Application of EGLN1 lactic acid as target spot in preparation of tumor diagnostic kit or therapeutic drug

Abstract

The invention discloses application of EGLN1 lactylation as a target spot in preparation of a tumor diagnosis kit or a therapeutic drug, and belongs to the technical field of biology. The invention discloses that lysine at the 402 site of EGLN1 protein is the lactylation site for the first time, the EGLN1 K402 lactylation level in tumor tissues is remarkably higher than that in para-carcinoma tissues, and EGLN1 K402 lactylation modification is related to tumor occurrence and development, so that EGLN1 K402 lactylation can be used as a tumor diagnosis or prognosis evaluation marker and is applied to development of a tumor diagnosis or prognosis evaluation kit. The detection of the EGLN1 K402 lactylation level can guide the treatment of tumor patients, and can be used for screening candidate drugs for treating tumors. When the compound for inhibiting EGLN1 K402 lactic acid modification is combined with a chemical effect drug, the chemotherapy sensitivity of the drug can be remarkably improved, the occurrence and development of tumors can be inhibited, and a new thought is provided for the development of anti-tumor drugs.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of an EGLN1 lactic acid modification site as a target in the preparation of a tumor diagnosis kit or a therapeutic drug. Background Art

[0002] Protein lactylation is one of the important mechanisms of protein post-translational modification. This modification changes the structure and function of the protein by covalently binding lactic acid molecules to specific amino acid residues of the protein. This modification plays a vital role in cell biology, affecting the stability, activity, localization and interaction of proteins with other molecules. Lactic acid modification is particularly active in tumor cells. In tumor tissues, in order to meet the high energy demand of tumor tissues, multiple metabolic modes coexist in tumor tissues. Metabolic reprogramming is a characteristic of cancer. Even if there is sufficient oxygen, tumor cells rely on glycolysis as an energy source, that is, aerobic glycolysis. Tumor cells produce a large amount of lactic acid during metabolism. These lactic acids not only participate in regulating the metabolic process of tumor cells, but may also affect the growth and differentiation of tumor cells through lactylation modification.

[0003] Egl-9 Family Hypoxia Inducible Factor 1 (EGLN1) catalyzes the post-translational formation of 4-hydroxyproline in the hypoxia-inducible factor (HIF) α protein. HIF is a transcriptional complex that plays a central role in mammalian oxygen homeostasis. This protein functions as a cellular oxygen sensor, and under normal oxygen concentrations, prolyl hydroxylation modification is a key regulatory event that targets HIF subunits for proteasomal destruction via the von Hippel-Lindau ubiquitination complex; under hypoxic conditions, hydroxylation is attenuated, allowing HIF to escape degradation, leading to their translocation to the nucleus, heterodimerization with HIF1B, and increased expression of hypoxia-inducible genes. EGLN1 is the most important isozyme under normoxia and participates in various processes affected by hypoxia, such as angiogenesis in the retina and cardiac function, by regulating the stability of HIF1.

[0004] Studies have shown that EGLN1 plays a certain role in some tumors as an independent molecule. For example, Xing Liu et al. reported that EGLN1 is a key mediator of oxygen enhancement of antiviral innate immune response (Oxygen enhances antiviral innate immunity through maintenance of EGLN1-catalyzed proline hydroxylation of IRF3. Nat Commun, 2024, 15: 3533.). Jiang et al. reported that EGLN1 promotes tumor growth and metastasis in breast cancer metabolic adaptation (A mitochondrial EglN1-AMPKα axis drives breast cancer progression by enhancing metabolic adaptation to hypoxic stress. Embo j, 2023, 42: e113743.). Xu et al. reported that EGLN1, as a proline hydroxylase, participates in catalyzing the binding of pVHL to HIF-1α, and then promotes tumor development (Prolonged hypoxia alleviates prolyl hydroxylation-mediated suppression of RIPK1 to promote necroptosis and inflammation. Nat Cell Biol, 2023, 25: 950-962.). In short, EGLN1 plays an important role in the occurrence and development of cancer.

[0005] The research team of the present invention found that the lactic acid modification level of EGLN1 is correlated with the occurrence and development of tumors, which has not been reported so far. Therefore, in-depth research on the lactic acid site of EGLN1 is expected to provide valuable biomarkers for the diagnosis and prognosis analysis of tumors. Summary of the invention

[0006] The purpose of the present invention is to provide a site where EGLN1 undergoes lactic acid modification, which can be used as a target for developing a tumor diagnosis or prognosis evaluation kit, or for screening and developing tumor therapeutic drugs.

[0007] To achieve the above object, the present invention adopts the following technical solution: The present invention found that the 402nd lysine of EGLN1 protein is its lactylation site through protein modification mass spectrometry detection and point mutation verification. The lactylation modification level in tumor tissue was detected by immunohistochemical staining and Western blot, and it was found that the lactylation modification level of EGLN1 K402 site in tumor tissue was significantly higher than that in adjacent tissue. After mutation of this site, the lactylation expression of EGLN1 and the biological functions of its downstream related molecules were changed, thereby affecting the occurrence and development of tumors.

[0008] Therefore, the present invention provides the use of the lactic acid modification site of EGLN1 as a detection target in the preparation of a tumor diagnosis kit, wherein the amino acid sequence of the tumor EGLN1 is shown in SEQ ID NO.1, wherein the lysine at position 402 is lactic acid modified.

[0009] The present invention follows up tumor patients undergoing chemotherapy, records relevant information such as efficacy evaluation and survival time, and detects the lactylation modification level of the EGLN1 K402 site. The results show that changes in the lactylation level of EGLN1 K402 can reflect the patient's response to chemotherapy to a certain extent; high EGLN1 K402 lactylation level is associated with tumor progression and chemotherapy resistance, and EGLN1 K402 lactylation has potential value as a prognostic marker.

[0010] Therefore, the present invention provides the use of the lactylation modification site of EGLN1 as a detection target in the preparation of a tumor prognosis evaluation kit.

[0011] Furthermore, the kit includes an antibody against the lactic acid modification site of EGLN1. The antibody is used to detect the sample to be tested to determine whether the lysine 402 of EGLN1 in the sample to be tested is lactic acid modified, or to quantify the lactic acid level. The detection method that can be used in the present invention is not limited to this.

[0012] Furthermore, the amino acid sequence of the hapten polypeptide used to prepare the antibody is ARAKVKYLTGEK, wherein the lysine at position 6 is lactylated. Studies have shown that the antibody prepared by selecting positions 397-408 (ARAKVK(Lac)YLTGEK) of EGLN1 in the antigenic region can specifically recognize the lactylated modification at position 402 in EGLN1.

[0013] The antibody described in the present invention may be a polyclonal antibody or a monoclonal antibody, and may be prepared by techniques known in the art.

[0014] Furthermore, the antibody is a polyclonal antibody, and the preparation method includes: firstly synthesizing a hapten polypeptide having an amino acid sequence of CARAKVKYLTGEK and a lactic acid modification of lysine at position 7, coupling it with hemocyanin to prepare an antigen, and then obtaining serum by animal immunization, and separating and purifying the serum to obtain the antibody. The present invention adds cysteine ​​to the N-terminus of the hapten polypeptide, and uses a thiol group to couple it with a carrier protein (such as hemocyanin).

[0015] Furthermore, the tumor is colorectal cancer or breast cancer.

[0016] The present invention provides a kit for diagnosing or evaluating prognosis of colorectal cancer, comprising an antibody against the lactic acid modification site of EGLN1, wherein the amino acid sequence of EGLN1 is shown in SEQ ID NO.1, wherein the lysine at position 402 is lactic acid modified.

[0017] The present invention also provides an application of a lactylation modification site of EGLN1 as a target in screening tumor therapeutic drugs, wherein the amino acid sequence of the tumor EGLN1 is shown in SEQ ID NO.1, wherein the lysine at position 402 is lactylation modified; and the tumor therapeutic drug targets and inhibits the lactylation of the lysine at position 402 of EGLN1 in tumor cells.

[0018] Studies have shown that changes in the lactylation level of EGLN1 K402 reflect the therapeutic effect of the drug to a certain extent. Therefore, when administering drugs in cell models or animal models, the lactylation level of EGLN1 K402 is detected to characterize the efficacy of the compound and to screen compounds with anti-tumor activity.

[0019] Furthermore, the tumor is colorectal cancer.

[0020] The present invention also provides the use of a compound that inhibits lactic acid modification of EGLN1 in the preparation of a drug for treating tumors, wherein the amino acid sequence of the tumor EGLN1 is shown in SEQ ID NO.1, wherein the lysine at position 402 is lactic acid modified. The drug achieves the purpose of anti-tumor by inhibiting the lactic acid modification of the lysine at position 402 of EGLN1 in tumor cells.

[0021] Furthermore, the tumor is colorectal cancer.

[0022] Furthermore, the compound inhibiting lactic acid modification of EGLN1 is any one of a polypeptide consisting of a transmembrane peptide and a peptide extending 5-10 amino acids upstream and downstream of lysine 402 of the EGLN1 protein, a lactate production inhibitor, and 2-deoxy-D-glucose.

[0023] Under the action of the cell-penetrating peptide, after entering the cell, the polypeptide simulates the endogenous EGLN1 K402 site and competitively binds to the endogenous EGLN1 lactase, thereby inhibiting the lactation of the endogenous EGLN1 K402 site. Preferably, the polypeptide consists of a cell-penetrating peptide segment and a peptide segment with an amino acid sequence of ERARAKVKYLTGEKG.

[0024] The cell-penetrating peptide may be, but is not limited to, a TAT peptide. Preferably, the amino acid sequence of the polypeptide is YGRKKRRQRRRAERARAKVKYLTGEKG.

[0025] Furthermore, the compound that inhibits the lactic acid modification of EGLN1 is used in combination with a chemotherapeutic drug to prepare an anti-tumor drug, and the chemotherapeutic sensitivity of the chemotherapeutic drug is increased by the combination of drugs, thereby enhancing the anti-tumor effect.

[0026] Furthermore, the drug also includes oxaliplatin. The combination of the compound that inhibits the lactic acid modification of EGLN1 and oxaliplatin can significantly enhance the chemotherapy sensitivity of oxaliplatin and improve the anti-tumor effect.

[0027] The present invention has the following beneficial effects: The present invention discloses for the first time that the lysine 402 of the EGLN1 protein is its lactylation site. The lactylation level of EGLN1 K402 in tumor tissue is significantly higher than that in adjacent cancer tissue. The lactylation modification of EGLN1 K402 is related to the occurrence and development of tumors. Therefore, the lactylation of EGLN1 K402 can be used as a tumor diagnosis or prognosis assessment marker and applied to the development of tumor diagnosis or prognosis assessment kits. Detection of the lactylation level of EGLN1 K402 can guide the treatment of tumor patients and can be used to screen candidate drugs for treating tumors. The combination of compounds that inhibit the lactylation modification of EGLN1 K402 and chemotherapeutic drugs can significantly improve the chemotherapy sensitivity of the drug and inhibit the occurrence and development of tumors. The present invention provides a new idea for the development of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Anti-EGLN1 K402 for dot blot detection Lac Polyclonal antibody specificity results.

[0029] Figure 2 Anti-EGLN1 K402 for Western Blot Analysis Lac Specificity of polyclonal antibodies.

[0030] Figure 3 Western Blot was used to verify that TAT peptide blocked EGLN1 K402 lactylation.

[0031] Figure 4For EGLN1 K402 Lac Results of polyclonal antibody immunohistochemistry.

[0032] Figure 5 EGLN1 K402 in colorectal cancer tissue and adjacent tissue Lac Immunohistochemistry results.

[0033] Figure 6 The results of Western blot detection of lactic acid modification levels in colorectal cancer tissues.

[0034] Figure 7 Western Blot was used to detect the lactylation level of EGLN1 K402 in PDX tumor tissue lysate.

[0035] Figure 8 K402 blocking peptide for EGLN1 enhances sensitivity of PDX models to oxaliplatin treatment.

[0036] Fig. 9 The combination of lactate production inhibitors (LDHi, 2DG) and oxaliplatin enhances the sensitivity of PDX models to oxaliplatin treatment.

[0037] Fig.10 The ROC curve was used to predict the effect of EGLN1 K402 lactylation modification level on the chemotherapy efficacy of colorectal cancer.

[0038] Fig.11 EGLN1 K402 lactylation modification level predicts the prognosis of colorectal cancer patients. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0041] Example 1: EGLN1 protein K402 Lac Peptide design and synthesis 1. Sequence analysis The amino acid sequence of human EGLN1 protein (Q9GZT9) was obtained from GenBank, and the amino acid sequence is shown in SEQ ID NO. 1. The human EGLN1 protein contains 426 amino acids. The characteristics of human EGLN1 protein were analyzed using DNAstar software, and the molecular weight of human EGLN1 protein was 46860 Daltons.

[0042] 2. Protein modification mass spectrometry detection Through protein modification mass spectrometry detection and point mutation verification, we found that the lysine 402 of EGLN1 is its lactylation site. Our previous work showed that after mutation of this site, the lactylation expression of EGLN1 and the biological functions of its downstream related molecules changed, thereby affecting the occurrence and development of tumors.

[0043] 3. Peptide synthesis The synthetic peptide sequence is used to prepare the antigen. After analysis, the antigen region is selected as 397-408aa. Cysteine ​​C is added to the N-terminus of the peptide. Specifically, the synthetic peptide sequences are: CARAKVK (Lac) YLTGEK (peptide I) and CARAKVKYLTGEK (peptide II). Peptide I is used for immunization after coupling with KLH; peptide II is used to prepare antigen affinity purification columns for detection and purification. The peptides are synthesized by Hangzhou Huaan Biotechnology.

[0044] Example 2: Anti-EGLN1 polypeptide (K402 Lac )Preparation, purification and identification of rabbit polyclonal antibodies 1. Peptide-carrier protein coupling The thiol-containing hapten polypeptide I prepared in Example 1 was dissolved in coupling buffer and added to 10 mg / mL KLH. The polypeptide and KLH were immediately mixed and then reacted at room temperature for 2 h. The conjugate was purified by gel chromatography desalting column.

[0045] 2. Animal immunization The coupled product was used to immunize two New Zealand white rabbits, and the serum titer of the rabbits after three immunizations was monitored. After Elisa verification, the immunization was strengthened and all the serum was collected. The rabbit serum with the best serum titer was selected for purification by affinity chromatography column.

[0046] Immunization method: animals are injected subcutaneously at multiple points, with 0.2 mL at each point. Immunization time: the second immunization is carried out on the 14th day after the first immunization, and the interval between the second and third immunizations is 7 days. On the 7th day after the third immunization, a small sample of serum is collected from the middle ear artery of the animal for testing. If the test is qualified, the animal will be given a booster immunization 7 days later, and whole blood can be collected 7 days after the booster immunization.

[0047] 3. Antibody purification The rabbit serum is added to the affinity chromatography column to purify the antibody. After the antibody is qualified, it is concentrated using an ultrafiltration concentration tube to reach a certain concentration and volume.

[0048] 4. Anti-EGLN1 (K402 Lac ) ELISA identification of polyclonal antibodies Synthetic EGLN1 K402 Lac The ELISA plates were coated with K402 polypeptide as the detection antigens, and the non-immune rabbit serum diluted 1:2000 was used as the negative control. The rabbit serum and the purified antibodies were diluted in multiples and tested by ELISA method to calculate the antibody titer. The results are shown in Table 1.

[0049] Table 1. EGLN1 (K402 Lac ) Antibody titer Dilution multiple Absorbance value of coated modified peptide Absorbance value of coated non-modified peptide 1:250 2.861 1.405 1:1000 2.326 0.626 1:4000 1.450 0.387 1:16000 0.620 0.055 1:64000 0.193 0.028 1:256000 0.058 0.011 1:1024000 0.024 0.002 Results: From the perspective of titer, the anti-EGLN1 (K402 Lac ) polyclonal antibody titer greater than 1:4000, EGLN1 (K402 Lac ) corresponds to an absorbance value of 1.45, while in the control group, under the same dilution conditions, the absorbance of the EGLN1 K402 non-modified peptide was only 0.387.

[0050] 5. Anti-EGLN1 (K402 Lac ) Immunodot assay Weigh 1 mg of EGLN1 K402 lactic acid modified peptide and non-modified peptide and dissolve them in 1 mL of 0.01 M PBS buffer. Take 5 μL of the dissolved peptide and spot it on the PVDF membrane. After drying, block the PVDF membrane with TBST blocking solution containing 5% skim milk powder for 1 h. Add the corresponding purified anti-EGLN1 K402 Lac The polyclonal antibody (dilution factor 1:1000) was incubated at room temperature for 2 h, then the membrane was washed five times (5 min / time). HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added, and the membrane was washed five times (5 min / time) after incubation at room temperature for 1 h. The final results were imaged by Jena UVP Chemstudio multifunctional imager.

[0051] The results are as follows Figure 1 As shown, EGLN1 K402 Lac The polyclonal antibodies almost all recognized the K402 lactic acid-modified peptides on the PVDF membrane, and could hardly recognize the K402 non-lactated peptides.

[0052] 6. Western-Blot Identification of Anti-Polyclonal Antibodies 6.1 Construction of expression vectors of wild-type EGLN1 and lysine mutation at position 402 of EGLN1 with HA tag (mutation of lysine to arginine). Specifically, Wuhan Miaoling Biotechnology was commissioned to construct HA-EGLN1 WT and HA-EGLN1 K402R plasmids, using pcDNA3.1 vector for construction. The wild-type nucleotide sequence of EGLN1 is shown in SEQ ID NO.2, the wild-type gene fragment of EGLN1 is cloned into the multiple cloning site of pcDNA3.1 vector to construct HA-EGLN1 WT plasmid, and then a point mutation kit is used to construct the mutant plasmid of HA-EGLN1 K402R, and the mutant nucleotide sequence is shown in SEQ ID NO.3.

[0053] 6.2 HEK293T cells were transiently transfected with HA-EGLN1 WT and HA-EGLN1 K402R plasmids, respectively. HEK293T cell proteins were collected 48 hours after transfection and purified by incubation with Anti-HA magnetic beads. The purified proteins were eluted and denatured, and then subjected to SDS-PAGE gel electrophoresis. After electrophoresis, they were transferred to PVDF membranes, blocked with skim milk powder, and purified anti-EGLN1 (K402 Lac ) polyclonal antibody (dilution factor 1:1000), incubate at 4°C overnight, wash the membrane five times (5 min / time), add HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000), incubate at room temperature for 2 hours, wash the membrane five times (5 min / time), and finally image the results using Jena UVP Chemstudio multifunctional imager.

[0054] The results are as follows Figure 2 As shown, anti-EGLN1 (K402 Lac ) The polyclonal antibody can detect a protein band of about 45 kDa in the HEK293T cell lysate, which is consistent with the molecular weight of the EGLN1 protein.

[0055] 6.3 HA-EGLN1 WT plasmid and HA-EGLN1 K402R plasmid were transfected into HEK293T cells respectively. One group transfected with WT plasmid was used as control group, and 30 mM sodium lactate solution was added to the other group and the group transfected with K402R plasmid to increase the lactylation level of EGLN1. After 24 h, HA-EGLN1 protein was precipitated using Anti-HA magnetic beads, and the lactylation level of EGLN1 K402 was detected by Western Blot.

[0056] The results are as follows Figure 2As shown, the lysine 402 of the EGLN1 protein expressed by cells transfected with the wild-type plasmid was obviously lactylated, and the lactylation level increased after stimulation with sodium lactate; the lactylation level of the EGLN1 protein expressed by cells transfected with the K402R mutant was significantly inhibited, and its lactylation level was reduced compared with the wild type despite the addition of sodium lactate.

[0057] 6.4 HEK 293T cells were transiently transfected with EGLN1 WT plasmid. After transfection, K402 TAT peptide (amino acid sequence: YGRKKRRQRRRAERARAKVKYLTGEKG, where YGRKKRRQRRRA is the TAT sequence that mediates the entry of peptides into cells. After entering the cells, TAT peptide can mimic the endogenous EGLN1 K402 site and compete with endogenous EGLN1 for binding to lactase, thereby inhibiting the lactylation of endogenous EGLN1 K402 site) was used to block its lactylation site. HEK293T cell proteins were collected 48 hours after transfection, and the EGLN1 protein was purified by incubation with Anti-HA magnetic beads. The purified protein was eluted and denatured, and then subjected to SDS-PAGE gel electrophoresis. After electrophoresis, it was transferred to a PVDF membrane, blocked with skim milk powder, and then added with purified anti-EGLN1 (K402 Lac ) polyclonal antibody (dilution factor 1:1000), incubate at 4°C overnight, wash the membrane five times (5 min / time), add HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000), incubate at room temperature for 2 hours, wash the membrane five times (5 min / time), and finally image the results using Jena UVP Chemstudio multifunctional imager.

[0058] The results are as follows Figure 3 As shown, the EGLN1 K402 site TAT peptide can successfully block the lactylation of the EGLN1 protein K402 site.

[0059] 7. Anti-EGLN1 (K402 Lac ) Immunohistochemical identification of polyclonal antibodies a. Place the colorectal tumor tissue pathological sections obtained by colonoscopy in a 60°C oven for 2 h, soak in xylene three times for 10 min each time, dehydrate with 100%, 95%, 80%, and 70% graded alcohol in turn for 5 min each time, and soak in deionized water for 3 min.

[0060] b. Thermal antigen repair: Add 1× sodium citrate antigen repair solution into a pressure cooker and heat to boiling. Place the dewaxed and hydrated paraffin sections on a high-temperature resistant plastic slide rack and slowly place them into the boiling buffer. Boil under high pressure for 15 minutes and rinse with tap water to cool to room temperature.

[0061] c. Block the activity of endogenous peroxidase: remove the slides from the antigen retrieval buffer, place them in 3% hydrogen peroxide solution for blocking, incubate them in the dark at room temperature for 15 min, wash them three times with PBS, 5 min each time, shake off and dry the liquid around the tissue, and place them flat in a humidified box.

[0062] d. Primary antibody incubation: Add EGLN1 (K402 Lac ) 100 μL of polyclonal antibody (1:200, diluted with immunohistochemistry primary antibody diluent), negative control plus 100 μL of primary antibody diluent, placed in a wet box, incubated at room temperature for 1 hour, washed 5 times with PBS, 3 min each time, shake off and dry the liquid around the tissue, and place it flat in a wet box.

[0063] e. Secondary antibody incubation: Add 100 μL of specific secondary antibody working solution onto the tissue, place in a wet box, incubate at room temperature for 20 min, wash 5 times with PBS, 3 min each time, shake off and dry the liquid around the tissue.

[0064] f. Add 100 μL of the pre-prepared color developer DAB working solution and control the color development under a light microscope. After the color development is complete, immerse the sample in distilled water to stop the color development.

[0065] g. Restain with hematoxylin for 5 minutes, soak in 1% hydrochloric acid alcohol for 30-60 seconds for differentiation, and rinse with tap water.

[0066] h. Soak in PBS for 1 min to turn blue, then rinse in tap water for 10 min.

[0067] i. Dehydrate and dry in 70%, 80%, 95%, and 100% alcohol gradients, 3 min each. Seal the slides with neutral gum and observe the results under a microscope.

[0068] The results are as follows Figure 4 As shown, EGLN1 (K402 Lac ) mainly appeared in the cell nucleus, which was consistent with the localization of EGLN1 and was a positive result; while using antibody diluent instead of EGLN1 (K402 Lac ) After detection with polyclonal antibodies, no specific staining was found in the cell nucleus, which was a negative result.

[0069] Example 3: Pan-Lactyl Lysine Antibody and EGLN1 (K402 Lac ) Polyclonal antibody detection of lactylation modification level in tumor tissue 1. Immunohistochemistry (IHC) to detect lactic acid modification levels in colorectal cancer tissues Colorectal cancer tissues obtained during surgery and their corresponding adjacent tissues were collected. This study was approved by the Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University and was conducted in accordance with the approved guidelines and regulations.

[0070] All samples were fixed in formalin, embedded in paraffin, and sectioned. Immunohistochemical staining was then used to detect the lactic acid modification level between tumor tissue and adjacent tissue (the primary antibody was Pan-Lactyl Lysine antibody), as well as the lactic acid modification of the EGLN1 K402 site (the primary antibody was EGLN1 (K402 Lac ) polyclonal antibody). This experiment aims to reveal the difference in lactylation modification between tumor tissues and normal tissues and the level of EGLN1 K402 lysine lactylation in tumor tissues.

[0071] The results are as follows Figure 5 As shown in Figure 2, the lactylation modification level in colorectal cancer tissues was significantly higher than that in the corresponding adjacent cancer tissues. Lac ) The detection results of polyclonal antibodies further confirmed that the lactylation modification level of EGLN1 K402 site in tumor tissues was also significantly higher than that in adjacent cancer tissues.

[0072] These results suggest that the lactate level in colorectal cancer tissues is significantly higher than that in adjacent adjacent tissues, indicating that lactylation modification may play an important role in the tumor microenvironment.

[0073] 2. Western blot detection of lactic acid modification level in colorectal cancer tissue We ground the colorectal cancer tissues and their corresponding adjacent cancer tissues collected during surgery, extracted tissue proteins, and performed Western blot to detect the level of lactic acid modification in tumor tissues.

[0074] The results are as follows Figure 6 As shown in the figure, the lactylation modification level in colorectal cancer tissues was significantly higher than that in the corresponding adjacent cancer tissues.

[0075] This result suggests that lactylation may have important biological significance in the colorectal cancer microenvironment.

[0076] Example 4: Targeted inhibition of EGLN1 K402 lactylation enhances oxaliplatin chemotherapy sensitivity 1. Determination of lactic acidification degree of EGLN1 K402 in patient-derived tumor xenograft model (PDX) Colorectal cancer tumor tissue was obtained by surgery and implanted into the armpits of immunodeficient nude mice in the form of fragments. When the tumor grew to a diameter of about 1-2 cm, the tumor was peeled off from the first-generation host (P0), and a portion of the tissue was minced to about 3 mm × 3 mm × 3 mm and implanted into new mice (P1). The other portion was preserved in cryopreservation solution for recovery and seed preservation. This process was repeated until the third generation (P3) was bred. P3 tumor tissue was frozen and ground to extract tissue protein, and SDS-PAGE gel electrophoresis was performed. After electrophoresis, it was transferred to a PVDF membrane, blocked with skim milk powder, and purified anti-EGLN1 (K402 Lac ) polyclonal antibody (dilution factor 1:1000), incubate at 4°C overnight, wash the membrane five times (5 min / time), add HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000), incubate at room temperature for 2 hours, wash the membrane five times (5 min / time), and finally image the results using Jena UVP Chemstudio multifunctional imager.

[0077] The results are as follows Figure 7 As indicated, anti-EGLN1 (K402 Lac ) The polyclonal antibody can detect a protein band of about 45 kDa in PDX tumor tissue lysate, which is consistent with the molecular weight of EGLN1 protein.

[0078] 2. EGLN1 K402 blocking peptide enhances chemotherapy sensitivity in human tumor xenograft models according to Figure 7 Results A PDX model with high lactylation modification of EGLN1 (#3) was selected and randomly divided into 6 groups, namely ① control group, ② K402R peptide group, ③ K402 peptide group, ④ Oxaliplatin group, ⑤ K402R peptide + Oxaliplatin group, ⑥ K402 peptide + Oxaliplatin group, and peptides targeting EGLN1 K402 were used to block the lactylation of EGLN1 K402. Note: The sequence of K402 peptide is: YGRKKRRQRRRAERARAKVKYLTGEKG, which can block the lactylation of EGLN1 K402 site, and the sequence of K402R peptide is: YGRKKRRQRRRAERARAKVRYLTGEKG, which cannot block the lactylation of K402 site and serves as the control.

[0079] Administration: Peptide was intraperitoneally injected, 5 mg / kg, once a day, for 2 weeks; oxaliplatin was intraperitoneally injected, 10 mg / kg, once a day, for one week. The peptide + oxaliplatin group started oxaliplatin treatment after one week of peptide treatment.

[0080] The weight of the mice, the size of the tumor tissue, and the response to chemotherapy were observed. After the treatment, the subcutaneous PDX tumor tissue of the mice was removed, photographed, and weighed. Before killing the mice, the whole blood of the mice was collected by eye bleeding, placed at room temperature for 2 hours, centrifuged at 1000×g for 20 minutes, and the supernatant was taken for biochemical analysis, such as mouse liver and kidney function.

[0081] The results are as follows Figure 8 As indicated, anti-EGLN1 (K402 Lac ) The polyclonal antibody can detect the lactylation degree of the EGLN1 K402 site in PDX tumor tissue, and the synthetic EGLN1 K402 blocking peptide can enhance the sensitivity of the PDX model to oxaliplatin treatment, as shown by the slower growth of tumor tissue in the K402 blocking peptide combined with oxaliplatin treatment group, smaller tumors, and lower weight than those in the oxaliplatin treatment group alone.

[0082] No obvious toxic reactions or abnormalities in other health indicators occurred during the treatment period, and HE results showed no significant effects on the mouse's heart, liver, spleen, lungs, kidneys and other organs. This result shows that the peptide used is safe and tolerable within the dosage range, providing a good biosafety foundation for subsequent research.

[0083] 3. Lactate production inhibitors (LDHi, 2DG) combined with oxaliplatin enhance chemotherapy sensitivity in human tumor xenograft models according to Figure 7 Results A PDX model with low-lactylation modification of EGLN1 (#1) was selected and randomly divided into 6 groups, namely ① control group, ② LDHi group, ③ 2-DG group, ④ Oxaliplatin group, ⑤ LDHi + Oxaliplatin group, ⑥ 2-DG + Oxaliplatin group. LDH inhibitor LDHi and glycolysis inhibitor 2-DG (LDHi and 2-DG were purchased from Sigma, with numbers: O2751 and D8375, respectively) were used to inhibit and reduce lactate production, aiming to inhibit EGLN1 K402 lactylation.

[0084] The weight of mice, tumor tissue size and response to chemotherapy were observed. After treatment, the subcutaneous PDX tumor tissue of mice was removed, photographed, recorded and weighed.

[0085] The results are as follows Fig. 9 As indicated, anti-EGLN1 (K402 Lac) polyclonal antibodies can detect the lactic acid level of EGLN1 K402 site in PDX tumor tissue. The anti-tumor efficacy of oxaliplatin was significantly enhanced by the use of lactate production inhibitors LDHi and 2-DG in combination with oxaliplatin. Specifically, the tumor growth rate in the LDHi or 2-DG combined with oxaliplatin treatment group was significantly lower than that in the oxaliplatin treatment group alone, and the tumor volume and weight were significantly reduced.

[0086] Example 5: IHC detection of EGLN1 K402 lactylation modification level in colorectal cancer tissues of patients to predict the efficacy of tumor chemotherapy 1. EGLN1 K402 lactylation modification level predicts the efficacy of chemotherapy in colorectal cancer Endoscopic tumor tissue samples and corresponding clinical data of patients with pathologically confirmed colorectal cancer were collected, including chemotherapy regimens, efficacy evaluation: divided into complete remission (CR), partial remission (PR), progressive disease (PD) and stable disease (SD), and patients using oxaliplatin chemotherapy were screened and followed up to record relevant information such as patient survival time.

[0087] The collected endoscopic tumor tissues were fixed with formalin, embedded in paraffin and sectioned. Anti-EGLN1 (K402 Lac ) polyclonal antibody for immunohistochemical staining, the staining results were observed under a microscope, and the lactylation level of the EGLN1 K402 site was quantified and scored (0-12 points). The diagnostic efficacy of the immunohistochemical score of EGLN1 K402 lactylation in predicting the efficacy of chemotherapy in patients with colorectal cancer was evaluated by ROC curve analysis. The results showed that the area under the curve (AUC) was 0.74, indicating that the score had good moderate predictive ability. Specifically, the AUC value ranged from 0.7 to 0.8, which is generally considered to be good for the effectiveness of diagnostic tests.

[0088] The results are as follows Fig.10 As shown in the figure, changes in EGLN1 K402 lactic acid levels can reflect the patient's response to chemotherapy (such as oxaliplatin) to a certain extent. By setting the optimal cutoff value, we can distinguish patients with high lactic acid and low lactic acid groups, thereby providing more targeted treatment strategies for the clinic. In addition, the immunohistochemical score of EGLN1 K402 lactic acid is a biomarker in the tumor microenvironment, laying the foundation for future personalized treatment and prognosis assessment.

[0089] 2. EGLN1 K402 lactylation modification level predicts the prognosis of colorectal cancer patients According to the IHC score of EGLN1 K402 lactic acidosis, the patients were divided into high lactic acidosis group and low lactic acidosis group for survival analysis, and the Kaplan-Meier survival curve was drawn.

[0090] The results are as follows Fig.11 As shown in Figure 2, patients with high lactic acidification levels have a significantly worse prognosis. Through the drawing and analysis of the Kaplan-Meier survival curve, it was observed that the survival time of patients in the high lactic acidification group was significantly shorter than that of patients in the low lactic acidification group.

[0091] This result further supports the value of EGLN1 K402 lactylation as a potential prognostic marker, suggesting that high lactylation levels may be associated with tumor progression and chemotherapy resistance, providing important information for clinical management. This finding emphasizes that in the treatment of colorectal cancer, assessing lactylation levels may have important clinical significance for patient prognosis.

[0092] In summary, the EGLN1 (K402 Lac ) The polyclonal antibody can specifically recognize the lactylated modification of lysine 402 of EGLN1 and can be used to detect the lactylation level of this site in tumor cells. At the same time, the EGLN1 K402 blocking peptide can target and block the lactylated modification of the EGLN1 K402 site. This invention not only provides a tool for exploring the chemotherapy resistance of tumor cells, but also provides certain help for the improvement of chemotherapy regimens in the later stage.

Claims

1. Use of the lactylation modification site of EGLN1 as a detection target in the preparation of a tumor diagnosis or prognosis evaluation kit, characterized in that: The amino acid sequence of EGLN1 of the tumor is shown in SEQ ID NO.1, wherein the lysine at position 402 is lactylated.

2. The use according to claim 1, characterized in that The kit includes an antibody against the lactylation modification site of EGLN1.

3. The use according to claim 2, characterized in that The amino acid sequence of the hapten polypeptide used to prepare the antibody is ARAKVKYLTGEK, wherein the lysine at position 6 is lactylated.

4. The use according to claim 2 or 3, characterized in that The antibody is a polyclonal antibody, and the preparation method comprises: firstly synthesizing a hapten polypeptide with an amino acid sequence of CARAKVKYLTGEK and a lactic acid-modified lysine at position 7, coupling it with hemocyanin to prepare an antigen, then obtaining serum through animal immunization, and separating and purifying the serum to obtain the antibody.

5. The use according to claim 1, characterized in that The tumor is colorectal cancer or breast cancer.

6. Application of the lactic acid modification site of EGLN1 as a target in screening tumor therapeutic drugs, characterized in that: The amino acid sequence of EGLN1 of the tumor is shown in SEQ ID NO.1, wherein the lysine at position 402 is lactylated; the tumor therapeutic drug targets and inhibits the lactylation of the lysine at position 402 of EGLN1 in tumor cells.

7. Use of a compound that inhibits lactic acid modification of EGLN1 in the preparation of a drug for treating tumors, characterized in that: The amino acid sequence of EGLN1 of the tumor is shown in SEQ ID NO.1, wherein the lysine at position 402 is lactylated.

8. The use according to claim 7, characterized in that The compound inhibiting EGLN1 lactic acid modification is any one of a polypeptide consisting of a transmembrane peptide segment and a peptide segment extending 5-10 amino acids upstream and downstream of the 402nd lysine of the EGLN1 protein, a lactate production inhibitor, and 2-deoxy-D-glucose.

9. The use according to claim 8, characterized in that The amino acid sequence of the polypeptide is YGRKKRRQRRRAERARAKVKYLTGEKG.

10. The use according to claim 8 or 9, characterized in that: The tumor includes colorectal cancer; the drug also includes oxaliplatin.

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