Application of EGLN1 lactylation as a target in the preparation of tumor diagnostic kits or therapeutic drugs

Abstract

The present invention discloses the use of EGLN1 lactylation as a target in the preparation of tumor diagnostic kits or therapeutic drugs, belonging to the field of biotechnology. The present invention firstly discloses that lysine at position 402 of the EGLN1 protein is its lactylation site. The lactylation level of EGLN1 K402 in tumor tissues is significantly higher than that in adjacent tissues. The lactylation modification of EGLN1 K402 is related to the occurrence and development of tumors. Therefore, EGLN1 K402 lactylation can be used as a tumor diagnosis or prognosis evaluation marker and applied to the development of tumor diagnosis or prognosis evaluation kits. Detecting 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 chemosensitivity of drugs and inhibit the occurrence and development of tumors. The present invention provides a new idea for the development of anti-tumor drugs.

Description

Technical Field

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

[0002] Protein lactylation is one of the important mechanisms of post-translational protein modification. This modification covalently binds lactic acid molecules to specific amino acid residues of proteins, thereby changing the structure and function of proteins. This modification method plays a crucial role in cell biology, affecting protein stability, activity, localization, and interactions with other molecules. Lactylation modification is particularly active in tumor cells. In tumor tissues, in order to meet the high energy demands of tumor tissues, multiple metabolic patterns coexist in tumor tissues. Metabolic reprogramming is a characteristic of cancer. Even under sufficient oxygen, tumor cells rely on glycolysis as an energy source, namely 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, Egl-9 Family Hypoxia Inducible Factor 1) catalyzes the post-translational formation of 4-hydroxyproline in hypoxia-inducible factor (HIF) α protein. HIF is a transcription complex that plays a central role in mammalian oxygen homeostasis. This protein acts as a cellular oxygen sensor. Under normal oxygen concentrations, prolyl hydroxylation modification is a key regulatory event that targets the HIF subunit for proteasomal destruction through the von Hippel-Lindau ubiquitination complex; under hypoxic conditions, the hydroxylation reaction weakens, enabling HIF to evade degradation, leading to their translocation to the nucleus, heterodimerization with HIF1B, and increased expression of hypoxia-inducible genes. EGLN1 is the most important isoenzyme under normoxia and participates in various processes affected by hypoxia, such as angiogenesis in retinal and cardiac functions.

[0004] Studies have shown that EGLN1 plays a certain role as an independent molecule in some tumors. For example, Xing Liu et al. reported that EGLN1 is a key mediator for the oxygen-enhanced antiviral innate immunity (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 the metabolic adaptation of breast cancer (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 prolyl 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 summary, EGLN1 plays an important role in the occurrence and development of cancer.

[0005] In the research of the research group of the present invention, it was found that the lactylation modification level of EGLN1 is correlated with the occurrence and development of tumors, and there is no relevant report yet. Therefore, in-depth study of the lactylation sites of EGLN1 is expected to provide valuable biomarkers for tumor diagnosis and prognosis analysis. Summary of the Invention

[0006] The purpose of the present invention is to provide the sites where EGLN1 undergoes lactylation modification, and use these as targets to develop tumor diagnosis or prognosis evaluation kits, or for screening and developing tumor therapeutic drugs.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Through protein modification mass spectrometry detection and point mutation verification, the present invention found that the 402nd lysine of the EGLN1 protein is its lactylation site. By using immunohistochemical staining and Western blot to detect the lactylation modification level in tumor tissues, it was found that the lactylation modification level of the EGLN1 K402 site in tumor tissues was significantly higher than that in adjacent tissues. After mutating 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.

[0009] Therefore, the present invention provides the application of the lactylation modification site of EGLN1 as a detection target in the preparation of a tumor diagnosis kit, wherein the amino acid sequence of EGLN1 in the tumor is as shown in SEQ ID NO.1, and the 402nd lysine has lactylation modification.

[0010] The present invention followed up tumor patients undergoing chemotherapy, recorded relevant information such as efficacy evaluation and survival time, and detected the lactylation modification level of the EGLN1 K402 site. The results showed that the change in the lactylation level of EGLN1 K402 could reflect the patient's response to chemotherapy to a certain extent; a high lactylation level of EGLN1 K402 was associated with tumor progression and chemotherapy resistance, and the lactylation of EGLN1 K402 had the potential value as a prognostic marker.

[0011] Therefore, the present invention provides the application of the lactylation modification site of EGLN1 as a detection target in the preparation of a tumor prognosis evaluation kit.

[0012] Furthermore, the kit includes an antibody against the lactylation modification site of EGLN1. Using the antibody to detect a test sample to determine whether the 402nd lysine of EGLN1 in the test sample has lactylation modification or to quantify the lactylation level. The detection methods that can be used in the present invention are not limited to this.

[0013] Furthermore, the amino acid sequence of the hapten polypeptide used to prepare the antibody is ARAKVKYLTGEK, and the 6th lysine has lactylation modification. Research shows that the antibody prepared by selecting the 397-408th position (ARAKVK(Lac)YLTGEK) of EGLN1 as the antigen region can specifically recognize the lactylation modification at the 402nd position of EGLN1.

[0014] The antibody in the present invention can be a polyclonal antibody or a monoclonal antibody, and can be prepared by known technical means in the art.

[0015] Furthermore, the antibody is a polyclonal antibody, and the preparation method includes: First, synthesize a hapten polypeptide with the amino acid sequence CARAKVKYLTGEK and the 7th lysine having lactylation modification, couple it with hemocyanin to prepare an antigen, then obtain serum through animal immunization, and isolate and purify the antibody from the serum. In the present invention, cysteine is added to the N-terminus of the hapten polypeptide, and the thiol group is used to couple with a carrier protein (such as hemocyanin).

[0016] Furthermore, the tumor is colorectal cancer or breast cancer.

[0017] The present invention provides a kit for diagnosing or prognosticating colorectal cancer, which includes an antibody against the lactylation modification site of EGLN1. The amino acid sequence of EGLN1 is as shown in SEQ ID NO.1, and the lysine at position 402 has lactylation modification.

[0018] The present invention also provides the application of the lactylation modification site of EGLN1 as a target in screening antitumor drugs. The amino acid sequence of EGLN1 in the tumor is as shown in SEQ ID NO.1, and the lysine at position 402 has lactylation modification; the antitumor drug targets and inhibits the lactylation of lysine at position 402 of EGLN1 in tumor cells.

[0019] Research shows that the change in the lactylation level of EGLN1 K402 reflects the therapeutic effect of drugs to a certain extent. Therefore, drugs are administered in a cell model or an animal model, and the lactylation level of EGLN1 K402 is detected to characterize the efficacy of the compound, so as to screen compounds with antitumor activity.

[0020] Furthermore, the tumor is colorectal cancer.

[0021] The present invention also provides the application of a compound that inhibits the lactylation modification of EGLN1 in the preparation of antitumor drugs. The amino acid sequence of EGLN1 in the tumor is as shown in SEQ ID NO.1, and the lysine at position 402 has lactylation modification. The drug inhibits the lactylation modification of lysine at position 402 of EGLN1 in tumor cells, thereby achieving the antitumor purpose.

[0022] Furthermore, the tumor is colorectal cancer.

[0023] Furthermore, the compound that inhibits the lactylation modification of EGLN1 is any one of a polypeptide composed of a transmembrane peptide segment and a peptide segment extending 5 - 10 amino acids upstream and downstream with the lysine at position 402 of the EGLN1 protein as the coordinate, a lactate production inhibitor, and 2-deoxy-D-glucose.

[0024] After the polypeptide enters the cell under the action of the cell-penetrating peptide, it mimics the endogenous EGLN1 K402 site and competitively binds to the endogenous lactic acidase with the endogenous EGLN1, thereby inhibiting the lactic acidification of the endogenous EGLN1 K402 site. Preferably, the polypeptide is composed of a cell-penetrating peptide segment and a peptide segment with the amino acid sequence ERARAKVKYLTGEKG.

[0025] The cell-penetrating peptide can be, but is not limited to, the TAT peptide. Preferably, the amino acid sequence of the polypeptide is YGRKKRRQRRRAERARAKVKYLTGEKG.

[0026] Furthermore, the compound that inhibits the lactic acidification modification of EGLN1 is used in combination with a chemotherapeutic drug for the preparation of an anti-tumor drug, and the chemosensitivity of the chemotherapeutic drug is improved through the combination of drugs, thereby enhancing the anti-tumor effect.

[0027] Furthermore, the drug also includes oxaliplatin. The combined use of the compound that inhibits the lactic acidification modification of EGLN1 and oxaliplatin can significantly enhance the chemosensitivity of oxaliplatin and improve the anti-tumor effect.

[0028] The beneficial effects of the present invention are as follows:

[0029] The present invention discloses for the first time that the 402nd lysine of the EGLN1 protein is its lactic acidification site. The lactic acidification level of EGLN1 K402 in tumor tissues is significantly higher than that in adjacent non-tumor tissues. The lactic acidification modification of EGLN1 K402 is related to the occurrence and development of tumors. Therefore, the lactic acidification of EGLN1 K402 can be used as a tumor diagnosis or prognosis evaluation marker and applied to the development of tumor diagnosis or prognosis evaluation kits. Detecting the lactic acidification level of EGLN1 K402 can guide the treatment of tumor patients and can be used to screen candidate drugs for treating tumors. The compound that inhibits the lactic acidification modification of EGLN1 K402 in combination with chemotherapeutic drugs can significantly improve the chemosensitivity of the drugs 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

[0030] Figure 1 For dot blot detection of the specificity of the anti-EGLN1 K402 Lac polyclonal antibody.

[0031] Figure 2 For Western Blot analysis of the specificity of the anti-EGLN1 K402 Lac polyclonal antibody.

[0032] Figure 3 For Western Blot verification of the inhibition of EGLN1 K402 lactic acidification by the TAT polypeptide.

[0033] Figure 4 For EGLN1 K402 Lac Results of polyclonal antibody immunohistochemistry

[0034] Figure 5 For EGLN1 K402 in colorectal cancer tissues and adjacent tissues Lac Immunohistochemistry results

[0035] Figure 6 Results of detecting the level of lactylation modification in colorectal cancer tissues by Western blot

[0036] Figure 7 For detecting the lactylation level of EGLN1 K402 in PDX tumor tissue lysate by Western Blot

[0037] Figure 8 For EGLN1 K402 blocking peptide to enhance the sensitivity of PDX model to oxaliplatin treatment

[0038] Figure 9 For lactate production inhibitor (LDHi, 2DG) combined with oxaliplatin to enhance the sensitivity of PDX model to oxaliplatin treatment

[0039] Figure 10 For the ROC curve to predict the effect of EGLN1 K402 lactylation modification level on the chemotherapy efficacy of colorectal cancer

[0040] Figure 11 For the EGLN1 K402 lactylation modification level to predict the prognosis of colorectal cancer patients Detailed implementation manners

[0041] The following further describes the present invention with specific embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement of the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0042] The test methods used in the following embodiments are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0043] Example 1: EGLN1 protein K402 Lac Design and synthesis of polypeptide

[0044] 1. Sequence analysis

[0045] The amino acid sequence of human EGLN1 protein (Q9GZT9) was obtained according to GenBank. The amino acid sequence is shown as SEQ ID NO.1, and the human EGLN1 protein contains 426 amino acids. The characteristics of the human EGLN1 protein were analyzed using DNAstar software, and the molecular weight of the human EGLN1 protein is 46,860 daltons.

[0046] 2. Protein modification mass spectrometry detection

[0047] Through protein modification mass spectrometry detection and point mutation verification, we found that lysine at position 402 of EGLN1 is its lactylation site. Our previous work showed that after mutating 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.

[0048] 3. Polypeptide synthesis

[0049] The synthetic polypeptide sequence was used to prepare the antigen. After analysis, the antigen region was selected as 397 - 408aa. Cysteine C was additionally added to the N - terminal of the polypeptide. Specifically, the synthetic polypeptide sequences were: CARAKVK(Lac)YLTGEK (polypeptide I) and CARAKVKYLTGEK (polypeptide II). Polypeptide I was conjugated with KLH for immunization; polypeptide II was used to prepare an antigen affinity purification column for detection and purification. The polypeptides were synthesized by Hangzhou Hua'an Biotech.

[0050] Example 2: Preparation, purification and identification of rabbit polyclonal antibody against EGLN1 polypeptide (K402 Lac )

[0051] 1. Conjugation of polypeptide with carrier protein

[0052] The thiol - containing hapten polypeptide I prepared in Example 1 was dissolved using conjugation Buffer and added to 10 mg / mL KLH. The polypeptide and KLH were immediately mixed evenly, and then reacted at room temperature for 2 h. The conjugate was purified through a gel filtration desalting column.

[0053] 2. Animal immunization

[0054] Two New Zealand white rabbits were immunized with the conjugate product, and the serum titers of the rabbits after three immunizations were monitored. After passing the Elisa verification, booster immunization was carried out and all sera were collected. The serum of the rabbit with the optimal serum titer was selected for purification by an affinity chromatography column.

[0055] The immunization method was as follows: The animals were injected subcutaneously at multiple points, 0.2 mL at each point. Immunization time: The second immunization was carried out 14 days after the first immunization, and the interval between the second immunization and the third immunization was 7 days. A small - sample serum was collected from the ear middle artery 7 days after the third immunization of the animals. After passing the detection, booster immunization was carried out 7 days later, and whole blood could be collected 7 days after the booster immunization.

[0056] 3. Antibody Purification

[0057] Rabbit serum was added to an affinity chromatography column to purify the antibody. After the antibody passed the inspection, an ultrafiltration concentrator tube was used to concentrate it to a certain concentration and volume.

[0058] 4. ELISA Identification of Polyclonal Antibody Against EGLN1 (K402 Lac )

[0059] Using the synthesized EGLN1 K402 Lac and K402 polypeptides as detection antigens, they were respectively coated on an ELISA plate. Unimmunized rabbit serum diluted 1:2000 was used as a negative control. The rabbit serum and the purified antibody were serially diluted, and the ELISA method was applied for detection to calculate the antibody titer. The results are shown in Table 1.

[0060] Table 1. Antibody Titer of EGLN1 (K402 Lac )

[0061] Dilution factor Absorbance value of the coated modified polypeptide Absorbance value of the coated unmodified polypeptide 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

[0062] Results: Analyzed from the titer, after antibody purification, the titer of the polyclonal antibody against EGLN1 (K402 Lac ) was greater than 1:4000. The absorbance value corresponding to EGLN1 (K402 Lac ) was 1.45, while under the same dilution factor in the control group, the absorbance of the non-modified polypeptide of EGLN1 K402 was only 0.387.

[0063] 5. Immunoblotting Detection of Anti-EGLN1 (K402 Lac )

[0064] Weighed 1 mg each of the EGLN1 K402 lactylation-modified polypeptide and the non-modified polypeptide and dissolved them in 1 mL of 0.01 M PBS buffer. 5 μL of the dissolved polypeptide was respectively spotted onto a PVDF membrane. After air-drying, the PVDF membrane was blocked with a TBST blocking solution containing 5% non-fat milk powder for 1 h. The corresponding purified polyclonal antibody against EGLN1 K402 Lac (dilution factor 1:1000) was added and incubated at room temperature for 2 h, then the membrane was washed five times (5 min each time). HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added and incubated at room temperature for 1 h, then the membrane was washed five times (5 min each time). The final result was imaged by a Jena UVP Chemstudio multi-functional imager.

[0065] The results are as Figure 1 shown, EGLN1 K402 LacThe polyclonal antibody basically recognizes all the K402 lactylation - modified polypeptides on the PVDF membrane and hardly recognizes the non - lactylated K402 polypeptides.

[0066] 6. Western - Blot identification of the polyclonal antibody

[0067] 6.1 Construct expression vectors of wild - type EGLN1 with an HA tag and EGLN1 lysine mutant at position 402 (lysine is mutated to arginine). Specifically, entrust Wuhan Miaoling Biology to construct HA - EGLN1 WT and HA - EGLN1 K402R plasmids, using the pcDNA3.1 vector for construction. The nucleotide sequence of wild - type EGLN1 is as shown in SEQ ID NO.2. Clone the wild - type EGLN1 gene fragment into the multiple cloning site of the pcDNA3.1 vector to construct the HA - EGLN1 WT plasmid, and then use a site - directed mutagenesis kit to construct the mutant plasmid of HA - EGLN1 K402R. The nucleotide sequence of the mutant is as shown in SEQ ID NO.3.

[0068] 6.2 Transiently transfect HEK293T cells with HA - EGLN1 WT and HA - EGLN1 K402R plasmids respectively. Collect the HEK293T cell proteins 48 hours after transfection, and incubate the proteins with Anti - HA magnetic beads to purify the EGLN1 protein. After elution and denaturation of the purified protein, perform SDS - PAGE gel electrophoresis, transfer the electrophoresis products to a PVDF membrane after electrophoresis, block with skim milk powder, and then add the purified anti - EGLN1 (K402 Lac ) polyclonal antibody (dilution factor 1:1000), incubate overnight at 4°C, wash the membrane five times (5 min each time), add the HRP - labeled goat anti - rabbit secondary antibody (dilution factor 1:50000), incubate at room temperature for 2 h, and then wash the membrane five times (5 min each time). The final result is imaged by the Jena UVP Chemstudio multi - functional imager.

[0069] The results are as Figure 2 shown. Using the anti - EGLN1 (K402 Lac ) polyclonal antibody, a protein band of about 45 kDa can be detected in the HEK293T cell lysate, which is consistent with the molecular weight of the EGLN1 protein.

[0070] 6.3 HA-EGLN1 WT plasmid and HA-EGLN1 K402R plasmid were transfected into HEK293T cells respectively. One group transfected with the WT plasmid was used as the control group, and 30 mM sodium lactate solution was added to the other group transfected with the WT plasmid and the group transfected with the K402R plasmid to increase the lactylation level of EGLN1. After 24 h, HA-EGLN1 protein was precipitated with Anti-HA magnetic beads, and the lactylation level of EGLN1 K402 was detected by Western Blot.

[0071] The results are as Figure 2 shown. Lysine at position 402 of the EGLN1 protein expressed in cells transfected with the wild-type plasmid showed obvious lactylation, and the lactylation level increased after stimulation with sodium lactate; the lactylation level of the EGLN1 protein expressed in cells transfected with the K402R mutant was significantly inhibited, and its lactylation level was reduced compared with the wild-type although sodium lactate was added.

[0072] 6.4 The EGLN1 WT plasmid was transiently transfected into HEK 293T cells. After transfection, the lactylation site was blocked with K402 TAT polypeptide (amino acid sequence: YGRKKRRQRRRAERARAKVKYLTGEKG, where YGRKKRRQRRRA is the TAT sequence that mediates the polypeptide into the cell. After the TAT polypeptide enters the cell, it can mimic the endogenous EGLN1 K402 site and competitively bind to the lactylation enzyme with endogenous EGLN1, thereby inhibiting the lactylation of the endogenous EGLN1 K402 site). HEK293T cell proteins were collected 48 hours after transfection, and EGLN1 protein was purified by incubating with Anti-HA magnetic beads and the protein. After the purified protein was eluted and denatured, SDS-PAGE gel electrophoresis was performed. After electrophoresis, it was transferred to a PVDF membrane. After blocking with skim milk powder, purified anti-EGLN1 (K402 Lac ) polyclonal antibody (dilution factor 1:1000) was added, and after incubating overnight at 4°C, the membrane was washed five times (5 min each time), HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added and incubated at room temperature for 2 h, and then the membrane was washed five times (5 min each time). The final result was imaged by a Jena UVP Chemstudio multifunctional imager.

[0073] The results are as Figure 3 shown. The TAT polypeptide at the EGLN1 K402 site can successfully block the lactylation of the EGLN1 protein at the K402 site.

[0074] 7. Immunohistochemical identification of anti-EGLN1 (K402 Lac ) polyclonal antibody

[0075] a. Place the pathological sections of colorectal tumor tissues obtained by colonoscopy in an oven at 60 °C and bake for 2 h. Immerse them in xylene three times, 10 min each time. Then hydrate and remove xylene successively with 100%, 95%, 80%, and 70% gradient alcohol, 5 min each time, and soak in deionized water for 3 min.

[0076] b. Heat antigen repair: Add 1× sodium citrate antigen repair solution to a pressure cooker and heat to boiling. Place the dewaxed and hydrated paraffin sections on a heat-resistant plastic slide rack and slowly put them into the boiling buffer. Keep boiling under high pressure for 15 min, then rinse with tap water and cool to room temperature.

[0077] c. Block the activity of endogenous peroxidase: Take out the slides from the antigen repair buffer, put them into a 3% hydrogen peroxide solution for blocking, incubate at room temperature in the dark for 15 min, then wash with PBS three times, 5 min each time. Shake off and dry the liquid around the tissue, and place them flat in a wet box.

[0078] d. Primary antibody incubation: Add 100 μL of polyclonal antibody against EGLN1 (K402 Lac )(1:200, diluted with immunohistochemical primary antibody diluent), and add 100 μL of primary antibody diluent for the negative control. Place them in a wet box and incubate at room temperature for 1 hour. Wash with PBS five times, 3 min each time. Shake off and dry the liquid around the tissue, and place them flat in a wet box.

[0079] e. Secondary antibody incubation: Drop 100 μL of specific secondary antibody working solution onto the tissue, place it in a wet box, incubate at room temperature for 20 min, then wash with PBS five times, 3 min each time. Shake off and dry the liquid around the tissue.

[0080] f. Drop 100 μL of pre-prepared chromogenic agent DAB working solution, control the chromogenesis under a light microscope. After complete chromogenesis, immerse it in distilled water to terminate chromogenesis.

[0081] g. Counterstain with hematoxylin for 5 min, soak in 1% hydrochloric acid alcohol for 30 - 60 s for differentiation, and rinse with tap water.

[0082] h. Immerse in PBS for 1 min to blue back, and rinse with tap water for 10 min.

[0083] i. Dehydrate and dry with 70%, 80%, 95%, and 100% gradient alcohol, 3 min each. Mount with neutral gum and observe the results under the microscope.

[0084] The results are as Figure 4 shown. EGLN1 (K402 Lac ) mainly appears in the cell nucleus, which is consistent with the localization of EGLN1 and is a positive result; while using antibody diluent instead of EGLN1 (K402 LacAfter detection with polyclonal antibodies, no specific staining was observed in the cell nucleus, indicating a negative result.

[0085] Example 3: Detection of lactylation modification levels in tumor tissues using Pan-Lactyl Lysine antibody and EGLN1 (K402 Lac polyclonal antibody)

[0086] 1. Detection of lactylation modification levels in colorectal cancer tissues by immunohistochemistry (IHC)

[0087] Colorectal cancer tissues and their corresponding adjacent tissues obtained during surgery 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.

[0088] All samples were fixed in formalin, paraffin-embedded, and sectioned. Subsequently, immunohistochemistry was used to detect the lactylation modification levels between tumor tissues and adjacent tissues (using Pan-Lactyl Lysine antibody as the primary antibody), as well as the lactylation modification of the EGLN1 K402 site (using EGLN1 (K402 Lac polyclonal antibody) as the primary antibody). This experiment aimed to reveal the differences in lactylation modification between tumor tissues and normal tissues and the level of lysine lactylation at the EGLN1 K402 site in tumor tissues.

[0089] The results were as Figure 5 shown. In colorectal cancer tissues, the lactylation modification level was significantly higher than that in the corresponding adjacent tissues. In addition, the detection results using EGLN1 (K402 Lac polyclonal antibody) further confirmed that the lactylation modification level at the EGLN1 K402 site in tumor tissues was also significantly higher than that in adjacent tissues.

[0090] These results suggest that the lactate level in colorectal cancer tissues is significantly higher than that in their adjacent tissues, indicating that lactylation modification may play an important role in the tumor microenvironment.

[0091] 2. Detection of lactylation modification levels in colorectal cancer tissues by Western blot

[0092] We ground the colorectal cancer tissues and their corresponding adjacent tissues collected during surgery, extracted tissue proteins, and performed Western blot to detect the lactylation modification levels in tumor tissues.

[0093] The results were as Figure 6 shown. In colorectal cancer tissues, the lactylation modification level was significantly higher than that in the corresponding adjacent tissues.

[0094] This result indicates that lactylation may have important biological significance in the colorectal cancer microenvironment.

[0095] Example 4: Targeted inhibition of EGLN1 K402 lactylation enhances the chemosensitivity of oxaliplatin

[0096] 1. Determination of the degree of lactylation at position K402 of EGLN1 in a patient-derived tumor xenograft model (PDX)

[0097] Colorectal cancer tumor tissues were obtained through surgery and implanted subcutaneously into the axilla of immunodeficient nude mice in the form of fragments. When the tumors grew to a diameter of about 1 - 2 cm, the tumors could be dissected from the first-generation host (P0). A part of the tissue was minced into pieces about 3 mm × 3 mm × 3 mm and implanted into new mice (P1), and the other part was stored with cryopreservation solution for resuscitation, preservation, and passage. This was repeated until the third generation (P3) was bred. The P3 tumor tissues were taken for cryogenic grinding to extract tissue proteins, and SDS-PAGE gel electrophoresis was performed. After electrophoresis, the proteins were transferred to a PVDF membrane. After blocking with skim milk powder, purified anti-EGLN1 (K402 Lac ) polyclonal antibody (dilution factor 1:1000) was added, and the membrane was incubated overnight at 4°C and then washed five times (5 min each time). HRP-labeled goat anti-rabbit secondary antibody (dilution factor 1:50000) was added and incubated at room temperature for 2 h, followed by washing the membrane five times (5 min each time). The final result was imaged by a Jena UVP Chemstudio multifunctional imager.

[0098] The results were as Figure 7 shown. A protein band of about 45 kDa could be detected in the PDX tumor tissue lysate with the anti-EGLN1 (K402 Lac ) polyclonal antibody, which was consistent with the molecular weight of the EGLN1 protein.

[0099] 2. Enhancement of the chemosensitivity of the patient-derived tumor xenograft model by the EGLN1 K402 blocking peptide

[0100] According to Figure 7One PDX model with high lactylation modification of EGLN1 (#3) was selected as the result and randomly divided into 6 groups: ① control group, ② K402R polypeptide group, ③ K402 polypeptide group, ④ Oxaliplatin group, ⑤ K402R polypeptide + Oxaliplatin group, ⑥ K402 polypeptide + Oxaliplatin group. The lactylation of EGLN1 K402 was blocked using a polypeptide targeting EGLN1 K402. Note: The sequence of the K402 polypeptide is: YGRKKRRQRRRAERARAKVKYLTGEKG, which can block the lactylation at the EGLN1 K402 site. The sequence of the K402R polypeptide is: YGRKKRRQRRRAERARAKVRYLTGEKG, which cannot block the lactylation at the K402 site and is used as a control.

[0101] Administration method: The polypeptide was administered by intraperitoneal injection at a dose of 5 mg / kg once a day for 2 weeks; Oxaliplatin was administered by intraperitoneal injection at a dose of 10 mg / kg once a day for 1 week. The polypeptide + Oxaliplatin group started using Oxaliplatin after 1 week of polypeptide treatment.

[0102] The body 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 dissected, photographed, and weighed. Before sacrificing the mice, 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 and detection, such as the liver and kidney functions of the mice.

[0103] The results were as Figure 8 shown. The polyclonal antibody against EGLN1 (K402 Lac ) could detect the degree of lactylation at the EGLN1 K402 site in the PDX tumor tissue, and the synthesized EGLN1 K402 blocking peptide could enhance the sensitivity of the PDX model to Oxaliplatin treatment, manifested as slower growth of the tumor tissue in the K402 blocking peptide combined with Oxaliplatin treatment group compared with the group treated with Oxaliplatin alone, smaller tumors, and lower weights.

[0104] No obvious toxic reactions or abnormalities in other health indicators occurred during the treatment. The HE results showed no obvious effects on organs such as the heart, liver, spleen, lungs, and kidneys of the mice. This result indicates that the polypeptide used is safe and tolerable within the dose range, providing a good basis for biological safety for subsequent research.

[0105] 3. Enhancement of the chemosensitivity of human-derived tumor xenograft models by lactate production inhibitor (LDHi, 2DG) combined with Oxaliplatin

[0106] According to Figure 7One PDX model with low lactic acid modification of EGLN1 (#1) was selected as the result and randomly divided into 6 groups: ① control group, ② LDHi group, ③ 2-DG group, ④ Oxaliplatin group, ⑤ LDHi + Oxaliplatin group, and ⑥ 2-DG + Oxaliplatin group. The LDH inhibitor LDHi and the glycolysis inhibitor 2-DG (both LDHi and 2-DG were purchased from sigma, with the numbers O2751 and D8375 respectively) were used to inhibit and reduce lactic acid production, aiming to inhibit the lactic acidification of EGLN1 K402.

[0107] The body 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 dissected, photographed, and weighed.

[0108] The results were as Figure 9 shown. The polyclonal antibody against EGLN1 (K402 Lac ) could detect the degree of lactic acidification at the EGLN1 K402 site in the PDX tumor tissue. The combined treatment of the lactic acid production inhibitors LDHi and 2-DG with oxaliplatin significantly enhanced the anti-tumor efficacy of oxaliplatin. Specifically, the tumor growth rate in the LDHi or 2-DG combined with oxaliplatin treatment group was significantly lower than that in the group treated with oxaliplatin alone, and the tumor volume and weight were significantly reduced.

[0109] Example 5: Prediction of the efficacy of tumor chemotherapy by detecting the level of lactic acid modification of EGLN1 K402 in colorectal cancer tissues of patients by IHC

[0110] 1. Prediction of the efficacy of colorectal cancer chemotherapy by the level of lactic acid modification of EGLN1 K402

[0111] Endoscopic tumor tissue samples and corresponding clinical data of colorectal cancer patients diagnosed by pathology were collected, including chemotherapy regimens and efficacy evaluations: complete remission (CR), partial remission (PR), disease progression (PD), and disease stability (SD). Patients treated with oxaliplatin chemotherapy were screened out and followed up, and relevant information such as the survival time of the patients was recorded.

[0112] The collected endoscopic tumor tissues were fixed with formalin and embedded in paraffin for sectioning; the anti-EGLN1 (K402 LacImmunohistochemical staining was performed with polyclonal antibodies, and the staining results were observed under a microscope. The lactylation level at the EGLN1 K402 site was quantitatively scored (on a 0 - 12 scale). Through ROC curve analysis, the diagnostic efficacy of the immunohistochemical score of EGLN1 K402 lactylation in predicting the chemotherapy efficacy of colorectal cancer patients was evaluated. The results showed that the area under the curve (AUC) was 0.74, indicating that this score had good moderate predictive ability. Specifically, an AUC value in the range of 0.7 to 0.8 is generally considered to indicate good effectiveness of a diagnostic test.

[0113] The results were as Figure 10 shown. The change in the lactylation level of EGLN1 K402 could, to a certain extent, reflect the response of patients to chemotherapy (such as oxaliplatin). By setting the optimal cut-off value, we were able to distinguish between patients in the high-lactylation group and the low-lactylation group, thus providing a more targeted treatment strategy for clinical practice. In addition, the immunohistochemical score of EGLN1 K402 lactylation serves as a biomarker in the tumor microenvironment, laying the foundation for future individualized treatment and prognosis assessment.

[0114] 2. Prediction of the prognosis of colorectal cancer patients by the lactylation modification level of EGLN1 K402

[0115] According to the IHC scores of EGLN1 K402 lactylation of the enrolled patients, they were divided into a high-lactylation group and a low-lactylation group for survival analysis, and Kaplan-Meier survival curves were plotted.

[0116] The results were as Figure 11 shown. Patients with a high lactylation level had significantly poorer prognoses. Through the plotting and analysis of the Kaplan-Meier survival curves, it was observed that the survival time of patients in the high-lactylation group was significantly shorter than that of patients in the low-lactylation group.

[0117] This result further supported the value of EGLN1 K402 lactylation as a potential prognostic biomarker, suggesting that a high lactylation level 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, evaluating the lactylation level may have important clinical significance for patient prognosis.

[0118] In summary, the polyclonal antibody of EGLN1 (K402 Lac ) provided by the present invention can specifically recognize the lactylation modification of lysine at the 402nd position of EGLN1 and can be used to detect the lactylation level at this site in tumor cells. At the same time, the EGLN1 K402 blocking peptide can target and block the lactylation modification at the EGLN1 K402 site. This invention not only provides a tool for exploring the study of chemotherapy resistance in tumor cells but also provides certain help for the improvement research of later chemotherapy regimens.

Claims

1. Use of an antibody for detecting lactate-modified EGLN1 in the preparation of a tumor prognosis evaluation kit, characterized in that, The amino acid sequence of the EGLN1 is as shown in SEQ ID NO.1, where the lysine at position 402 is lactylated, and the tumor is colorectal cancer.

2. The application according to claim 1, characterized in that The amino acid sequence of the hapten polypeptide used for preparing the antibody is ARAKVKYLTGEK, where the lysine at position 6 is lactylated.

3. The application according to claim 1, characterized in that The antibody is a polyclonal antibody, and the preparation method includes: first synthesizing a hapten polypeptide with an amino acid sequence of CARAKVKYLTGEK and the lysine at position 7 being lactylated, coupling it with hemocyanin to prepare an antigen, then obtaining serum through animal immunization, and separating and purifying the antibody from the serum.

4. Use of EGLN1 K402 blocking peptide combined with oxaliplatin in the preparation of a medicament for treating tumors, characterized in that, The EGLN1K402 blocking peptide is YGRKKRRQRRRAERARAKVKYLTGEKG, the amino acid sequence of the EGLN1 of the tumor is as shown in SEQ ID NO.1, where the lysine at position 402 is lactylated, and the tumor is colorectal cancer.

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