Tagln2-k40 succinylated monoclonal antibody and application thereof

Abstract

This invention discloses a TAGLN2-K40 succinylated monoclonal antibody and its applications, belonging to the field of biotechnology. This invention addresses the significant upregulation of succinylation modification of TAGLN2 protein at position 40 (lysine 40) in glioblastoma (GBM) and TMZ-resistant cells, leading to TMZ chemotherapy failure. This invention provides a monoclonal antibody targeting this specific protein modification of TAGLN2-K40 succinylation. This monoclonal antibody exerts its antitumor effect through multiple mechanisms (blocking signaling, inducing ferroptosis, and promoting degradation), and has synergistic potential with existing standard therapy (TMZ). It offers a promising new strategy for overcoming GBM chemotherapy resistance and has excellent application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a monoclonal antibody targeting the succinylation modification of lysine at position 40 of the TAGLN2 protein and its use in the diagnosis, prevention and / or treatment of diseases characterized by TAGLN2-K40 succinylation (especially malignant tumors such as glioblastoma). Background Technology

[0002] Glioblastoma (GBM) is the most malignant and has the worst prognosis among primary central nervous system tumors. Currently, temozolomide (TMZ) is the first-line chemotherapy drug for GBM treatment, but the vast majority of patients eventually develop chemotherapy resistance, leading to treatment failure. Therefore, elucidating the molecular mechanisms of TMZ resistance and finding effective reversal strategies are crucial scientific issues that urgently need to be addressed in the clinical treatment of GBM.

[0003] In recent years, ferroptosis (a novel type of programmed cell death driven by iron-dependent lipid peroxidation) has been shown to be closely related to tumor therapy resistance. Studies have shown that inducing ferroptosis can enhance the killing effect of TMZ on GBM cells, but GBM cells themselves develop strong ferroptosis resistance, the specific mechanism of which has not been fully elucidated. Post-translational modifications of proteins, especially succinylation, play a key role in regulating cellular metabolism, signal transduction, and stress responses, but their role in tumor ferroptosis resistance is still poorly studied.

[0004] TAGLN2 (Transgelin-2) is an actin-binding protein that plays a role in cytoskeleton reorganization, cell migration, and signal transduction. Existing research indicates that TAGLN2 is highly expressed in various tumors and promotes tumor progression; however, whether its function is finely regulated by post-translational modifications, and how this regulation affects tumor biological behavior, remains poorly understood. In particular, as a non-enzymatic scaffold protein, TAGLN2 lacks the traditional small-molecule inhibitor binding "pocket," posing a significant challenge to the development of direct-targeting drugs for this protein.

[0005] Previous research by the inventors revealed that succinylation of lysine 40 in the TAGLN2 protein (TAGLN2-K40 succinylation) was significantly upregulated in GBM tissues and TMZ-resistant cells. This modification, by inhibiting PP2A phosphatase activity, activates the AKT / β-catenin signaling axis, thereby upregulating the transcription of the key negative regulator of ferroptosis, GPX4, ultimately conferring strong ferroptosis resistance to GBM cells, leading to TMZ chemotherapy failure. Furthermore, TAGLN2-K40 succinylation also stabilizes the TAGLN2 protein itself, inhibiting its MARCH1-mediated ubiquitination degradation, forming a positive feedback loop that further exacerbates the malignant phenotype.

[0006] Monoclonal antibody drugs, due to their high specificity, high affinity, and favorable pharmacokinetic properties, have become an important pillar of targeted cancer therapy. Currently, there are no reports of monoclonal antibodies or related products exhibiting high affinity and high specificity for the specific succinylated epitope TAGLN2-K40. Developing such antibodies could not only serve as a key tool for studying the biological function of this modification but also hold promise for providing a novel, immunotherapeutic approach to overcoming TMZ resistance in tumors such as GBM. Summary of the Invention

[0007] In view of this, the object of the present invention is to provide a monoclonal antibody capable of specifically recognizing succinylation modification of lysine at position 40 of the TAGLN2 protein (TAGLN2-K40 succinylation) and the use of the antibody in the preparation of products for the diagnosis, prevention and / or treatment of diseases (especially malignant tumors) associated with TAGLN2-K40 succinylation.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a monoclonal antibody that specifically recognizes the succinylation modification of lysine at position 40 of the TAGLN2 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0010] Secondly, the present invention provides the encoding gene of the above-mentioned monoclonal antibody.

[0011] Thirdly, the present invention provides a pharmaceutical composition comprising the above-mentioned monoclonal antibody.

[0012] Based on the above technical solution, the pharmaceutical composition further comprises temozolomide (TMZ).

[0013] Fourthly, the present invention provides the application of the above-mentioned monoclonal antibody and the encoding gene of the monoclonal antibody in the preparation of reagents for diagnosing diseases characterized by TAGLN2-K40 succinylation.

[0014] Based on the above technical solution, the disease further includes glioblastoma.

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

[0016] Fourthly, the present invention provides the use of the above-mentioned monoclonal antibody, the encoding gene of the monoclonal antibody, and the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases characterized by TAGLN2-K40 succinylation.

[0017] Based on the above technical solution, the disease further includes 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 drug further includes pharmaceutically acceptable excipients.

[0020] Based on the above technical solution, the pharmaceutically acceptable excipients further include fillers, binders, disintegrants, and emulsifiers.

[0021] 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.

[0022] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention provides the first monoclonal antibody targeting the specific pathological modification of TAGLN2-K40 succinylation, filling a technological gap in this field.

[0023] 2) Clear mechanism and precise targeting: The development of this antibody is based on the complete analysis of the molecular pathway of TAGLN2-K40 succinylation driving TMZ resistance and ferroptosis resistance, which enables precise intervention at key oncogenic modification nodes.

[0024] 3) Overcoming “undruggability”: For non-enzymatic scaffold proteins like TAGLN2, which are traditionally “undruggable”, this invention achieves specific targeting of their functional modification epitopes through antibody technology, providing a new paradigm for the development of similar targets.

[0025] 4) Great therapeutic potential: This antibody can exert anti-tumor effects through multiple mechanisms (blocking signaling, inducing ferroptosis, and promoting degradation), and has synergistic potential with existing standard therapy (TMZ), providing a promising new strategy for overcoming GBM chemotherapy resistance.

[0026] 5) Wide range of applications: In addition to treatment, this antibody is also a powerful research tool and diagnostic probe, with multiple applications. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0028] Figure 1 The image shows the fluorescence intensity of green fluorescent protein (GFP) in U87 stably transfected cells and the results of Western blot analysis of Flag tag expression.

[0029] Figure 2 The image shows the fluorescence intensity of green fluorescent protein (GFP) in U251 stably transfected cells and the results of Western blot analysis of Flag tag expression.

[0030] Figure 3 The figure shows the effect of TAGLN2 K40 succinylation after treatment with erlastin or DMSO on the viability of U87 and U251 cells in CCK-8 assays.

[0031] Figure 4 Figure showing the results of Prussian blue staining for detecting iron deposits in U87 and U251 cells after TAGLN2-K40 succinylation.

[0032] Figure 5 Figure showing the results of the kit's detection of the effect of TAGLN2-K40 succinylation on malondialdehyde (MDA) in U87 and U251 cells.

[0033] Figure 6 Figure showing the results of the kit's detection of the effect of TAGLN2-K40 succinylation on reactive oxygen species (ROS) in U87 and U251 cells.

[0034] Figure 7 Figure showing the effect of TAGLN2-K40 succinylation on glutathione (GSH) in U87 and U251 cells, as determined by the kit.

[0035] Figure 8 The figure shows the effect of TAGLN2 K40 succinylation on GPX4 mRNA expression in U87 and U251 cells, as well as the results of Western blot analysis on the protein levels of β-catenin and GPX4.

[0036] Figure 9 To verify that GPX4 is a downstream target gene of β-catenin in chromatin immunoprecipitation experiments.

[0037] Figure 10 To detect the effect of TAGLN2-K40 succinylation on the nuclear localization of β-catenin in U87 and U251 cells using immunofluorescence.

[0038] Figure 11 The figure shows the effect of TAGLN2 K40 succinylation on the expression of p-AKT and p-β-catenin (S552) proteins in U87 and U251 cells.

[0039] Figure 12 The results of verifying the binding relationship between TAGLN2 and PP2A for HDOCK are shown in the figure.

[0040] Figure 13 To verify the binding relationship between PP2A and TAGLN2 protein using co-immunoprecipitation and to detect the enzyme activity of PP2A using substrate colorimetric method.

[0041] Figure 14 Immunohistochemical staining and GEPIA analysis of TAGLN2-K40 succinylation levels in glioma samples of different grades, recurrent glioma samples, and normal brain tissue samples, as well as the results of post-treatment survival analysis of glioma patients.

[0042] Figure 15 The results of CCK8 assays were used to detect the drug sensitivity of U87 and U251 cells to short-term stimulation with temozolomide (TMZ), and to detect the succinylation levels of TAGLN2 and TAGLN2-K40 in U87 and U251 cells after short-term TMZ stimulation. The results of CCK8 assays were also used to detect the drug sensitivity of U87 and U251 cells to TMZ after TAGLN-K40 succinylation.

[0043] Figure 16 The diagram shows the results of using the CCK8 method to detect the effects of TAGLN2 on cell death pathways.

[0044] Figure 17 TAGLN2 for WB analysis K40WT and TAGLN2 K40R Figure showing the results of TAGLN2 protein levels in China.

[0045] Figure 18 Figure showing the results of the thymosin assay to detect the effect of TAGLN2 K40 succinylation on TAGLN2 protein expression.

[0046] Figure 19 The figure shows the results of detecting the effects of the proteasome inhibitor MG132 and the autophagy and Toll-like receptor (TLR) inhibitor chloroquine on the level of TAGLN2 protein in cells.

[0047] Figure 20 To predict the ubiquitination site of the TAGLN2 protein and detect TAGLN2 using the Phosphosite website WT With TAGLN2 K40R The results of the difference in ubiquitination levels in cells.

[0048] Figure 21The graph shows the results of a correlation analysis of MARCH1 and TAGLN2 on the GEPIA website.

[0049] Figure 22 This figure shows the results of Western blot analysis to detect the level of TAGLN2 protein after interference with MARCH1.

[0050] Figure 23 To investigate the effect of MARCH1 on protein expression, a quantitative analysis was performed to interfere with MARCH1 to detect differences in ubiquitination in U87 and U251 cells.

[0051] Figure 24 The results are from the SDS-PAGE analysis of the monoclonal antibody.

[0052] Figure 25 Figure showing the effects of CPP-TAGLN2K40succ on the uptake of cell-penetrating peptide CPP-TAGLN2K40succ by U87 and U251 cells, as well as its influence on the viability and protein expression of normal NHA, U87, and U251 cells.

[0053] Figure 26 Figure showing the expression of TAGLN2-K40 succinylation, TAGLN2, β-catenin, and GPX4 proteins after uptake of the penetrating peptide CPP-TAGLN2K40succ into U87 and U251 cells.

[0054] Figure 27 The figure shows the effect of immunofluorescence detection on the localization of β-catenin in the cell nucleus after U87 and U251 cells take up the penetrating peptide CPP-TAGLN2K40succ.

[0055] Figure 28 The figure shows the effect of the cell-penetrating peptide CPP-TAGLN2K40succ on cell viability after short-term administration of temozolomide to CCK-8 assays of U87 and U251 cells.

[0056] Figure 29 The figure shows the effect of cell-penetrating peptide CPP-TAGLN2K40succ on the viability of GL261 cells and the results of TAGLN2-K40 succinylation and TAGLN2 protein expression.

[0057] Figure 30 The results are shown in the figure, which is used to detect whether CPP-TAGLN2K40succ can penetrate into the brain of animals by immunofluorescence and to detect the change in iron ion level after drug administration by Prussian blue staining.

[0058] Figure 31Immunohistochemical results of Ki67, TAGLN2-K40 sulfation, and GPX4 levels in tumor tissue after administration of CPP-TAGLN2K40succ. Detailed Implementation

[0059] The present invention will be described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0060] Experimental Example 1: Succinylation of TAGLN2-K40 can inhibit ferroptosis. 1. Materials: Glioma cell lines: U87 and U251 were purchased from ATCC.

[0061] 2. Method: 2.1 Cell Culture U87 and U251 cells were cultured using conventional tumor cell culture methods.

[0062] 2.2 TAGLN2-K40 overexpression of TAGLN2 K40WT and desuccinylated TAGLN2 K40R Lentiviral cell construction The entire genome was synthesized and the vector constructed by Gemma Genetics, and the sequence was verified to be correct. The recombinant plasmid was packaged into lentivirus and used to infect host cells (U87 and U251 cells), successfully constructing a stable overexpression of TAGLN2. K40WT With TAGLN2 K40R The cell lines were used for subsequent experimental research.

[0063] 2.3 The effect of erastin, a promoter of ferroptosis, on TAGLN2 was determined by the CCK8 assay. K40WT and TAGLN2 K40R Effects on cell viability Cells were seeded at a rate of 3 × 10³ cells / well in 96-well plates. When the cell density reached 70%, the cells were cultured for 48 h in media containing 10 μMerastin and media without erastin, respectively. After discarding the medium, 100 μL of serum-free medium containing 10% CCK-8 was added to each well, and the cells were incubated for 1 h. The absorbance at 450 nm was measured using a microplate reader to calculate cell viability.

[0064] 2.4 Prussian Blue-Nuclear Solid Red Staining Detection of TAGLN2 K40WT Group and TAGLN2 K40R Iron ion levels in group cells 1) Cell sample processing: Fix cells with fixative for more than 10 minutes, wash with distilled water for 2 minutes, and repeat twice.

[0065] 2) Prepare an acidic potassium ferrocyanide solution (1:1); 3) Staining: Add 50 μl of acidic potassium ferrocyanide solution to each slide to completely cover it, place it in a humidified chamber and stain at room temperature for 1 hour, immerse it in distilled water and wash it on a shaker for 5 minutes, and air dry; add 50 μl of nuclear solid red staining solution to stain for 5-10 minutes, then immerse the slide in distilled water and wash it on a shaker for 5 minutes, and observe it under a microscope.

[0066] 2.5 Detection of TAGLN2 K40WT Group, TAGLN2 K40R The levels of malondialdehyde (MDA), reactive oxygen species (ROS), and reduced glutathione (GSH) in cells were used to assess the extent of ferroptosis. 1) MDA content determination First, a standard curve was constructed, followed by MDA determination of the samples. Adherent cells were digested with trypsin, centrifuged at 1000 rpm for 3 min, and the cell pellet was collected. The pellet was resuspended in PBS and transferred to a 1.5 mL centrifuge tube, centrifuged at 3000 rpm at 4°C for 10 min, and the supernatant was discarded. An appropriate amount of RIPA lysis buffer containing 1% phosphatase inhibitor and 1% protease inhibitor was added, and the mixture was incubated at 4°C for 30 min for lysis. The protein supernatant was collected after centrifugation at 10,000 g–12,000 g for 10 min, and protein concentration was determined using BCA. Using protein lysis buffer or PBS as blank controls, add 100 µL of blank or sample to a 1.5 mL EP tube, add 200 µL of MDA detection working solution and mix well. Heat in a 100℃ water bath for 15 min, cool to room temperature, centrifuge at 1000g for 10 min, and add 200 µL of supernatant to a 96-well plate. Set up three replicates for each concentration. Measure the OD value at 532 nm using a microplate reader. Substitute the values ​​into the standard curve to calculate the MDA content in the sample, expressed as μM / mg protein.

[0067] 2) ROS detection: Intracellular ROS levels were detected and visualized using fluorescent probes for oxidative stress in live cells and fluorescence microscopy.

[0068] 3) Determination of GSH (reduced glutathione) content (1) Construction of standard curve: Dilute the 10 mM GSSG stock solution standard with protein removal reagent to a concentration of 15 μM GSSG solution, and then serially dilute to 10, 5, 2, 1, and 0.5 μM GSSG standard solutions. Take 10 µL of each of the six concentrations (15, 10, 5, 2, 1, and 0.5 μM) and add 150 µL of total glutathione detection working solution. Mix well and incubate at 25ºC for 5 min. Set up three replicates for each concentration. Add 50 µL of 0.5 mg / mL NADPH solution, mix well, and incubate at 25ºC for 25 min. Measure the OD value at 412 nm using a microplate reader. Plot a standard curve with the concentration of the standard and the OD value on the x and y axes, respectively. Use this curve when measuring total glutathione.

[0069] Add diluted GSH scavenging auxiliary solution to the standard at a ratio of 5:1 and mix well. Then add GSH scavenging reagent working solution to the standard at a ratio of 20:1, mix well, and incubate at 25ºC for 60 min. After the reaction, take 10 µL and add it to 150 µL of total glutathione detection working solution and mix well. Incubate at 25ºC for 5 min. Set up three replicates for each concentration. Add 50 µL of 0.5 mg / mL NADPH solution and mix well. Incubate at 25ºC for 25 min. Measure the OD value at 412 nm using a microplate reader. Plot a standard curve with the concentration of the standard and the OD value on the x and y axes, respectively. Use this curve when determining the GSSG content in the sample.

[0070] (2) Determination of total glutathione in samples Adherent cells were digested with trypsin, centrifuged at 1000 rpm for 3 min, and the cell pellet was collected. The cells were resuspended in PBS, centrifuged at 3000 rpm for 4°C for 10 min, the supernatant was discarded, and the cell pellet was obtained. Three times the volume of protein removal reagent was added to the cell pellet and mixed well. The sample was subjected to two rapid freeze-thaw treatments using liquid nitrogen and a 37°C water bath, and then incubated at 4°C for 5 min. Centrifuge at 10000 g for 10 min at 4 ºC, collect the supernatant, dilute the sample appropriately with protein removal reagent, and add 10 µL of the diluted sample to a 96-well plate. Add 150 µL of total glutathione detection working solution and mix well. Incubate at 25 ºC for 5 min, then add 50 µL of 0.5 mg / mL NADPH solution and mix well. Incubate at 25 ºC for 25 min, and then measure the OD value at 412 nm using a microplate reader. Substitute the values ​​into the standard curve to calculate the GSSG content. Total glutathione content (μM) = GSSG (μM) × 2. The total glutathione content is expressed as nmol / mg prot.

[0071] (3) GSSG determination of the sample: The sample diluted with protein removal reagent in step (2) above was mixed with diluted GSH removal auxiliary solution at a ratio of 5:1. Then, the sample was mixed with GSH removal reagent working solution at a ratio of 20:1. The mixture was incubated in a 25ºC incubator for 60 min. After the reaction, 10 µL was added to 150 µL of total glutathione detection working solution and mixed. The mixture was incubated at 25°C for 5 min. 50 µL of 0.5 mg / mL NADPH solution was added and mixed. The mixture was incubated at 25ºC for 25 min. The OD value was measured at 412 nm using an ELISA reader. The GSSG content was calculated by substituting the OD value into the standard curve and expressed as nmol / mg prot.

[0072] (4) Calculation of GSH content in sample: The GSH content in sample is calculated based on the total glutathione content and GSSG content obtained above. GSH = total glutathione - GSSG content × 2.

[0073] The results are as follows Figure 1-2 As shown, TAGLN2 K40WT Group, TAGLN2 K40R The cell line for this group was successfully constructed, such as Figure 3 As shown, in respectively to TAGLN2 K40WT and TAGLN2 K40R After adding the ferroptosis promoter erastin to glioma cells, cell viability was assessed, and the results showed that TAGLN2... K40R More sensitive to erastin; such as Figure 4-7 As shown, TAGLN2 K40WT The iron ion level, ROS, and MDA level were all lower than those of TAGLN2. K40R GSH levels are higher than TAGLN2. K40R Group.

[0074] Conclusion: TAGLN2-K40 succinylation can inhibit ferroptosis in gliomas.

[0075] Experimental Example 2: TAGLN2-K40 succinylation induces ferroptosis resistance by upregulating GPX4 via β-catenin. 1. Detection of TAGLN2 by q-RT PCR K40WT Group, TAGLN2 K40R Differences in GPX4 mRNA among group cells (1) Total RNA extraction: Total RNA was extracted using a standard kit and the OD value of the samples was measured.

[0076] (2) Reverse transcription reaction: Prepare the genomic DNA removal system, mix well, and react at 42℃ for 2 min; then add 4 μL of 5×HiScript II Select qRT SuperMix II directly to the EP tube, mix well, and continue the reaction in the gene amplification instrument at 50℃ for 15 min; 85℃ for 5 s to obtain the reverse transcription product.

[0077] Table 1. Dosage of each component in reverse transcription

[0078] (3) qRT-PCR reaction The reaction system is shown in Table 2 (the relevant primer sequences are shown in Table 3), and the reaction is carried out according to the procedure in Table 4.

[0079] Table 2 Amplification System Table

[0080] Table 3 Primer sequence listing

[0081] Table 4 qRT-PCR reaction procedure

[0082] After amplification, use 2 -△△Ct The relative expression levels of the target gene in each group were calculated using the method.

[0083] 2. Western blot (WB) detection of TAGLN2 K40WT Group, TAGLN2 K40R GPX4 protein level in the group (1) Sample extraction: Adherent cells were digested with trypsin, centrifuged at 1000 rpm for 3 min, and the cell pellet was collected. The cells were resuspended in PBS and transferred to a centrifuge at 3000 rpm and 4 °C for 10 min. The supernatant was discarded and the cell pellet was obtained. Cell lysis buffer was prepared according to RIPA containing 1% protease inhibitor and 1% phosphatase inhibitor. The prepared lysis buffer was added according to the volume of cell pellet, mixed well, and sonicated. The mixture was placed at 4 °C for 30 min and centrifuged at 4 °C and 12000 rpm for 15 min. The supernatant was collected as the total protein in the cell sample.

[0084] (2) Protein quantification: The protein concentration of the sample was detected by the BCA method.

[0085] (3) SDS-PAGE gel electrophoresis (4) Immunological reaction: 5% skim milk powder was used as protein blocking solution and blocked for 2 h. Then, appropriate protein bands were cut according to the molecular weight of the target protein. Primary antibody was added and incubated overnight at 4°C for 12-18 h. The bands were washed 3 times with TBST for 15 min each time. Secondary antibody was incubated at room temperature for 2 h. Then, the bands were washed 3 times under the above conditions, developed, and quantitatively analyzed and statistically analyzed by ImageJ.

[0086] 3. Chromatin Immunoprecipitation (ChIP) assay to verify the effect of β-catenin on TAGLN2. K40WT Group, TAGLN2 K40R Regulation of GPX4 in cells 1) Optimization of sample ultrasonic treatment conditions (1) Culture the cells in a 10cm culture dish with 10ml of cell culture medium. Under the condition that the expected protein β-catenin binds to the DNA of the GPX4 gene promoter region, add an appropriate amount of formaldehyde directly to the cell culture medium, mix well, and incubate at 37℃ for 10 minutes to crosslink the target protein and the corresponding genomic DNA.

[0087] (2) Add 5-10 ml of PBS containing 1 mM PMSF pre-cooled in an ice bath and wash the cells; (3) Add 5-10 ml of PBS containing 1 mM PMSF pre-chilled on ice to further wash the cells; (4) Add 1 ml of PBS containing 1 mM PMSF pre-cooled in an ice bath, scrape off the cells with a cell scraper, collect them into centrifuge tubes, count the cells, and dispense them into tubes containing approximately 1 million cells each.

[0088] (5) Centrifuge at 4℃, 800-1000 g for 1-2 minutes, and discard the supernatant.

[0089] (6) The 1 million cell pellet from the previous step was resuspended in 0.2 ml of SDS Lysis Buffer containing 1 mM PMSF and incubated on ice for 10 minutes to fully lyse the cells.

[0090] (7) Ultrasonic treatment is used to cut genomic DNA, causing most of the DNA to break into 200-1000 bp.

[0091] (8) Add 8 μL of 5M NaCl to 0.2 ml of the ultrasonically treated sample and mix well. Heat at 65°C for 4 hours, add an equal volume of Tris-equilibrated phenol, vortex, and then centrifuge at 12000 g for 5 minutes at 4°C. Transfer the supernatant to another centrifuge tube.

[0092] (9) Add an equal volume of chloroform, vortex, then centrifuge at 12000 g for 5 minutes at 4°C, and transfer the supernatant to another centrifuge tube.

[0093] (10) Perform agarose gel electrophoresis to observe the effect of sonication on the shearing of genomic DNA.

[0094] 2) Chromatin immunoprecipitation (1) The sample to be tested was operated according to the above ultrasonic treatment steps. The final experimental conditions were 10 seconds of ultrasonic treatment each time, 10 seconds of rest, and a total ultrasonic power of 20 times, with a power of 40W.

[0095] (2) The ultrasonically treated sample was centrifuged at 12000 g for 5 minutes at 4℃, and the supernatant (0.2 ml) was transferred to a 2 ml centrifuge tube and placed in an ice bath.

[0096] (3 Add 1.8 ml of ChIP Dilution Buffer containing 1 mM PMSF.)

[0097] (4) Take 20 μL of sample as input for subsequent detection. Add 70 μL of Protein A+G Agarose / Salmon Sperm DNA to the remaining approximately 2 ml of sample and mix slowly by rotating or shaking at 4°C for 30 minutes.

[0098] (5) Centrifuge at 4℃ and 1000g for 1 minute, and transfer the supernatant to a new 2ml centrifuge tube.

[0099] (6) Add an appropriate amount of primary antibody, and mix slowly by rotating or shaking at 4°C overnight.

[0100] (7) Add 60 μL of Protein A+G Agarose / Salmon Sperm DNA and mix slowly by rotating or shaking at 4°C for 60 minutes to precipitate the protein or corresponding complex recognized by the primary antibody.

[0101] (8) Centrifuge at 4℃ and 1000g for 1 minute to remove the liquid. Then wash the precipitate sequentially with Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer, and TE Buffer (all reagents used are Beyotime P2078 kit). The volume of washing solution used each time is 1 ml. Each time, slowly rotate or shake at 4℃ for 3-5 minutes. Then centrifuge at 4℃ and 1000g for 1 minute to remove the liquid.

[0102] 3) PCR amplification of the target gene sequence (1) Add 250 μL of Elution buffer (1% SDS, 0.1M NaHCO3), vortex to mix, and elute at room temperature for 3-5 minutes; centrifuge at 1000 g for 1 minute, transfer the supernatant to a new centrifuge tube, add another 250 μL of Elution buffer to the precipitate, vortex to mix, and elute at room temperature for 3-5 minutes; centrifuge at 1000 g for 1 minute, remove the supernatant, and combine the supernatants, totaling about 500 μL of supernatant.

[0103] (2) Add 20 μL of 5M NaCl to 500 μL of supernatant, mix well, and heat at 65°C for 4 hours.

[0104] (3) Add 1 μL of 5M NaCl to 20 μL of the input sample, mix well, and heat at 65°C for 4 hours.

[0105] (4) Add 10 μL of 0.5M EDTA, 20 μL of 1M Tris pH 6.5 and 1 μL of 20 mg / ml proteinase K to approximately 520 μL of sample, mix well and incubate at 45°C for 60 minutes; add an equal volume of Tris to balance phenol, vortex vigorously to mix well, then centrifuge at 12000 g for 5 minutes at 4°C, and transfer the supernatant to another centrifuge tube.

[0106] (5) Add an equal volume of chloroform, vortex, then centrifuge at 12000 g for 5 minutes at 4°C. Transfer the supernatant to another centrifuge tube, add 20 μg of glycogen or yeast tRNA, add 1 / 10 volume of 3M NaAc (pH 5.2), and then add 2.5 times the volume of anhydrous ethanol. Mix well and precipitate at -70°C for at least 1 hour. Centrifuge at 12000-14000 g for 10 minutes at 4°C, and discard the supernatant. Wash the precipitate with about 1 ml of 70% ethanol. Centrifuge at 12000 g for 10 minutes at 4°C, discard the supernatant, and centrifuge at 12000 g for 1 minute at 4°C, then discard the liquid.

[0107] (6) Resuspend the DNA precipitate with a small amount of water, mix water, Taq enzyme, primers and template in a certain proportion, put them into a PCR instrument for PCR amplification, and then perform agarose gel electrophoresis again.

[0108] 4. Immunofluorescence (IF) detection of TAGLN2 K40WT Group, TAGLN2 K40R Differences in protein localization of β-catenin in the group With 4×10 4Cells were cultured in cell / well slides for 24 h, fixed with 4% paraformaldehyde, washed, permeabilized with 0.1% Triton X-100 in PBS solution, washed, and blocked with 5% BSA for 1 h; incubated with primary antibody, stained with DAPI, and mounted; images were acquired using an upright fluorescence microscope after being placed in the dark for 12 h.

[0109] The results are as follows Figure 8 As shown, experiments revealed that it is similar to TAGLN2. K40WT Compared to the group, TAGLN2 K40R The mRNA level of GPX4 was significantly reduced in the two groups; further analysis of β-catenin showed no significant difference in β-catenin levels between the two groups. Given that β-catenin can enhance GPX4 transcriptional activity, the chromatin immunoprecipitation assay results are as follows... Figure 9 As shown, GPX4 is a downstream target gene of β-catenin in gliomas. However, immunofluorescence reveals that TAGLN2... K40R The amount of β-catenin in the nucleus of the group was reduced ( Figure 10 This indicates that the succinylation modification enhances ferroptosis resistance by regulating GPX4 expression, and immunofluorescence shows that TAGLN2-K40 succinylation modification affects the nuclear localization of β-catenin.

[0110] Experimental Example 3: AKT-mediated phosphorylation of β-catenin at Ser552 (S552) significantly promotes its nuclear translocation. 1) TAGLN2 was detected by Western blotting. K40WT Group and TAGLN2 K40R Protein levels of AKT, phosphorylated AKT (p-AKT), and S552 phosphorylated β-catenin (p-β-catenin S552) in the group cells were determined by Western blotting, as described in Example 2.

[0111] 2) The HDOCK website (http: / / hdock.phys.hust.edu.cn / ) predicts the potential binding of TAGLN2 to the PP2A protein. The protein structures of TAGLN2 and PP2A were downloaded from the PDB database and uploaded to the HDOCK database for binding prediction.

[0112] 3) Verify their interaction using co-immunoprecipitation (Co-IP) experiments. (1) Sample processing: Collect cells at a ratio of 1.0 × 10⁻⁶. 5Add 20-30 µL of binding buffer per cell, along with a protease inhibitor (PMSF at a final concentration of 1 mM), mix well, and incubate on ice for 10 min. Centrifuge to collect the supernatant (4°C, 14000 g, 10 min) and incubate on ice for later use.

[0113] (2) Pretreatment of magnetic beads (3) Antibody binding reaction: The pretreated magnetic bead suspension was magnetically separated, and the supernatant was discarded. 200 µL of antibody working solution was added, and the suspension was resuspended and placed in a rotary mixer. After 15 min, magnetic separation was performed, and the supernatant was collected and placed on ice for subsequent detection. 200 µL of binding buffer was added to the EP tube for washing. The magnetic bead-antibody complex was gently dispersed by pipetting, and then magnetic separation was performed. The supernatant was discarded, and the EP tube was removed from the magnetic separator. The washing was repeated once.

[0114] (4) Antigen precipitation reaction: Add 200 µL of antigen sample, and pipette to disperse the antigen and magnetic bead-antibody complex evenly; place the EP tube in a reverse mixer and invert for 10 min to ensure sufficient binding of antigen and antibody; perform magnetic separation, collect the supernatant and place it on ice for subsequent detection; add 200 µL of washing buffer to the EP tube and wash, pipette to disperse the magnetic bead-antibody-antigen complex evenly, then perform magnetic separation and discard the supernatant; remove the EP tube from the magnetic separator and repeat the washing twice. Finally, add 200 µL of washing buffer, use a pipette to transfer the magnetic bead-antibody-antigen complex suspension to a new 1.5 mL EP tube, and perform magnetic separation, discarding the supernatant. Remove the EP tube from the magnetic separator, add 25 µL of 1×SDS-PAGE Loading Buffer and mix well, heat at 95℃ for 5 min; then perform magnetic separation and collect the supernatant for SDS-PAGE detection.

[0115] 4) Substrate colorimetric method for detecting PP2A enzyme activity The total volume of the reaction system was 50 μL, containing 50 mM Tris-HCl (pH 7.5) (ST780-100ml), 10 mM MgCl2 (Beyotime: R0058-1ml), 1 mM DTT (Beyotime ST043-1g), the substrate p-nitrophenyl phosphate (PNPP) (Meilun Biotechnology), and protein samples. After incubation at 37℃ for 30 min, the reaction was terminated by adding 0.5 M NaOH, and the absorbance was measured at 650 nm. Each group included wells treated with the PP2A inhibitor (LB-100 Beyotime: SF1082-10mM) as a control. The PP2A-specific enzyme activity was represented by the difference in absorbance between the experimental and control wells, after deducting non-specific phosphatase activity.

[0116] like Figure 11 As shown, with TAGLN2 K40WT Compared to the group, TAGLN2 K40R The levels of p-AKT and S552 phosphorylated β-catenin (p-β-catenin S552) protein in the group were significantly reduced; Figure 12-13 Experimental results confirmed the direct binding of TAGLN2 to PP2A. Further substrate colorimetric analysis showed that succinylation of TAGLN2 at the Lys40 (K40) site significantly inhibited the enzymatic activity of PP2A. This indicates that succinylation of TAGLN2 at the Lys40 (K40) site promotes AKT-mediated phosphorylation of β-catenin at the Ser552 site, thereby enhancing cellular resistance to ferroptosis. Furthermore, the interaction between TAGLN2 and PP2A proteins may regulate the enzymatic activity of PP2A. TAGLN2-K40 succinylation enhances AKT phosphorylation by regulating the enzymatic function of PP2A, thereby promoting nuclear translocation of β-catenin.

[0117] Experimental Example 4: High levels of TAGLN2-K40 succinylation and TAGLN2 overexpression promote glioma cell resistance to temozolomide (TMZ). 1. Materials Clinical glioma tissue samples were obtained from the Northern Theater General Hospital of the Chinese People's Liberation Army. The clinical tissue samples obtained included a normal group, three cases each of grade II and grade III-IV gliomas, and three cases of recurrent gliomas.

[0118] 2. Methods 1) Immunohistochemical (IHC) detection of glioma pathological grade and TAGLN2 succinylation level at Lys40 site in patients. After tissue trimming, embedding, sectioning, and baking, the sections were dewaxed, rehydrated, and subjected to antigen retrieval. The retrieval sections were placed at room temperature and, after cooling, washed with PBS for 5 min x 3 times. The tissue areas were then circled with a histochemical pen. Endogenous peroxidase inhibitors were incubated at room temperature for 10 min, followed by washing with PBS for 5 min x 3 times. Non-specific staining inhibitors were incubated at room temperature for 10 min, excess water was removed, and the antibody was diluted to a suitable concentration with PBS and incubated overnight at 4°C. After 12 h, the sections were washed with PBS for 5 min x 3 times. Biotin-labeled IgG polymers were incubated at room temperature for 10 min, followed by washing with PBS for 5 min x 3 times. Streptomycin-peroxidase was incubated at room temperature in the dark for 10 min, followed by washing with PBST for 5 min x 3 times.

[0119] 2) Patient prognosis and survival were analyzed using the GEPIA2 database and the TAGLN2 gene input for survival analysis.

[0120] 3) Western blot analysis confirmed the succinylation level of TAGLN2-K40 in TMZ-resistant glioma cells; Western blotting was performed using the same procedure as in Experiment 2.

[0121] 4) CCK8 assay to detect the effect of TAGLN2-K40 succinylation on cell proliferation in glioma cells CCK-8 Experiment: The operating procedure is the same as that of the CCK-8 experiment in Experiment Example 1.

[0122] 5) Use different inhibitors of cell death pathways to determine the cell death pathways affected by TAGLN2. The effect of TAGLN2 on the cell death pathway was verified by CCK8 assay. Ferrostatin-1 (10 μM), TTM (10 μM), Z-VAD (10 μM), and Nec-1 (10 μM) were selected as cell death inhibitors, and cell viability was detected. The experimental protocol was the same as in Experiment 1.

[0123] like Figure 14 As shown, the pathological grade of gliomas in patients is positively correlated with the succinylation level of TAGLN2 at the Lys40 site. Patients with recurrent gliomas show high expression of TAGLN2-K40 succinylation; patient prognostic survival data indicate that patients with high TAGLN2 expression have a poorer prognosis. Figure 15 The results showed that the TAGLN2-K40 succinylation level was increased in TMZ-resistant glioma cells, and high TAGLN2 succinylation was associated with enhanced cell survival and proliferation. Figure 16 The results showed that the ferroptosis inhibitor Ferrostatin-1 (Fer-1) significantly reversed si-TAGLN2-induced cell death. This indicates that patients with high-grade glioma pathology and recurrent gliomas exhibit high levels of TAGLN2-K40 succinylation and TAGLN2 overexpression, and that TAGLN2 primarily promotes TMZ resistance by inhibiting ferroptosis. These findings suggest that TAGLN2 and its K40 site succinylation modification are key regulators of TMZ chemotherapy resistance in gliomas.

[0124] Experimental Example 5: Succinylation of TAGLN2-K40 significantly enhances the expression level of TAGLN2 protein. 1) Comparison of wild-type TAGLN2 (TAGLN2) by Western blot analysis K40WT ) and K40 mutant (TAGLN2) K40R Protein expression levels Protein immunoblotting: The procedure is the same as that for protein immunoblotting in Experiment Example 2.

[0125] 2) Effect of TAGLN2-K40 succinylation on the stability of TAGLN2 protein Fresh culture medium containing the protein synthesis inhibitor actinomycin (CHX, 10 μM) was added to the cultured cells, and the cells were incubated in a 37°C, 5% CO2 incubator. Cells were collected at 0 h, 4 h, and 8 h according to the time gradient. After lysis, the supernatant was collected, the protein concentration was determined, and a Western blotting assay was performed, following the same procedure as in Experiment 2.

[0126] 3) Adding protein degradation pathway inhibitors to determine protein degradation pathways Fresh culture medium containing ubiquitin-proteasome pathway inhibitor (MG132, 10 μM) and autophagy-lysosome pathway inhibitor (CQ, 10 μM) was added to the cultured cells, and the cells were incubated. The cells were then collected and subjected to Western blotting, following the same procedure as in Experiment 2.

[0127] The results are as follows Figure 17 As shown, TAGLN2 WT The protein expression level in group A was significantly higher than that in group B. K40R Group; Figure 18 The results show that TAGLN2 K40R The degradation rate of TAGLN2 protein was significantly accelerated in this group; Figure 19 The results showed that the expression of TAGLN2 protein was significantly increased after the addition of MG132. This indicates that TAGLN2 succinylation modification can inhibit the degradation of TAGLN2 via the ubiquitin-proteasome pathway, improve protein stability, and thus significantly increase the expression level of TAGLN2 protein.

[0128] Experimental Example 6: Succinylation of TAGLN2-K40 inhibits MARCH1-mediated TAGLN2 ubiquitination. 1) Predict the ubiquitination sites of the TAGLN2 protein using the Phosphosite website. Enter TAGLN2 into the Phosphosite website to predict ubiquitination sites.

[0129] 2) Detection of TAGLN2 via Co-IP K40WT Group, TAGLN2 K40R Differences in ubiquitination among cells Add fresh culture medium containing ubiquitin-proteasome pathway inhibitor (MG132, 10 μM) to the cultured cells, culture for 4 h, collect the cells, and perform co-immunoprecipitation (Co-Ip) experiment: the operation procedure is the same as the co-immunoprecipitation (Co-IP) experiment in Experiment Example 2.

[0130] 3) GST pull-down assay to investigate proteins that interact with TAGLN2. Purified GST-bait protein was incubated with glutathione agarose beads at 4°C for 1-2 hours to form a "bead-bait" complex. Cell lysate was pre-cleaned with glutathione agarose beads for 1 hour, and the supernatant was collected. The "bead-bait" complex was then incubated with the pre-cleaned supernatant at 4°C for 2-4 hours to capture the interacting proteins. After removing unbound proteins, Western blotting was performed using the same procedure as in Experiment 2.

[0131] 4) GEPIA analysis showed a correlation between TAGLN2 and MARCH1. On the website, select multigene analysis, then correlation analysis, and input TAGLN2 and MARCH1 for Gene A and B respectively to perform Pearson correlation analysis.

[0132] 5) Co-IP verification of the interaction between TAGLN2 and MARCH1 Co-immunoprecipitation (Co-ip) assay: The procedure is the same as the Co-immunoprecipitation (Co-Ip) assay in Experiment Example 2.

[0133] 6) Detect TAGLN2 protein and ubiquitination levels after MARCH1 interference (si-RNA). siRNA transfection: siRNA was synthesized by Kintop (Wuhan) Biotechnology Co., Ltd.

[0134] Table 5. siRNA-March1 and siRNA-CPT1A sequences

[0135] When the cells reach 70% confluency, prepare solution A (opti-MEM, Lipofectamine 3000) and solution B (opti-MEM, siRNA). Let them stand for 5 minutes, then add solution B to solution A, mix well, and let them stand for 15 minutes. During the standing time, replace the DMEM complete culture medium in the well plate with opti-MEM culture medium. After the standing time is complete, slowly and evenly add the A / B mixture to opti-MEM. After culturing for 12 hours, replace the transfection culture medium with normal culture medium and continue culturing for 48-72 hours before proceeding with subsequent experiments.

[0136] Protein immunoblotting: The procedure is the same as that for protein immunoblotting in Experiment Example 2.

[0137] Ubiquitination level detection: The operation steps are the same as those for ubiquitination detection described above.

[0138] The results are as follows Figure 20 As shown, Phosphosite website predictions indicate that the K40 site is where ubiquitination occurs. Experimental data show that the ubiquitination level in the K40 mutant group was significantly increased compared to the wild type. Figure 21As shown, TAGLN2 K40R The mutant exhibited significantly stronger binding to the E3 ubiquitin ligase MARCH1 than the wild-type. GEPIA analysis showed a negative correlation between TAGLN2 and MARCH1. Figure 22 The results confirmed that succinylation of TAGLN2 at the K40 site interferes with its binding to MARCH1. Figure 23 The results showed that inhibiting MARCH1 significantly reduced the ubiquitination level of TAGLN2 protein, while its expression level significantly increased. These findings indicate that succinylation at the TAGLN2-K40 site can effectively block the MARCH1-mediated ubiquitination process.

[0139] Example 7: Preparation of a monoclonal antibody that specifically recognizes the succinylation modification of lysine 40 in the TAGLN2 protein (TAGLN2-K40 succinylation) 1. Immunization: Using peptide-KLH, the antigen dose was 60ug / mouse for the initial immunization and 30ug / mouse for subsequent immunizations. Four SPF-grade BALB / c female mice were subcutaneously immunized, numbered 1#, 2#, 3#, and 4#. Blood was collected from the orbital sinus after immunization to measure serum titer. Immunotitable titer assay: Peptide-BSA, 2ug / ml, was used for coating overnight at 4℃; 5% milk was used for blocking at 37℃ for 2 hours; serum was serially diluted 2-fold starting from 200-fold. The blank control was PBS, and the negative control was a 200-fold dilution of negative serum. Mice #2 were selected for cell fusion experiments.

[0140] 2. Cell fusion experiment: Mouse spleen cells and SP2 / 0 cells were fused using the PEG method. After fusion, the cells were screened and cultured in a semi-solid culture medium (containing HAT).

[0141] 3. Selecting clones: Select 8 plates of 93 single cell clones and culture them in 96-well cell culture plates.

[0142] 4. Single-clonal cell screening: Discard all cell supernatant from the 96-well cell culture plate, add 200 μL / well of 20% fetal bovine IMDM medium (containing HT) for the first screening.

[0143] 5. Second screening of monoclonal cells: The selected clones were coated with modified peptide-BSA and screened a second time using ELISA (primary antibody: cell culture supernatant, secondary antibody: goat anti-mouse IgG / HRP, chromogenic solution: TMB chromogenic solution, stop solution: 0.5M sulfuric acid) to obtain positive hybridoma cell lines.

[0144] 6. Monoclonal Cell Screening (3-stage): Eight positive cell lines were coated again with modified and unmodified peptide-BSA and screened a third time using ELISA, yielding seven positive hybridoma cell lines. Subclass identification revealed seven IgG-type positive hybridoma cell lines. Finally, the 3E8 monoclonal antibody secreted by the hybridoma cell line with sample number 3E8 was selected. The amino acid sequences of the heavy chain variable region and light chain variable region of this antibody were analyzed by Hubei Qiangyao Biotechnology Co., Ltd., as shown in Table 6. The SDS-PAGE results of the 3E8 monoclonal antibody are shown below. Figure 24 As shown.

[0145] Table 6. Amino acid sequences of the heavy chain variable region and light chain variable region of the 3E8 monoclonal antibody.

[0146] Experimental Example 8: The cell-penetrating peptide of the anti-TAGLN2-K40 succinylated antibody (CPP-TAGLN2K40succ) significantly inhibited the expression of TAGLN2 protein in glioma cells and significantly enhanced the sensitivity of TMZ cells to the drug. 1. Materials: Cell lines: NHA, U87, U251; The cell-penetrating peptide (CPP-TAGLN2K40succ) of TAGLN2-K40 succinylated antibody was purchased from Shanghai Qiangyao Company. The sequence of the cell-penetrating peptide (antibody) is: 5-FITC-(Acp)-YGRKKRRQRRRC-monoclonal antibody.

[0147] 2. Method: 2.1 Verification of the cellular uptake capacity of CPP-TAGLN2K40succ and its effect on cell viability 1) Cellular uptake capacity of CPP-TAGLN2K40succ: 24 hours in advance, place U87 or U251 cells in microcircular slides in 24-well plates, 4 × 10⁶ cells per well. 4 Cells were cultured in complete medium containing 10% fetal bovine serum for 24 hours until cell confluence reached 60%–80%. The old medium was discarded, and cells were incubated in complete medium containing fluorescently labeled CPP-TAGLN2K40succ (10 mM) for 48 hours. Cells were washed three times with PBS and fixed with 4% paraformaldehyde. DAPI staining solution was added and incubated in the dark for 5 minutes. DAPI staining solution was discarded, and cells were washed twice. Fluorescence was observed under a fluorescence microscope to determine whether fluorescence signals were present in the cells.

[0148] 2) CCK-8 experiment: The operation steps are the same as those of the CCK-8 experiment in Experiment Example 1.

[0149] 2.2 Verification of the effect of CPP-TAGLN2K40succ on the cell viability of normal astrocytes (NHA) and glioma cells U87 and U251. The effect of CPP-TAGLN2K40succ on the viability of NHA, U87, and U251 cells was detected using a CCK-8 assay. Groups: Blank group: CCK-8 solution; Control group: cells + CCK-8 solution; Experimental group: cells + CPP-TAGLN2K40succ + CCK-8 solution (cells in each group were NHA, U87, and U251 cells); the procedure was the same as the CCK-8 assay in Example 1.

[0150] 2.3 Verification of the effects of CPP-TAGLN2K40succ on TAGLN2-K40 succinylation and TAGLN2 protein in normal astrocytes (NHA) and glioma cells U87 and U251. Add fresh culture medium containing CPP-TAGLN2K40succ (10 mM) to the cultured cells, incubate for 48 hours, and collect the cells for Western blotting: the procedure is the same as the Western blotting in Experiment 2.

[0151] 2.4 Verification of the effect of CPP-TAGLN2K40succ on ferroptosis-related proteins The cultured cells were added to fresh medium containing CPP-TAGLN2K40succ (10 mM) and incubated for 48 hours. The cells were then collected and subjected to Western blotting to detect the expression of β-catenin and GPX4 proteins. The procedure was the same as that for Western blotting in Experiment 2.

[0152] 2.5 The effect of CPP-TAGLN2K40succ on the cellular localization of β-catenin was verified by immunofluorescence. Add fresh culture medium containing CPP-TAGLN2K40succ (10 mM) to the cultured cells, incubate for 48 hours, and perform immunofluorescence, following the same procedure as in Experiment 2.

[0153] 2.6 Investigating the effect of CPP-TAGLN2K40succc on TMZ chemosensitivity using the CCK8 assay CCK-8 Experiment: The operating procedure is the same as that of the CCK-8 experiment in Experiment Example 1.

[0154] U87 and U251 cells were treated with gradient concentrations (0, 500, 1000, 1500, 2000, 2500, 3000 μM) of TMZ and a fixed concentration of CPP (10 mM), respectively. Each group was set up in triplicate. After incubation at 37°C for 48 hours, 100 µL of CCK-8 culture medium with a final concentration of 10% prepared in serum-free medium was added to each well. The wells were then incubated in an incubator for 1 h. The absorbance of the plates was measured at 450 nm using a microplate reader to assess cell viability.

[0155] Figure 25 The study demonstrated the uptake capacity of CPP-TAGLN2K40succ in glioma cells, and that CPP-TAGLN2K40succ selectively reduced glioma cell viability while having no effect on normal astrocytes. Figure 26 The results showed that CPP-TAGLN2K40succ significantly reduced TAGLN2-K40 succinylation levels, total TAGLN2 protein content, and the expression of ferroptosis-related proteins; Figure 27 As shown, the nuclear translocation of β-catenin is reduced after CPP-TAGLN2K40succ treatment; Figure 28 The combination of CPP-TAGLN2K40succc and TMZ significantly enhanced the cytotoxicity of TMZ in U87 and U251 cells, showing superior efficacy compared to either drug alone. This indicates that CPP-TAGLN2K40succc significantly reduced TAGLN2-K40 succinylation levels and total TAGLN2 protein content, downregulated GPX4 protein levels, maintained total β-catenin expression but reduced nuclear translocation, thereby promoting ferroptosis in glioma cells; CPP-TAGLN2K40succc also enhanced the therapeutic sensitivity of TMZ.

[0156] Experimental Example 9: Combination therapy of TAGLN2-K40 succinylated antibody cell-penetrating peptide with TMZ enhances in vitro and in vivo antitumor efficacy. 1. Materials: GL261 cells, purchased from ATCC; C57 mice, purchased from Beijing Huafukang Experimental Animal Technology Co., Ltd.

[0157] 2. Methods 2.1 Detection of the effect of CPP-TAGLN2K40succ on the proliferation ability of GL261 cells CPP-TAGLN2K40succ was administered at concentrations of 0, 10, 20, and 40 mM, and the results were detected by CCK8 assay. The experimental procedure was the same as in Experiment 1.

[0158] 2.2 The effects of CPP-TAGLN2K40succ on the succinylation level of TAGLN2-K40 and the TAGLN2 protein level in GL261 cells were detected. The experimental procedure was the same as that described in Experiment Example 2, including Western blotting.

[0159] 2.3 An orthotopic glioma model was established by intracranial injection of GL261 cells into C57 mice to evaluate the antitumor effects of CPP-TAGLN2K40succc alone or in combination with TMZ. 1) GL261 cells were inoculated near the anterior skull of mice; 2) One week after successful tumor grafting in mice, drug treatment was administered. The animal experiment was divided into four groups: control group, CPP-TAGLN2K40succ group (0.2 mg / kg), TMZ group (25 mg / kg), and combination drug administration group (CPP-TAGLN2K40succ 0.2 mg / kg + TMZ 25 mg / kg). The drugs in each group were administered intraperitoneally at the corresponding doses, three times a week for two consecutive weeks. Brain tissue samples were then collected for efficacy evaluation.

[0160] 3) Immunofluorescence assay to detect whether CPP-TAGLN2K40succ can enter the brain After obtaining mouse brain tissue sections, immunofluorescence detection was performed.

[0161] 4) Detection of the effect of CPP-TAGLN2K40succ on iron ions in gliomas using Prussian blue staining method First, glioma tissue samples were collected, then stained with Prussian blue, and finally observed under a microscope.

[0162] 5) Immunohistochemical detection of the therapeutic effect of CPP-TAGLN2K40succ The same immunohistochemistry experimental protocol as in Experiment Example 4.

[0163] The results are as follows Figure 29 As shown, CPP-TAGLN2K40succ significantly reduced the survival rate of GL261 cells, the level of TAGLN2-K40 succinylation modification, and the content of TAGLN2 protein. Figure 30 The results showed that CPP-TAGLN2K40succ significantly enriched iron ions after entering the brain. Figure 31 The results showed that the combination therapy significantly reduced TAGLN2-K40 succinylation levels, accompanied by downregulation of GPX4 expression and a decrease in the proliferation marker Ki-67. This indicates that CPP-TAGLN2K40succ also promotes ferroptosis in mice both in vitro and in vivo, and that CPP-TAGLN2K40succ enhances the efficacy of TMZ by inhibiting TAGLN2-K40 succinylation.

Claims

1. A monoclonal antibody that specifically recognizes the succinylation modification of lysine at position 40 of the TAGLN2 protein, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

2.

2. The encoding gene of the monoclonal antibody according to claim 1.

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal antibody of claim 1.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutical composition contains temozolomide (TMZ).

5. The use of the encoding genes of the monoclonal antibody of claim 1 and the monoclonal antibody of claim 2 in the preparation of reagents for diagnosing diseases characterized by TAGLN2-K40 succinylation.

6. The application according to claim 5, characterized in that, The diseases mentioned include glioblastoma.

7. The use of the monoclonal antibody of claim 1, the encoding gene of the monoclonal antibody of claim 2, or the pharmaceutical composition of claim 3 or 4 in the preparation of a medicament for the prevention and / or treatment of diseases characterized by TAGLN2-K40 succinylation.

8. The application according to claim 7, characterized in that, The diseases mentioned include glioblastoma.

9. The application according to claim 7, characterized in that, The drug contains pharmaceutically acceptable excipients.

10. The application according to claim 7, characterized in that, The dosage forms of the drugs include tablets, granules, oral liquid preparations, drops, injectable preparations, and capsule preparations.

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