prognosis evaluation of glioblastoma

Abstract

The application discloses application of succinyl modification of a PGAM1 protein K225-K228 site in diagnosis, treatment or prognosis evaluation of glioblastoma, and belongs to the technical field of biomedical science and molecular biology. The application proves through experiments that the succinyl modification of the PGAM1 protein K225-K228 site is specifically highly expressed in GBM, the modification level is positively correlated with tumor malignancy grading, and can be used for evaluating the malignant degree of GBM in an ex vivo sample and prognosis of a patient; cell function experiments prove that the succinyl modification of the site positively regulates GBM proliferation, invasion and promotes glycolysis reprogramming, and blocking the modification of the site can inhibit the malignant phenotype of the tumor, and the succinyl level of the PGAM1 protein K225-K228 site can be used for diagnosis, treatment or prognosis evaluation of glioblastoma, and has important clinical application value.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical and molecular biology, specifically relating to the application of succinylation modification of the K225-K228 sites of the PGAM1 protein in the diagnosis, treatment or prognostic assessment of glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is the most common and most malignant primary tumor of the central nervous system, accounting for approximately 80% of all primary malignant intracranial tumors. Currently, the standard treatment for GBM is maximal surgical resection combined with radiotherapy and temozolomide (TMZ) chemotherapy. However, due to the invasive growth of the tumor, complete surgical eradication of the lesion is difficult, and temozolomide resistance is becoming increasingly prominent, resulting in a median survival of only about 12-15 months and a 5-year survival rate of less than 20%. Therefore, in-depth understanding of the molecular mechanisms of GBM malignant progression and treatment resistance, and the discovery of novel diagnostic biomarkers and therapeutic targets, are of significant clinical importance for improving patient prognosis.

[0003] Phosphoglycerate mutase 1 (PGAM1) is a key enzyme in the glycolysis pathway, catalyzing the conversion of 3-phosphoglycerate to 2-phosphoglycerate. It is highly expressed in various malignant tumors and is associated with poor prognosis. Previous studies have shown that PGAM1 expression is significantly upregulated in glioma tissues and can promote tumor proliferation and invasion by enhancing glycolytic flux or interacting with cytoskeletal proteins. In recent years, protein lysine succinylation has attracted widespread attention as a novel post-translational modification. The introduced succinylation group can significantly alter protein charge and spatial conformation, thereby regulating protein function. However, whether PGAM1 undergoes functional succinylation modification, and whether this modification participates in the regulation of glioma malignant progression, has not been reported domestically or internationally. Existing research has not elucidated the association between the level of succinylation modification at specific lysine sites of PGAM1 and the clinicopathological features of gliomas, nor has it explored the application value of this modification site as a diagnostic biomarker or potential therapeutic target for gliomas.

[0004] Previous studies have shown that the key glycolytic enzyme PGAM1 is abnormally expressed in gliomas and participates in regulating malignant processes such as tumor proliferation and invasion. However, the function and clinical significance of the specific succinylation modification of the PGAM1 protein at sites K225-K228 in gliomas have not yet been reported in current research.

[0005] Currently, there is a lack of effective biomarkers and detection methods in this field for the auxiliary diagnosis, malignancy grading, and prognostic assessment of gliomas based on the succinylation modification status at this site. Addressing the dual needs of glioma diagnosis and treatment and the development of targeted drugs, this invention proposes using succinylation modification at the PGAM1 K225-K228 site as a biomarker for glioma diagnosis and prognostic assessment. Simultaneously, this site can also serve as a novel drug target for anti-glioma drugs, laying the foundation for the subsequent development of diagnostic reagents and targeted drugs. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide the application of succinylation modification of the PGAM1 protein at the K225-K228 site in the diagnosis, treatment, or prognostic assessment of glioblastoma. This invention experimentally demonstrates that PGAM1 K225-K228 succinylation modification is specifically and highly expressed in GBM, and the modification level is positively correlated with the tumor malignancy grade, which can be used for the assessment of GBM malignancy and patient prognosis in in vitro samples. Cell function experiments demonstrate that succinylation modification at this site positively regulates GBM proliferation and invasion and promotes glycolytic reprogramming, and blocking modification at this site can inhibit the malignant phenotype of tumor.

[0007] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a biomarker for the diagnosis, treatment or prognostic assessment of glioblastoma, wherein the biomarker is succinylation modification of the PGAM1 protein at sites K225 and / or K228.

[0008] Based on the above technical solution, the glioblastoma further includes U87 and U251 cell lines.

[0009] Secondly, the present invention provides the application of the above-mentioned biomarkers as therapeutic targets in the preparation of drugs for treating glioblastoma.

[0010] Based on the above technical solution, the glioblastoma further includes U87 and U251 cell lines.

[0011] Based on the above technical solution, the drug further includes pharmaceutically acceptable excipients.

[0012] Based on the above technical solution, the pharmaceutically acceptable excipients further include fillers, binders, disintegrants, and emulsifiers.

[0013] Based on the above technical solution, the dosage form of the drug further includes tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

[0014] Thirdly, the present invention provides the application of the above-mentioned biomarkers in the preparation of reagents or kits for diagnosing glioblastoma.

[0015] Based on the above technical solution, the glioblastoma further includes U87 and U251 cell lines.

[0016] Based on the above technical solution, the detection methods of the reagents or kits further include immunoprecipitation, immunohistochemistry, Western blotting, and immunofluorescence.

[0017] Fourthly, the present invention provides the application of the above-mentioned biomarkers in the preparation of reagents or kits for prognostic assessment of glioblastoma.

[0018] Based on the above technical solution, the glioblastoma further includes U87 and U251 cell lines.

[0019] Based on the above technical solution, the detection methods of the reagents or kits further include immunoprecipitation, immunohistochemistry, Western blotting, and immunofluorescence.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention experimentally verified that the succinylation level of the PGAM1 protein at the K225-K228 sites is significantly upregulated in glioma tissues and glioma cells, and is positively correlated with the pathological grade of glioma. Detection of the succinylation level at this site using immunohistochemistry, immunoprecipitation, and Western blotting can effectively distinguish glioma patients from normal individuals and assess the malignancy and prognosis of gliomas. Further functional experiments confirmed that succinylation mimicking this site (K225E-K228E mutation) significantly promotes the proliferation, migration, and invasion of glioma cells, and enhances PGAM1 enzyme activity and glycolytic flux; while desuccinylation mimicking mutations (K225R-K228R) significantly inhibit the above malignant phenotypes. Therefore, the succinylation level of the PGAM1 protein at the K225-K228 sites can serve as an independent biomarker for glioma diagnosis and prognostic assessment, possessing significant clinical application value. Attached Figure Description

[0021] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0022] Figure 1 This is a diagram of the PGAM1 gene structure.

[0023] Figure 2 This is a protein spatial structure diagram of PGAM1.

[0024] Figure 3The image shows the expression levels of PGAM1 in glioma tissues of different grades in Example 1. In the image, A is an immunohistochemical image and B is a statistical graph of the average optical density value.

[0025] Figure 4 Figure 1 shows the overall succinylation levels of glioma U87 and U251 cells, normal astrocytes, cancerous tissue, and adjacent cancerous tissue in Example 1. Figure A is a Western blot diagram of the overall succinylation levels of glioma U87 and U251 cells and normal astrocytes; Figure B is a statistical graph of Figure A; Figure C is a Western blot diagram of the overall succinylation levels of cancerous tissue and adjacent cancerous tissue; and Figure D is a statistical graph of Figure C.

[0026] Figure 5 The image shows the results of PGAM1 succinylation levels in glioma U87 and U251 cells and normal astrocytes in Example 1.

[0027] Figure 6 In Example 2, Vector and PGAM1 were transfected respectively. WT PGAM1 K225E-K228E PGAM1 K225R-K228R Viral transfection efficiency of four glioma U87 cell lines.

[0028] Figure 7 In Example 2, Vector and PGAM1 were transfected respectively. WT PGAM1 K225E-K228E PGAM1 K225R-K228R Viral transfection efficiency of four glioma U251 cell lines.

[0029] Figure 8 In Example 2, Vector and PGAM1 were transfected respectively. WT PGAM1 K225E-K228E PGAM1 K225R-K228R The expression level of Flag-Tag on the PGAM1 sequence in four types of glioma U87 cells (A) and glioma U251 cells (B).

[0030] Figure 9 This study examines the effect of succinylation of PGAM1K225-K228 sites on the proliferation capacity of U87 cells in Example 3.

[0031] Figure 10 This study examines the effect of succinylation of PGAM1K225-K228 sites on the proliferation capacity of U251 cells in Example 3.

[0032] Figure 11This is a statistical result graph showing the effect of succinylation of PGAM1 at K225-K228 sites on the proliferation ability of U87 cells (A) and U251 cells (B) in Example 3.

[0033] Figure 12 This study examines the effect of succinylation of PGAM1 at K225-K228 sites on the migration ability of U87 cells in Example 3.

[0034] Figure 13 This describes the effect of succinylation of PGAM1 at K225-K228 sites on the migration ability of U251 cells in Example 3.

[0035] Figure 14 The figure shows the statistical results of the effect of PGAM1 K225-K228 succinylation on the migration ability of U87 and U251 cells in Example 3. In the figure, A is the cell migration result of U87 cells at 24h, B is the cell migration result of U87 cells at 48h, C is the cell migration result of U251 cells at 24h, and D is the cell migration result of U251 cells at 48h.

[0036] Figure 15 The effect of succinylation of PGAM1 at K225-K228 sites on the invasive ability of U87 cells (A) and U251 cells (B) in Example 3.

[0037] Figure 16 This is a statistical result graph showing the effect of succinylation of PGAM1 at K225-K228 sites on the invasive ability of U87 cells (A) and U251 cells (B) in Example 3.

[0038] Figure 17 The image shows the effect of succinylation of PGAM1 at K225-K228 sites on the growth capacity of gliomas in mice in Example 4. In the image, A is a picture of a tumor-bearing mouse, B is a picture of a glioma, C is the volume of the glioma, and D is the weight of the glioma.

[0039] Figure 18 The effect of succinylation of PGAM1 at K225-K228 sites on the proliferation ability of gliomas in mice in Example 4 is shown in A, where Ki67 detection and CD31 detection are used.

[0040] Figure 19 The effects of succinylation of PGAM1 at K225-K228 sites on the binding pocket activity and enzyme activity of PGAM1 protein in Example 5 are shown in Figure A, which compares the binding pocket activity of PGAM1 protein before and after succinylation; Figure B compares the enzyme activity of PGAM1 in U87 cells that have been stably transformed; and Figure C compares the enzyme activity of PGAM1 in U251 cells that have been stably transformed.

[0041] Figure 20 The effect of succinylation of PGAM1 at K225-K228 sites on extracellular acidification rate (ECAR) in Example 5 is shown in Figure A, where A is a glycolytic function curve and B is a graph showing the results of basal glycolysis and glycolytic capacity.

[0042] Figure 21 The effect of succinylation of PGAM1 at K225-K228 sites on oxygen consumption (OCR) in Example 5 is shown in Figure A, where A is a mitochondrial respiration curve and B is a graph showing the results of basal respiration, maximal respiration, and ATP production.

[0043] Figure 22 The effect of succinylation of PGAM1 at K225-K228 sites on glucose consumption and lactate production in U87 and U251 cells in Example 5 is shown. In this example, A represents lactate production in U87 and U251 cells, and B represents glucose consumption in U87 and U251 cells.

[0044] Figure 23 The expression levels of ACTA2 in normal tissues and gliomas in Example 6 are shown in (A) and the effect of high ACTA2 expression on prognosis is shown in (B).

[0045] Figure 24 The correlation between ACTA2 expression and glioma grade in Example 6 (A) and the effect of PGAM1 expression on ACTA2 expression (B).

[0046] Figure 25 The correlation between ACTA2 expression and glioma grade in human glioma tissue in Example 6.

[0047] Figure 26 The effect of succinylation of PGAM1 at K225-K228 sites on ACTA2 expression level in Example 6 is shown in Figure A, where A is a Western blot band diagram and B is a statistical graph of Western blot results.

[0048] Figure 27 Vector and PGAM1 in Example 6 WT PGAM1 K225E-K228E PGAM1 K225R-K228R The effect of ACTA2 distribution on four glioma U87 cell lines.

[0049] Figure 28 Vector and PGAM1 in Example 6 WT PGAM1 K225E-K228E PGAM1 K225R-K228R The effect of ACTA2 distribution on four glioma U251 cell lines.

[0050] Figure 29 The co-localization of ACTA2 and PGAM1 in the U87 cell line (A) and U251 cell line (B) in Example 6 is shown.

[0051] Figure 30 The effect of succinylation of PGAM1 at sites K225-K228 in Example 7 on the binding ability of ACTA2 to PGAM1.

[0052] Figure 31 This study describes the effect of succinylation of PGAM1 at K225-K228 sites on the expression level of F-actin in U87 cells, as shown in Example 8.

[0053] Figure 32 This study describes the effect of succinylation of PGAM1 at K225-K228 sites on the expression level of F-actin in U251 cells, as shown in Example 8. Detailed Implementation

[0054] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0055] Unless otherwise specified in the examples, standard conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0056] Example 1: Expression and succinylation level of PGAM1 in glioma tissue (1) Collection of clinical samples Nine glioma tissue samples were collected from different grades (high-grade and low-grade) and paired adjacent normal tissue, three from each grade. All samples were obtained from the Northern Theater General Hospital of the Chinese People's Liberation Army, with approval from the hospital's ethics committee and informed consent from the patients. Immediately after surgery, the samples were divided into two portions: one portion was stored at -80°C for protein extraction, and the other portion was fixed in 4% paraformaldehyde for 48 hours before paraffin embedding.

[0057] (2) Detection of PGAM1 expression The fixed tissue was dehydrated using a gradient of ethanol (50% ethanol for 30 minutes, 60% ethanol for 30 minutes, 75% ethanol overnight, 95% ethanol for 30 minutes, anhydrous ethanol I for 30 minutes, and anhydrous ethanol II for 30 minutes), then cleared with xylene (xylene I for 5 minutes and xylene II for 30 minutes), and then immersed in xylene-paraffin (1:1) for 30 minutes, followed by sequential immersion in paraffin I for 2 hours, paraffin II for 2 hours, and paraffin III for 2 hours. Finally, it was embedded into paraffin blocks using an embedding machine. Sections with a thickness of 5 μm were cut using a paraffin microtome, spread in a 40°C water bath, retrieved using a detachable slide, and dried at 60°C for 2 hours. The slides were sequentially immersed in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% ethanol for 5 minutes, 90% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, 50% ethanol for 5 minutes, and distilled water for 5 minutes (repeated 3 times) for dewaxing and rehydration. The slides were then placed in sodium citrate buffer (pH 6.0, one packet of powder dissolved in 1000 mL of distilled water), and microwaved on high until boiling, then on medium heat for 15 minutes (adding buffer every 5 minutes). The slides were then allowed to cool naturally to room temperature to complete antigen retrieval. The tissue area was circled with an immunohistochemical pen, and endogenous peroxidase inhibitor was added. The slides were incubated at room temperature for 10 minutes, then washed three times with PBS (pH 7.4) for 3 minutes each time. A non-specific staining inhibitor was added, and the slides were incubated at room temperature for 10 minutes, then discarded. Add anti-PGAM1 antibody (Proteintech, 16126-1-AP, diluted 1:200 with antibody dilution buffer), incubate overnight at 4°C in a humidified chamber, then wash three times with PBS for 3 minutes each time. Add biotin-labeled IgG polymer, incubate at room temperature for 10 minutes, then wash three times with PBS. Add streptomycin-peroxidase, incubate at room temperature for 10 minutes, then wash three times with PBS. Freshly prepared DAB chromogenic solution (50 μL each of reagents 1, 2, and 3 added sequentially to 1 mL of distilled water) is added to the tissue, and the color development is observed under a microscope, usually for 2-5 minutes, then stopped by running water. Counterstain with hematoxylin for 15 seconds, rinse with running water for 10 minutes to achieve blue return. Dehydrate and clear the tissue sequentially with 70%, 80%, 90%, and 100% ethanol for 10 seconds each, and xylene I and xylene II for 5 minutes each, then mount with neutral resin. Take microscopic images and analyze the average optical density using Image-ProPlus 6.0 software.

[0058] (3) Detection of total succinylation level Normal human astrocytes (NHA), glioma cells U87 and U251 (all purchased from ATCC) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and incubated at 37°C in a 5% CO2 incubator. When the cells reached 80%-90% confluence, the medium was discarded, and the cells were washed twice with pre-cooled PBS. Cells were scraped off with a cell scraper and collected into centrifuge tubes. The cells were centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. An appropriate amount of RIPA lysis buffer (containing 1% protease inhibitor PMSF and 1% phosphatase inhibitor before use) was added, and the cells were lysed on ice for 30 minutes, vortexing once every 10 minutes. Cells were then disrupted using an ultrasonic disruptor at 200 W, sonicated for 2 seconds followed by a 5-second interval, for a total of 3 times. The cells were then centrifuged at 4°C and 12000×g for 30 minutes, and the supernatant was collected. Protein quantification was performed using the BCA protein concentration assay kit: BSA standard (25 mg / mL) was diluted to 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.025, and 0 mg / mL. 20 μL of the standard or the sample to be tested was added to a 96-well plate, and 200 μL of BCA working solution (solution A:solution B = 50:1) was added to each well. The plate was incubated at 37°C for 30 minutes, and the absorbance at 562 nm was measured using a microplate reader. A standard curve was plotted, and the protein concentration of the sample to be tested was calculated.

[0059] Take an equal volume of protein (30 μg), add 5×SDS-PAGE loading buffer, and boil at 95℃ for 5 minutes. Prepare a 10% separating gel and a 5% stacking gel (separating gel formulation: 7.9 mL distilled water, 6.7 mL 30% acrylamide, 5 mL 1.5M Tris-HCl pH 8.8, 200 μL 10% SDS, 200 μL 10% AP, 12 μL TEMED; stacking gel formulation: 6.8 mL distilled water, 1.7 mL 30% acrylamide, 1.25 mL 1.5M Tris-HCl pH 6.8, 100 μL 10% SDS, 100 μL 10% AP, 10 μL TEMED). Perform electrophoresis in a vertical electrophoresis tank, starting at 80 V for 30 minutes, then adjusting to 130 V until the bromophenol blue reaches the bottom. Wet transfer was used, with a constant current of 200 mA and transfer in an ice bath for 1.5 hours to transfer proteins to PVDF membranes (activated with methanol for 15 seconds before use). The membranes were blocked with 5% skim milk at room temperature for 2 hours, then incubated overnight at 4°C with pansuccinylation antibody (PTM-419, 1:1000, Jingjie Biotechnology). The next day, the membranes were washed three times with TBST (TBS containing 0.1% Tween-20) for 10 minutes each time, then incubated for 1 hour with HRP-labeled goat anti-rabbit secondary antibody (1:5000) at room temperature, followed by three washes with TBST. ECL chemiluminescence reagent was added, and the membranes were exposed using a gel imaging system. The same method was used to detect glioma tissues of different grades and paired adjacent normal tissues.

[0060] (4) Detection of PGAM1 succinylation level Immunoprecipitation was used to detect succinylation levels: NHA, U87, and U251 cells were collected (approximately 1 × 10⁻⁶ cells). 7 Wash twice with pre-cooled PBS, add 500 μL IP lysis buffer (containing 1 mM PMSF), lyse on ice for 30 minutes, sonicate (parameters same as (3) detection of total succinylation level), centrifuge at 4℃ and 14000×g for 20 minutes, and take the supernatant; take 50 μL of supernatant as input. Take 500 μL of supernatant, add 2 μg of anti-PGAM1 antibody (Proteintech, 16126-1-AP), slowly rotate and incubate overnight at 4℃, add 40 μL of Protein A+G magnetic beads the next day, continue incubation for 4 hours, separate with a magnetic rack, discard the supernatant, wash 6 times with IP washing buffer, 200 μL each time, add 30 μL of 1×SDS loading buffer, boil at 95℃ for 5 minutes, perform Western blot (steps same as (3) detection of total succinylation level), and detect with anti-PGAM1 antibody and anti-succinylation antibody respectively.

[0061] The results are as follows Figure 5As shown, the experiment found that the succinylation level of PGAM1 protein in glioma cells was significantly higher than that in normal astrocytes, confirming that PGAM1 protein succinylation is significantly upregulated in gliomas.

[0062] Example 2: Construction of a PGAM1-stabilized human glioma cell line (1) Construction of lentiviral vector PGAM1 wild-type (WT) cDNA and mutant sequences K225E-K228E (simulated succinylation) and K225R-K228R (simulated desuccinylation) were cloned into the lentiviral vector LV5 (containing the EF-1a promoter, GFP tag, and Flag tag), synthesized by Suzhou Gemma Gene Co., Ltd., with a viral titer of 1×10⁻⁶. 8 TU / mL.

[0063] (2) Lentiviral transfection Take U87 and U251 cells in the logarithmic growth phase, and use 2×10 4 Cells were seeded per well in a 24-well plate, with 400 μL of LMEM complete medium added to each well. The plates were incubated at 37°C and 5% CO2 for 24 hours. The medium was then discarded, and 200 μL of fresh complete medium was added to each well. The viral volume was calculated based on MOI=30 (viral volume (μL) = MOI × cell number / viral titer (TU / mL) × 1000). The calculated volume of lentivirus solution (Vector, WT, K225E-K228E, and K225R-K228R corresponded to different wells) was added, and polybrene was added to a final concentration of 5 μg / mL. A control group (virus-free) was set up. The mixture was gently mixed, and the plates were incubated at 37°C for 24 hours. The culture medium was then replaced with normal complete medium.

[0064] (3) Screening for puromycin Determination of the minimum lethal concentration (MLC) of puromycin: Untransfected U87 and U251 cells were seeded in 24-well plates. When the cell density reached 80%, culture medium containing 0, 1, 2, 4, 6, 8, and 10 μg / mL puromycin, respectively, was added. After 48 hours, the minimum concentration at which all cells died was observed, and the MLC for both U87 and U251 was determined to be 2 μg / mL. 72 hours after lentivirus transfection, the medium was replaced with complete culture medium containing 2 μg / mL puromycin. The selection medium was changed every 3 days for 2 weeks. During this period, the GFP positivity rate was observed using an inverted fluorescence microscope. When the positivity rate reached >90%, the culture was expanded.

[0065] (4) Verification of transfection efficiency The percentage of GFP-positive cells was calculated by taking images under bright field and blue excitation light (488 nm) using an inverted fluorescence microscope. Cell proteins from each group were collected (using the same method as in Example 1) and detected by Western blot using anti-Flag antibody (Proteintech, 66008-4-Ig, 1:3000).

[0066] The results are as follows Figure 8 As shown in the figure, the experiment found that transfection with PGAM1... WT PGAM1 K225E-K228E PGAM1 K225R-K228R Both U87 and U251 cells stably expressed Flag-Tag, while the Vector group did not, proving that the stable cell line was successfully constructed.

[0067] Example 3: The proliferation, migration, and invasion capabilities of glioma cells after PGAM1 succinylation. (1) Cell proliferation capacity detection The stable U87 and U251 cells from the above groups were treated at a rate of 4 × 10⁶. 3 Cells were seeded in 96-well plates with 100 μL of culture medium per well and incubated at 37°C for 24 hours. Following the EdU imaging kit procedure, 100 μL of 2× EdU working solution (10 mM EdU stock solution diluted to 100 μM with complete culture medium) was added to each well to a final concentration of 50 μM, and incubated for 2 hours. The culture medium was discarded, and 100 μL of 4% paraformaldehyde was added to each well for fixation at room temperature for 15 minutes, followed by washing twice with PBS. 100 μL of 0.5% Triton X-100 was added to each well for permeation for 20 minutes, followed by washing twice with PBS. Click reaction solution (containing 430 μL 1× reaction buffer, 20 μL CuSO4, 0.5 μL Cy3-azide, and 50 μL Click additive per 500 μL) was added to each well, and incubated at room temperature in the dark for 30 minutes. The solution was aspirated, and 100 μL of 3% BSA was added to each well for washing three times with PBS. 100 μL of [unclear text - possibly a continuation of the previous sentence] was added to each well. Stain nuclei with Hoechst 33342 (1:2000 dilution) for 15 minutes, wash 3 times with PBS; take pictures using an inverted fluorescence microscope (Cy3 channel and DAPI channel), randomly select 5 fields of view from each well, and calculate the proportion of EdU positive cells (number of EdU positive cells / number of DAPI-stained cells × 100%).

[0068] (2) Cell migration ability detection Stable U87 and U251 cells from each group were cultured at a rate of 3 × 10⁻⁶. 5Cells were seeded per well in 6-well plates with 2 mL of complete culture medium per well and incubated at 37°C until complete confluence (approximately 24 hours). A straight vertical scratch was made at the bottom of each well using a 200 μL sterile pipette tip. Cells were gently washed three times with PBS to remove detached cells, and 2 mL of serum-free DMEM was added to each well. Images were taken at the same location under an inverted microscope at 0, 24, and 48 hours. The scratch area was measured using ImageJ software, and the wound healing rate (%) was calculated as: (scratch area at 0 hours - scratch area at each time point) / scratch area at 0 hours × 100%.

[0069] (3) Detection of cell invasion ability Matrigel was thawed overnight at 4°C, diluted 1:5 with pre-chilled serum-free DMEM, and 50 μL of the diluted gel was vertically added to the upper chamber of a Transwell chamber (8 μm pore size). The cells were then incubated at 37°C for 3 hours to solidify. Stable-transfected U87 and U251 cells from each group were trypsinized, centrifuged (1000 rpm, 5 min), resuspended in serum-free DMEM, and their density adjusted to 5 × 10⁶ cells / mL. 5 cells / mL, take 100 μL of cell suspension (5 × 10⁻⁶ cells / mL). 4 Add cells to the upper chamber, and add 600 μL of DMEM medium containing 20% ​​FBS to the lower chamber. Incubate at 37°C for 24 hours. Remove the chamber, wash twice with PBS, gently wipe away uninvaded cells from the upper chamber with a cotton swab, fix the chamber in methanol for 20 minutes, air dry, stain with 0.1% crystal violet for 20 minutes, and rinse with running water. Take pictures of 5 random fields of view under a microscope and count the number of invasive cells.

[0070] The results are as follows Figure 11 , Figure 14 and Figure 16 As shown, experiments revealed similarities with Vector and PGAM1. K225R-K228R Compared to the group, PGAM1 WT Group and PGAM1 K225E-K228E The proportion of EdU-positive cells, scratch healing rate, and number of Transwell-invading cells in the group were significantly increased, indicating that PGAM1 K225-K228 succinylation significantly promotes the proliferation, migration, and invasion of glioma cells.

[0071] Example 4: Effect of PGAM1 on the growth of gliomas in vivo (1) Construction of a subcutaneous ectopic xenograft model in nude mice The nude mice were purchased from Beijing Huafukang Experimental Animal Technology Co., Ltd. (Animal Quality Certificate No. 110322231100568971). Fourteen healthy male Balb-c nu mice, aged 6-8 weeks and weighing 18-20 g, were purchased. The nude mice were housed at the SPF-grade Experimental Animal Center of Shenyang Pharmaceutical University (housed under conditions of 25±2℃ and 40%-60% humidity, Experimental Unit Use License No.: SYXK (Liaoning) 2021-0009), and were approved by the Animal Ethics Committee of Shenyang Pharmaceutical University (Ethics Review No.: SYPU-IACUC-S2023-0306-101).

[0072] U87 stable cells (PGAM1) in logarithmic growth phase were collected. WT Group and PGAM1 K225R-K228R (Group 1), discard the culture medium, wash once with 5 mL pre-cooled PBS, add 1 mL 0.25% trypsin-EDTA, digest at 37°C for 2 minutes until cells become rounded and detach, add 2 mL DMEM containing 10% FBS to stop digestion. Transfer the cell suspension to a 15 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 minutes at room temperature, discard the supernatant, resuspend in 5 mL PBS, centrifuge and wash once again; discard the supernatant, resuspend the cells in serum-free DMEM, mix 20 μL of cell suspension with 20 μL of 0.4% trypan blue solution, count viable cells using a cell counting chamber, and adjust the cell concentration to 4 × 10⁶ cells / mL. 7 cells / mL. Thaw the Matrigel overnight at 4°C, keeping it on ice until use. In a 1.5 mL sterile EP tube, take an equal volume of cell suspension and gently pipette it into the Matrigel (1:1 volume ratio) to mix thoroughly (avoiding air bubbles). The final result should be 2 × 10⁶ cells / mL. 7 The mixed suspension of cells / mL was prepared, and all operations were performed under ice bath conditions in a laminar flow hood. The skin of the right axilla of nude mice was disinfected with 75% alcohol swabs. Using a 1 mL sterile syringe, 100 μL of the cell-Matrigel mixed suspension was drawn, air was expelled, and the syringe was inserted subcutaneously at a 30-degree angle and slowly injected. Small wheals were observed to form locally. The mice were fed for 30 days post-inoculation. Starting from day 3 post-inoculation, the long diameter (L) and short diameter (W) of the tumor were measured every 3 days using an electronic digital caliper, calculated using the formula V = L × W. 2 / 2 The tumor volume was calculated, and the weight of the nude mice was measured using an electronic balance. After 30 days of feeding, the nude mice were euthanized by cervical dislocation. The tumor tissue was completely dissected using sterile surgical scissors and forceps. Bloodstains were rinsed with PBS, dried with filter paper, and the tumor weight was measured and photographed. The dissected tumor tissue was fixed in 4% paraformaldehyde for 48 hours, followed by gradient ethanol dehydration (70%, 80%, 90%, 95%, and 100% for 1 hour each), xylene clearing (15 minutes × 2 times), paraffin embedding, and then 5 μm thick sections were cut using a paraffin microtome for later use.

[0073] (2) Detection of proliferation and angiogenesis capacity The above paraffin sections were subjected to HE staining. The sections were dewaxed with xylene (10 minutes × 2 times), rehydrated with a series of ethanol solutions (100%, 95%, 90%, 80%, and 70% ethanol for 5 minutes each), washed with distilled water for 2 minutes, stained with hematoxylin for 8 minutes, rinsed with running water for 10 minutes, differentiated with 1% hydrochloric acid ethanol for 5 seconds, rinsed with running water for 10 minutes, stained with eosin for 30 seconds, rinsed with running water for 2 minutes, dehydrated with a series of ethanol solutions, cleared with xylene, mounted with neutral resin, and observed and photographed under an upright microscope.

[0074] Immunohistochemical detection of Ki-67: After dewaxing and rehydration of paraffin sections, microwave antigen retrieval was performed using sodium citrate buffer (pH 6.0) (95-100℃, 15 minutes). After cooling, endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 minutes, followed by blocking with 5% bovine serum albumin for 30 minutes. Anti-Ki-67 antibody (Abcam, ab15580, 1:200) was added and incubated overnight in a humidified chamber at 4℃. The next day, HRP-labeled goat anti-rabbit secondary antibody (1:500) was added and incubated at room temperature for 30 minutes. DAB staining was performed, followed by hematoxylin counterstaining, dehydration, clearing, and mounting. Microscopic images were taken, and the percentage of Ki-67 positive cells (number of positive cells / total number of cells × 100%) was calculated using Image-Pro Plus 6.0 software.

[0075] Immunofluorescence detection of CD31: After dewaxing and rehydration of paraffin sections and antigen retrieval, the sections were permeabilized with 0.3% Triton X-100 for 15 minutes, blocked with 5% BSA for 30 minutes, and incubated overnight at 4°C with anti-CD31 antibody (Abcam, ab28364, 1:100). The next day, the sections were incubated with Alexa Fluor 488-labeled goat anti-rabbit secondary antibody (1:500) at room temperature in the dark for 1 hour, and the nuclei were stained with DAPI for 5 minutes. The sections were mounted with anti-fluorescence attenuation mounting medium, observed and photographed under a confocal microscope, and the density of CD31-positive microvessels was calculated (the number of CD31-positive vessels was counted per 200x field of view).

[0076] The results are as follows Figure 17-18 As shown, experiments revealed that it is related to PGAM1 K225R-K228R Compared to the group, PGAM1WT The tumor volume and weight of the group were significantly increased. HE staining showed deeply stained nuclei, microvascular proliferation, and a significant increase in the number of Ki-67 positive cells and the density of CD31 positive blood vessels. This indicates that PGAM1 promotes tumor growth, proliferation, and angiogenesis in vivo.

[0077] Example 5: Changes in enzyme activity and glycolytic metabolism after succinylation of PGAM1 at K225-K228 sites (1) Dynamic simulation The crystal structure of human PGAM1 (PDB ID: 4GPI) was downloaded from the RCSB PDB database. Molecular dynamics simulations were performed on wild-type and K225E-K228E mutant PGAM1 to analyze root mean square deviation (RMSD) and root mean square fluctuation (RMSF), and an activity pocket diagram was plotted.

[0078] (2) Detection of PGAM1 enzyme activity Collect stable U87 and U251 cells (Vector, WT, K225E-K228E, K225R-K228R) from the above groups, lyse them on ice for 30 minutes using RIPA lysis buffer containing 1% PMSF, sonicate (200 W, 2 seconds / 5 seconds interval, 3 times), centrifuge at 12000×g at 4℃ for 30 minutes, collect the supernatant, and quantify using the BCA method.

[0079] Prepare reaction solutions A and B: Reaction solution A contains 100 mM Tris-HCl (pH 8.0), 100 mM KCl, 5 mM MgCl2, 1 mM ADP, 0.2 mM NADH, 0.5 units / mL enolase, 0.5 units / mL pyruvate kinase M1, and 0.1 units / mL lactate dehydrogenase; reaction solution B contains 100 mM Tris-HCl (pH 8.0) and 2 mM 3-PG. Add 20 μg of total protein to each well of a 96-well plate (adjust to a total volume of 20 μL with lysis buffer), add 100 μL of reaction solution A, and incubate at 37°C for 20 minutes. Then add 50 μL of reaction solution B to each well. Immediately measure the absorbance at 340 nm using a microplate reader, reading every 30 seconds for a total of 5 minutes. Enzyme activity is expressed as the NADH consumption rate (ΔA). 340 The protein content is expressed as / min / μg protein.

[0080] (3) Detection of cell oxygen consumption rate and acidification rate Using Seahorse XFe96 cell culture plates, each group of stably transfected U87 cells was cultured at 2×10⁶ cells / mL. 4Cells were seeded per well with 80 μL of complete culture medium and incubated overnight (approximately 16-18 hours) at 37°C and 5% CO2, ensuring uniform cell adhesion. The original culture medium was discarded, and 180 μL of Seahorse XF basal medium (containing 2 mM GlutaMAX, pH adjusted to 7.4) was added to each well. The wells were then incubated in a CO2-free incubator (37°C) for 1 hour to equilibrate. Following the XF Glycolysis Stress Test Kit procedure, glucose (final concentration 10 mM), oligomycin (final concentration 1 μM), and 2-DG (final concentration 50 mM) were added to each well sequentially. The instrument automatically measured the ECAR value in mpH / min and calculated non-glycolytic acid production, glycolytic activity, and glycolytic capacity. Following the XF Cell Mito Stress Test Kit procedure, oligomycin (1 μM), FCCP (1 μM), and antimycin A / rotenone (0.5 μM) were added sequentially. The OCR value was measured in pmol / min, and basal respiration, ATP-related oxygen consumption, proton leakage, maximum respiration, and reserve respiration capacity were calculated.

[0081] (4) Detection of glucose consumption and lactic acid production Stable U87 and U251 cells from each group were injected at a dose of 1×10⁻⁶. 6 Cells were seeded in 6 cm culture dishes and collected after 24 hours of culture. The cells were washed twice with pre-cooled PBS, and 0.3 mL of homogenizing medium (0.1 M PBS, pH 7.4) was added. The cells were sonicated (parameters as described above for PGAM1 enzyme activity detection), centrifuged at 10000×g for 10 minutes at 4°C, and the supernatant was collected. The glucose content (mmol / g protein) was calculated using a glucose assay kit at 340 nm. The cell culture supernatant from each group was collected (after 24 hours), and the lactate concentration (mmol / L protein) was calculated using a lactate assay kit at 530 nm.

[0082] The results are as follows Figure 19-22 As shown, experiments revealed that succinylation modification of PGAM1 increased the overall amino acid fluctuation and enlarged the active pocket, and PGAM1 WT Group and PGAM1 K225E-K228E The PGAM1 enzyme activity in the group was significantly higher than that in the Vector group and the PGAM1 group. K225R -K228R In addition, the extracellular acidification rate, oxygen consumption rate, glucose consumption, and lactate production were significantly increased in the succinylation group. This indicates that PGAM1 succinylation promotes glioma metabolic reprogramming by enhancing enzyme activity and increasing glycolysis flux.

[0083] Example 6: Expression level and distribution of ACTA2 in glioma after succinylation of PGAM1 at K225-K228 sites. (1) Bioinformatics analysis Log in to GEPIA (http: / / gepia.cancer-pku.cn), enter "ACTA2", select "GBM" and "LGG" vs "Normal", and obtain expression box plots and overall survival curves. Log in to CGGA (http: / / www.cgga.org.cn) to query the expression of ACTA2 in gliomas of different grades, and log in to TIMER2.0 (http: / / timer.cistrome.org) to analyze the correlation between ACTA2 and PGAM1 expression.

[0084] (2) Verify the expression of ACTA2 The method is the same as the immunohistochemical (IHC) detection procedure in Example 1, and the primary antibody is anti-ACTA2 antibody (Proteintech, 55135-1-AP, 1:200).

[0085] (3) Detection of colocalization of PGAM1 and ACTA2 and distribution of ACTA2 Place sterile round coverslips into a 24-well plate and add 3×10⁻⁶ drops to each well. 4 U87 or U251 cells (normal culture, non-stable transformation) were cultured for 24 hours. The culture medium was discarded, and the cells were washed three times with PBS. 400 μL of pre-chilled acetone was added to each well for fixation for 20 minutes, followed by three 5-minute PBS washes. 0.1% Triton X-100 was added for permeation for 5 minutes, followed by three PBS washes. The cells were blocked with 5% BSA for 1 hour, simultaneously adding anti-PGAM1 antibody (1:100) and anti-ACTA2 antibody (1:100), and incubated overnight at 4°C. The cells were washed three times with PBS, and simultaneously adding Alexa Fluor 488-labeled goat anti-rabbit IgG and Alexa Fluor 594-labeled goat anti-mouse IgG (1:500), and incubated at room temperature in the dark for 1 hour. The cells were washed three times with PBS, and the nuclei were stained with DAPI for 5 minutes. The slides were mounted and photographed using a confocal microscope.

[0086] For the ACTA2 distribution detection, after each group of stable cells were smeared, fixed, permeabilized, and blocked according to the above steps, the distribution intensity of ACTA2 in the cytoplasm was observed by staining with only anti-ACTA2 antibody (1:100) and the corresponding Alexa Fluor 594 secondary antibody (1:500).

[0087] (4) Detection of total ACTA2 protein Proteins from stable U87 cells in each group were collected (using the same method as in Example 1). Western blot was performed using anti-ACTA2 antibody (Proteintech, 55135-1-AP, 1:2000) and β-actin was used as an internal control (Proteintech, 81115-1-RR, 1:5000).

[0088] The results are as follows Figure 23-29 As shown, experiments revealed that PGAM1 and ACTA2 are co-localized in the cytoplasm and perinuclear region; and with PGAM1 WT Group and PGAM1 K225E-K228E Compared to the group, PGAM1 K225R-K228R The cytoplasmic distribution of ACTA2 was significantly reduced in the group, while there was no significant difference in the total protein expression level of ACTA2 among the groups. This indicates that PGAM1 succinylation affects the cellular distribution of ACTA2, but does not change its total protein expression level.

[0089] Example 7: Binding of PGAM1 to ACTA2 after succinylation of PGAM1 at K225-K228 sites. PGAM1 and ACTA2 binding ability test Collect stable U87 cells from each group (Vector, WT, K225E-K228E, K225R-K228R), 1×10 7 Cells were added to 500 μL of IP lysis buffer (containing 1 mM PMSF), lysed on ice for 30 minutes, and then sonicated (200 W, 2 seconds / 5 seconds interval, 3 times). The cells were centrifuged at 14000×g for 20 minutes at 4°C, and the supernatant was collected. 50 μL of the supernatant was used as input. 500 μL of the supernatant was added to 2 μg of anti-PGAM1 antibody and incubated overnight at 4°C. The next day, 40 μL of Protein A+G magnetic beads were added and incubated at 4°C for 4 hours. Cells were separated using a magnetic rack, the supernatant was discarded, and the cells were washed 6 times with 200 μL of IP washing buffer each time. 30 μL of 1×SDS loading buffer was added, and the cells were boiled at 95°C for 5 minutes. Western blot was performed, and the cells were detected with anti-PGAM1 antibody and anti-ACTA2 antibody, respectively. Reverse Co-IP was performed to enrich the cells with anti-ACTA2 antibody (2 μg) and detected with anti-PGAM1 antibody.

[0090] The results are as follows Figure 30 As shown, experiments revealed that it is related to PGAM1 WT Group and PGAM1 K225E-K228E Compared to the group, PGAM1 K225R-K228R The binding affinity of PGAM1 to ACTA2 was significantly reduced in the group, indicating that succinylation of PGAM1 at K225-K228 sites can enhance the interaction between PGAM1 and ACTA2.

[0091] Example 8: Rearrangement of the F-actin cytoskeleton after succinylation of PGAM1 at K225-K228 sites Detection of F-actin cytoskeleton rearrangement Each group of U87 and U251 stable cells was treated with 3×10 4 Cells / wells were transferred to 24-well plates and cultured for 24 hours. The culture medium was discarded, and the cells were washed twice with PBS. 400 μL of 3.7% formaldehyde (without methanol) was added to each well, and the plates were fixed at room temperature for 15 minutes. The cells were then washed three times with PBS for 5 minutes each time. 400 μL of 0.1% Triton X-100 (prepared with PBS) was added to each well, and the plates were permeated for 5 minutes. The cells were then washed three times with PBS. Actin-Tracker Red-555 working solution (1:50 dilution) was prepared with PBS containing 5% BSA and 0.1% Triton X-100. 50 μL of the working solution was added to each well, and the plates were incubated at room temperature in the dark for 60 minutes. The plates were washed three times with PBS for 5 minutes each time, and the nuclei were stained with DAPI for 5 minutes. The plates were then mounted, and the distribution of F-actin and stress fiber formation were observed and photographed using a confocal microscope.

[0092] The results are as follows Figures 31-32 As shown, experiments revealed similarities with Vector and PGAM1. K225R-K228R Compared to the group, PGAM1 WT Group and PGAM1 K225E-K228E The expression of F-actin was significantly increased in the group, and F-actin was mostly concentrated at the cell edge and in the membranous protrusions, indicating that PGAM1 succinylation promotes F-actin polymerization and rearrangement and enhances the dynamic remodeling ability of the cytoskeleton.

[0093] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biomarker for the diagnosis, treatment, or prognostic assessment of glioblastoma, characterized in that, The biomarker is succinylation modification at sites K225 and / or K228 of the PGAM1 protein.

2. The use of the biomarker according to claim 1 in the preparation of a medicament for treating glioblastoma.

3. The application according to claim 2, characterized in that, The glioblastomas mentioned include the U87 and U251 cell lines.

4. The application according to claim 2, characterized in that, The drug contains pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients include fillers, binders, disintegrants, and emulsifiers.

6. The application according to claim 2, characterized in that, The dosage forms of the drugs include tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

7. The use of the biomarker of claim 1 in the preparation of reagents or kits for diagnosing glioblastoma.

8. The application according to claim 7, characterized in that, The glioblastomas mentioned include U87 and U251 cell lines; the detection methods of the reagents or kits include immunoprecipitation, immunohistochemistry, Western blotting, and immunofluorescence.

9. The use of the biomarker of claim 1 in the preparation of reagents or kits for prognostic assessment of glioblastoma.

10. The application according to claim 9, characterized in that, The glioblastomas mentioned include U87 and U251 cell lines; the detection methods of the reagents or kits include immunoprecipitation, immunohistochemistry, Western blotting, and immunofluorescence.

Images