Application of ENO1 and YWHAZ in multiple myeloma

Abstract

The invention provides application of ENO1 and YWHAZ in multiple myeloma. The application comprises application of an ENO1 inhibitor and bortezomib in preparation of a medicine for treating and / or preventing the multiple myeloma. The biomarker for diagnosing the multiple myeloma, provided by the invention, has relatively high sensitivity and specificity and can be used for safely and effectively diagnosing the multiple myeloma, and meanwhile, the invention also provides a method for jointly detecting the biomarker capable of predicting the prognosis of a patient with the multiple myeloma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and specifically to the application of ENO1 and YWHAZ in multiple myeloma. Background Art

[0002] Multiple myeloma (MM) is a blood system tumor caused by the clonal proliferation of malignant plasma cells. The proteasome inhibitor bortezomib is currently one of the most effective and commonly used drugs for the treatment of MM, and is also the core of a variety of combined treatment regimens. However, drug resistance seriously affects the clinical efficacy of myeloma and often leads to relapse and death of patients.

[0003] ENO1 (Q-enolase) is a conserved glycolytic enzyme that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate. Increasing evidence suggests that in addition to its main function in glycolysis, ENO1 is also a multifunctional tumor-related protein that participates in a variety of physiological processes, including growth control, hypoxia tolerance, and autoimmune activity. Therefore, it is of great clinical significance to explore and identify its downstream targets and elucidate the molecular mechanism of chemotherapy resistance caused by abnormal ENO1 expression.

[0004] YWHAZ, also known as 14-3-3ζ, belongs to the 14-3-3 protein family and is a central hub protein in signal transduction pathways and tumor progression. It regulates mitophagy by interacting with key mitophagy proteins such as Parkin. In addition, YWHAZ interacts with several apoptosis-related proteins, suggesting that YWHAZ plays a key role in regulating apoptosis and promoting cell adaptability to environmental stress.

[0005] Multiple myeloma is the second most common malignant tumor of the blood system and has no obvious symptoms in the early stage of the disease. As multiple myeloma progresses, it can lead to serious consequences, including repeated infections, bone pain, osteoporosis, pathological fractures, hypercalcemia, bone marrow failure, and renal insufficiency. Since multiple myeloma has no obvious symptoms in the early stages of the disease, it is difficult to detect and treat the disease in the early stages, thus missing the best treatment time; at the same time, the current diagnosis of multiple myeloma is mainly performed through bone marrow puncture, which causes great trauma and pain to patients. Therefore, it is extremely important to develop a new, safe and effective method for diagnosing multiple myeloma. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an application of ENO1 and YWHAZ in multiple myeloma, including the application of ENO1 inhibitor and bortezomib in the preparation of a drug for treating and / or preventing multiple myeloma. The biomarker for diagnosing multiple myeloma provided by the present invention has high sensitivity and specificity, and can safely and effectively diagnose multiple myeloma. At the same time, the present invention also provides a method for combined detection of biomarkers that can predict the prognosis of multiple myeloma patients.

[0007] A first aspect of the present invention provides a use of an ENO1 inhibitor and bortezomib in the preparation of a medicament for treating and / or preventing multiple myeloma.

[0008] In a preferred embodiment, the ENO1 inhibitor is AP-III-a4, or an RNA interference agent for ENO1.

[0009] In a preferred embodiment, the shRNA primers in the RNA interference reagent are as shown in SEQ ID NOs: 3 and 4, or as shown in SEQ ID NOs: 5 and 6.

[0010] In a preferred embodiment, the drug has the effect of increasing the sensitivity of multiple myeloma to BTZ.

[0011] In a preferred embodiment, the drug has the effect of increasing the sensitivity of multiple myeloma to BTZ.

[0012] In a preferred embodiment, the drug has a toxic effect on multiple myeloma cells.

[0013] In a preferred embodiment, the drug has the effects of increasing the apoptosis level of multiple myeloma cells and reducing the Ki-67 level.

[0014] Another aspect of the present invention provides use of an ENO1 inhibitor, a YWHAZ inhibitor and bortezomib in preparing a medicament for treating and / or preventing multiple myeloma.

[0015] In a preferred embodiment, the ENO1 inhibitor is as defined in the use described in the first aspect of the present invention.

[0016] That is, the ENO1 inhibitor is AP-III-a4, or is an RNA interference agent of ENO1.

[0017] In a preferred embodiment, the shRNA primers in the RNA interference reagent are as shown in SEQ ID NOs: 3 and 4, or as shown in SEQ ID NOs: 5 and 6.

[0018] In a preferred embodiment, the YWHAZ inhibitor is an RNA interference agent of YWHAZ.

[0019] Another aspect of the present invention further provides a use of an ENO1 inhibitor and a YWHAZ inhibitor in the preparation of a drug for reducing drug resistance of multiple myeloma cells or reducing mitochondrial autophagy ability of multiple myeloma cells;

[0020] In a preferred embodiment, the drug resistance is caused by the administration of bortezomib.

[0021] In a preferred embodiment, the ENO1 inhibitor and the YWHAZ inhibitor are as defined in the above application.

[0022] Another aspect of the present invention further provides use of a marker combination in the preparation of a drug for predicting and / or diagnosing multiple myeloma, wherein the marker combination comprises ENO1 protein and YWHAZ protein.

[0023] In a specific embodiment, subjects with low levels of ENO1 and low levels of YWHAZ have longer progression-free survival.

[0024] Another aspect of the present invention provides a use of a reagent for determining a marker combination in preparing a kit for determining the prognosis of multiple myeloma in a subject, wherein the marker combination includes ENO1 protein and YWHAZ protein.

[0025] In a preferred embodiment, the reagent is used to detect the expression level of the marker combination, such as the protein expression level and / or the mRNA transcription level.

[0026] In a preferred embodiment, the reagent is a biomolecular reagent that specifically binds to the biomarker or specifically hybridizes to the nucleic acid encoding the biomarker.

[0027] In a preferred embodiment, the biomolecule reagent is a primer, a probe and / or an antibody.

[0028] In a preferred embodiment, the expression levels of ENO1 protein and YWHAZ protein are obtained from the biological sample of the subject.

[0029] In a preferred embodiment, the biological sample is selected from one or more of peripheral venous blood, lymph node, and bone marrow fluid.

[0030] In a preferred embodiment, the biological sample is at least 1-10 mL of peripheral venous blood, such as 3 mL of peripheral venous blood.

[0031] In a preferred embodiment, the biological sample is at least 1-10 mL of bone marrow fluid, such as 1 mL of bone marrow fluid.

[0032] Another aspect of the present invention further provides a reagent for detecting a marker combination, wherein the marker combination comprises ENO1 protein and YWHAZ protein, and the reagent is defined in the application described in the above aspect of the present invention.

[0033] On the other hand, the present invention also provides a prognosis prediction model for multiple myeloma, which includes a detection module and an analysis and judgment module; the detection module detects the level of the marker combination in the biological sample to be tested, and transmits the level data to the analysis and judgment module; the analysis and judgment module judges the level data of the marker combination, and outputs a prediction or diagnosis result: the sample has a good prognosis or a poor prognosis; wherein the marker combination includes ENO1 protein and YWHAZ protein.

[0034] In a preferred embodiment, the biological sample is as defined in the application described in the above aspects of the present invention.

[0035] Another aspect of the present invention further provides a computer-readable storage medium for implementing the functions of the prognosis prediction model as described in the above aspect of the present invention, wherein the computer-readable storage medium stores a computer program.

[0036] On the other hand, the present invention also provides an electronic device, which includes a memory and a processor, wherein the memory stores a computer program, and is characterized in that the processor is used to execute the computer program to implement the function of the prognosis prediction model as described in the above aspect of the present invention.

[0037] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0038] Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0039] The positive and progressive effect of the present invention is that since multiple myeloma has no obvious symptoms in the early stage of the disease, it is difficult to detect and treat the disease in the early stage, thus missing the best treatment time. At present, the diagnosis of MM is mainly performed by bone marrow puncture, which will cause great trauma and pain to the patient. Therefore, it is of great significance to develop a new safe and effective method for diagnosing multiple myeloma. The present invention proposes a biomarker for diagnosing multiple myeloma, which has high sensitivity and specificity and can diagnose multiple myeloma safely and effectively; at the same time, a method for combined detection of biomarkers that can predict the prognosis of multiple myeloma patients is proposed. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1:The expression of ENO1 in different stages of MM development (upper figure) and the expression of ENO1 in bone marrow tissues of healthy volunteers and MM patients (lower figure), where MGUS refers to monoclonal gammopathy of unidentified significance, and PCL refers to plasma cell leukemia.

[0041] Figure 2 : Differential expression of ENO1-related mRNA levels and YWHAZ-related mRNA levels in bone marrow tissues of healthy volunteers and MM patients.

[0042] Figure 3 : Effects of ENO1 and YWHAZ expression on the progression-free period of MM patients.

[0043] Figure 4 :Inhibitory effect of ENO1i and BTZ combination treatment: (A) H929 and 8226 cells were treated with the indicated concentrations of ENOi (AP-Ⅲ-a4, 0.2-1.6 μM) and different concentrations of BTZ (1-6 μM) alone or in combination for 72 h. Cell viability was assessed using CCK-8 assay. Dose-response matrix (inhibition) of ENOi and BTZ, the inhibition rate was positively correlated with the degree of redness, and drug interactions were displayed based on the ZIP model. (B) Flow cytometry was used to detect apoptosis in MM cells treated with ENO1i (AP-Ⅲ-a4) and BTZ. (C) After 72 h of ENO1i (AP-Ⅲ-a4), BTZ and combination treatment, the survival rate of MM patient cells was evaluated. Pt# represents the patient number. Pt#1 and Pt#4 were diagnosed with relapsed / refractory multiple myeloma (RRMM). Data are presented as the mean ± SD of three independent experiments. (D) The medication process is shown in the figure: MM subcutaneous tumor-bearing mice were treated with ENO1i (10 mg / kg), BTZ (1 mg / kg) or a combination of the two, and saline was used as a control to obtain representative images of each group of tumors (n = 5 mice / group). (E) Tumor size was measured every two days, and tumor volume was shown by growth curve. (F) Tumor weight was detected in different treatment groups. (G) Mean and SD of mouse body weight over time (mean body weight SD). (H) Tumor tissues of tumor-bearing mice were treated with ENO1 inhibitor, bortezomib alone or in combination, and Ki-67 and TUNEL staining were performed. (Original magnification: ×400), *P<0.05, **P<0.01 and ***P<0.001.

[0044] Figure 5: ENO1 interacts with YWHAZ and promotes its protein stability. (A) Total cell lysates were extracted from H929 cells, purified and resolved on SDS-PAGE. Coomassie Brilliant Blue-stained gels showed distinct bands, and the bands were retrieved and analyzed by mass spectrometry. (B) Venn diagram showing that 195 proteins were specifically pulled down in the IP group, excluding 97 proteins that were co-crossed between the IgG and IP groups. (C) Identified YWHAZ peptides are shown. (D) Endogenous YWHAZ in MM cells was immunoprecipitated using anti-ENO1 antibody and rabbit IgG as a nonspecific control. (E) HEK293T cells were co-transfected with Flag-YWHAZ, HA-ENO1, or both plasmids. Cell lysates were immunoprecipitated using anti-Flag magnetic beads and analyzed by immunoblotting with anti-HA or anti-Flag. (F) Colocalization between YWHAZ (green) and ENO1 (red) was analyzed in H929 cells by confocal microscopy (scale bar: 5 μm). (G) Co-immunoprecipitation was performed to investigate the region where ENO1 specifically binds to YWHAZ. (H) In the case of ENO1 knockdown, YWHAZ and ENO1 protein levels were analyzed by Western blotting. (I) Downregulation of ENO1 increases the degradation of YWHAZ. Western blotting detected the changes in YWHAZ in H929 cells after treatment with 10 μg / mL CHX for the indicated time.

[0045] Figure 6 :YWHAZ is acetylated by HDAC6, and the acetylation level of YWHAZ affects mitophagy: (A) The identified HDAC6 peptides are shown. (B) Immunoblotting of anti-HDAC6 immunoprecipitates in H929 cells. (C) Immunoblotting of cell lysates or anti-YWHAZ immunoprecipitates of H929 cells treated with MG132 (10 μM) for 2 h with control solvent or HDAC6i. (D) Immunoblotting of Flag-YWHAZ in HEK293T cells transfected with Flag vector, Flag YWHAZ, and Flag K138R. (E) Representative transmission electron microscopy images showing the mitochondrial ultrastructure of MM cells in control, YWHAZ-OE, and YWHAZ mutants, with red arrows indicating autophagosomes (scale bar: 2 μm).

[0046] Figure 7: ENO1 regulates mitophagy through YWHAZ: (A) Immunoblot analysis of autophagy-related proteins was detected in MM shCtrl and shENO1 cells treated with or without BTZ, and in cells transiently transfected with or without vector or YWHAZ plasmid. (B) Co-localization of LC3B (green) and TOMM20 (red) in shCtrl and shENO1 cells transiently transfected with or without empty vector or YWHAZ plasmid under CCCP (10 μM) stimulation was analyzed by confocal microscopy (scale bar: 5 μm). (C) Cell viability was assessed after H929 shCtrl and shENO1 cells were treated with different concentrations of BTZ and transiently transfected with or without vector or YWHAZ plasmid. (D) TUNEL assay of H929 shENO1 cells treated with or without BTZ and transiently transfected with vector or YWHAZ plasmid. (E) Apoptosis of shENO1 MM cells transiently transfected with vector or YWHAZ plasmid and treated with or without BTZ (upper row). Percent apoptosis of shENO1 MM cells with or without CCCP pretreatment and treated with or without BTZ (lower row). (F) Colocalization between LC3B (green) and TOMM20 (red) was analyzed by confocal microscopy in shCtrl and shENO1 cells transiently transfected with or without vector or YWHAZ plasmid under CCCP (10 μM) stimulation. (G) Western blotting analysis of autophagy-related proteins detected in shCtrl and shENO1 cells transiently transfected with or without BTZ and treated with or without vector or YWHAZ plasmid. (H) Western blotting analysis of apoptosis-related proteins detected in shCtrl and shENO1 cells transiently transfected with or without BTZ and treated with or without vector or YWHAZ plasmid, *P<0.05, **P<0.01, ***P<0.001.

[0047] Figure 8 Indicates that ENO1 regulates YWHAZ protein levels, independent of its glycolytic activity. (A) Correlation analysis between ENO1 and YWHAZ. (B) Immunoprecipitation of endogenous ENO1 in MM cells using anti-YWHAZ antibody (purchased from Proteintech) and rabbit IgG as a nonspecific control. (C) Relative expression of YWHAZ mRNA after ENO1 knockout. DETAILED DESCRIPTION

[0048] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0049] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0050] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0051] In the present invention, ENO1 protein refers to Q-enolase, and the YWHAZ protein is also called 14-3-3ζ, which belongs to the 14-3-3 protein family.

[0052] In a specific embodiment of the present invention, the ENO1 protein is of human type, and its information can be retrieved from the UniProt database, and its entry number is P06733. The YWHAZ protein is of human type, and its information can be retrieved from the UniProt database, and its entry number is P63104.

[0053] In the present invention, the multiple myeloma (MM) is a hematological cancer characterized by clonal proliferation of plasma cells in the bone marrow and is usually associated with monoclonal components in serum and / or urine. The main symptoms of the disease include bone pain, anemia, infection, bleeding, pathological fractures, renal insufficiency, neurological symptoms, hypercalcemia and amyloidosis. According to the revised diagnostic criteria of the International Myeloma Working Group (IMWG), the diagnosis of multiple myeloma needs to meet the following conditions: bone marrow examination shows that the proportion of plasma cells is ≥10% or biopsy confirms plasmacytoma. Monoclonal immunoglobulins or light chain fragments are present in serum or urine. One or more punch-like osteolytic bone lesions can be seen on bone radiological examinations (such as X-ray, CT, MRI or PET-CT).

[0054] The RNA interference reagent of the present invention is a tool used in RNA interference (RNAi) experiments, and mainly achieves gene silencing through double-stranded RNA (dsRNA) or small interfering RNA (siRNA).

[0055] The RNA interference reagent of the present invention is not limited to: siRNA, shRNA or liposome transfection reagent.

[0056] The siRNA is one of the most commonly used RNA interference agents, and is usually prepared by synthesis or in vitro transcription. After the siRNA enters the cell through transfection, it is processed into mature siRNA under the action of Dicer enzyme and participates in the subsequent gene silencing process.

[0057] The shRNA is a short hairpin RNA, which is another common RNA interference agent. The shRNA is introduced into cells through a viral vector, transcribed into mature siRNA in the cells, and then participates in gene silencing.

[0058] The liposome transfection reagent: such as siLentFect TM and siPORT TM NeoFX TM Etc. These reagents can efficiently deliver siRNA into cells and are the key to achieving effective RNA interference.

[0059] In a preferred embodiment, the shRNA primers in the RNA interference reagent for ENO1 of the present invention are as shown in SEQ ID NOs: 3 and 4, or as shown in SEQ ID NOs: 5 and 6.

[0060] Ki-67 described in the present invention refers to a nuclear protein, which is a protein encoded by the MKI67 gene, belongs to a member of chromosome mitosis-related proteins, and is related to ribosomal RNA transcription. Ki-67 is widely expressed in the G1, S, G2 and mitosis phases of the cell cycle, but is not expressed in the G0 phase of the quiescent phase. In addition, Ki-67 is often used as a tumor marker to evaluate the prognosis and diagnostic value of tumors.

[0061] Unless otherwise indicated, technical terms are used in accordance with conventional usage. Definitions of common terms in molecular biology are readily available, for example, Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341) and other similar technical references.

[0062] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0063] Overview of Materials and Methods

[0064] Clinical samples

[0065] Bone marrow samples were obtained from multiple myeloma patients after written informed consent was obtained from the Department of Hematology, Shanghai Oriental Hospital (Shanghai, China). The protocol for the collection and use of clinical samples was approved by the Ethics Committee of Shanghai Oriental Hospital.

[0066] Cells, antibodies, and reagents

[0067] Human multiple myeloma cell lines NCI-H929, RPMI-8226, and HEK293T cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured at 37°C in a humidified atmosphere containing 5% CO2 using RPMI-1640 or Dulbecco's modified Eagle's medium (catalogue no. 16000-044; Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum. Preparation of BTZ-resistant cell lines H929R and 8226R5 reference article Hu, L., et al., A novel M phase blocker, DCZ3301 enhances the sensitivity of bortezomib inresistant multiple myeloma through DNA damage and mitotic catastrophe. Journal of Experimental & Clinical Cancer Research: CR, 2020. 39 (1): p. 105; or article Xie, Y., et al., Preclinical validation and phase I trial of 4-hydroxysalicylanilide, targeting ribonucleotide reductase mediated dNTP synthesis in multiple myeloma. Journal of Biomedical Science, 2022. 29 (1): p. 32. In order to maintain the drug resistance of BTZ-resistant cell lines, H929R and 8226R5 were cultured with 20 nM BTZ. Primary antibodies including cleaved caspase-8, cleaved caspase-9, cleaved caspase-3, p62, HA-tag, CDK4, CDK6, CyclinD1, β-actin, and DYKDDDDK tag protein (Flag) were purchased from Cell Signaling Technology (Beverly, MA, USA); LC3B and ENO1 were purchased from Abcam (Cambridge, MA, UK); Parkin and YWHAZ were purchased from Proteintech (Wuhan, Hubei, China). Chemotherapeutic agents AP-III-a4, oligomycin, nicotinamide (NAM), trichostatin A (TSA), rabbit statin A hydrochloride, and cycloheximide (CHX) were purchased from MedChemExpress (Monmouth Junction, NJ, USA).Bortezomib and puromycin were purchased from Sigma (St. Louis, MO, US).

[0068] RNA interference, plasmids and transfection

[0069] Lipofectamine was used according to the manufacturer's instructions. TM All transfections were performed using 3000 (Invitrogen, L3000015). Table 1 below lists the corresponding primer sequences for ENO1 knockout. ENO1 protein information was retrieved from the UniProt database, and its entry number is P06733.

[0070] Table 1 Corresponding primer sequences for ENO1 knockout

[0071]

[0072] To specifically silence ENO1 using shRNA, the inventors followed the following experimental steps:

[0073] 1) Design of shRNA sequence: Design of shRNA sequence targeting specific region of ENO1 mRNA transcript, see Table 1.

[0074] 2) Construction of shRNA expression vector: The designed shRNA sequence was cloned into the PGMLV-hU6-MCS-CMV-Puro vector backbone and a selectable marker (puro resistance) to identify transfected cells.

[0075] 3) Transfection of shRNA vector: According to the manufacturer's instructions, the ENO1-shRNA expression vector was transfected into MM cells using Lipofectamine 3000 transfection reagent.

[0076] 4) Screening and verification of transfected cells: Use puromycin to screen transfected cells. Use real-time quantitative PCR (qPCR) and immunoblot analysis to verify the knockdown efficiency of ENO1 mRNA and protein levels in transfected cells, respectively. Ensure that shRNA can effectively reduce the expression of ENO1 compared with control cells transfected with empty vector.

[0077] 5) Functional studies: Functional assays were performed to evaluate the biological consequences of ENO1 gene knockout.

[0078] Cytotoxicity assay, apoptosis and cell cycle assay

[0079] Cell toxicity was assessed using cell counting kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, CA, USA) was used to determine apoptosis by staining with FITC Annexin V and propidium iodide (PI). For cell cycle determination, cells were subjected to serum-free starvation for 24 h, then PI (BD Biosciences, CA, USA) staining was performed, and flow cytometry was used for analysis. FlowJo software (TreeStar, Ashland, OR, USA) was used for data analysis. All experiments were repeated three times.

[0080] TUNEL assay

[0081] Apoptosis was assessed by TUNEL staining according to the manufacturer's instructions. Briefly, cells were treated with 0.3% TritonX-100 (10 min) and TUNEL reaction mixture (60 min) at 37°C. Cell nuclei were stained with DAPI (1:1000). Images were acquired using a fluorescent microscope, and apoptotic cells appeared red.

[0082] Immunofluorescence

[0083] Cells were placed on slides, fixed with 4% paraformaldehyde, blocked with bovine serum albumin (BSA), and incubated with primary antibodies at 4°C overnight, followed by incubation with secondary antibodies. Cell nuclei were stained with DAPI (C1002, Beyotime Biotechnology, Shanghai, China) for 10 minutes at room temperature. Images were taken with a Zeiss LSM900 or a fluorescence microscope (AX10 VERT A1, Zeiss, Germany). Colocalization of ENO1 and YWHAZ analysis was calculated by ImageJ software.

[0084] Immunoprecipitation and co-immunoprecipitation analysis

[0085] The enriched proteins were incubated with 2 μg of antibodies (anti-acetyl-lysine, anti-HA, anti-ENO1, anti-Flag, and anti-YWHAZ), and 30 μL of magnetic beads were added to the mixture. The mixture was then incubated overnight at 4°C in a vertical rotating mixer. Subsequently, the magnetic beads were heated at 100°C for 5 minutes until the proteins were separated from the magnetic beads. The amount of protein was analyzed by western blotting.

[0086] Animal studies

[0087] Nude mice (BALB / c) were housed in cages in a specific pathogen-free room at 25°C ± 1°C with a 12-h light / dark cycle and fed with autoclaved food and water. In the subcutaneous tumor-bearing nude mouse model, tumor growth was monitored every 2 days and the formula (a × b 2 ) / 2, where a and b represent the maximum and minimum diameters of the tumor, respectively. The whole tumor was fixed with 4% paraformaldehyde before dehydration and paraffin embedding. The sections were stained with H&E according to standard protocols and counted by microscopic counting analysis. Survival was assessed using Kaplan-Meier curve analysis.

[0088] Statistical analysis

[0089] Data were expressed as mean ± SEM, and statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad Software Inc., CA, USA). The differences between two groups were compared using t-test, and multiple groups were compared using analysis of variance (ANOVA). Survival analysis was performed using the Kaplan–Meier method, and statistical significance was assessed using log-rank. A threshold of P < 0.05 was considered statistically significant.

[0090] Example 1 Expression of glycolytic enzyme ENO1 in multiple myeloma cells and controls

[0091] The specific process of the experiment is:

[0092] A1: Protein extraction: Collect 3 mL of venous blood samples from multiple myeloma patients and healthy controls (three subjects each) to be tested, and centrifuge at 1000 rpm for 5 min. Wash twice with PBS and centrifuge at 1000 rpm for 5 min. Aspirate the supernatant with a pipette, add protein lysis buffer (RIPA) containing protease and phosphatase inhibitors to the precipitate, resuspend the cell pellet by blowing, and lyse on ice for 30 min. Ultrasonicate in an ultrasonic disruptor for 5 s, 3 times. Then centrifuge at 4°C, 12000 rpm for 20 min. After centrifugation, transfer the supernatant and record the supernatant volume.

[0093] A2: Determination of protein concentration (BCA method): 1. Preparation of BCA working solution: Mix solution A and solution B at a volume ratio of 50:1. Each sample requires 200μL of AB mixed solution and is calculated according to the amount of the sample. 2. Standard protein: Dilute the BSA standard (2mg / mL) with ultrapure water in a transparent 96-well plate, and set the concentration (mg / mL) of the standard in wells 1-8 as follows: 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, that is, add 0, 1, 2, 4, 8, 16, 20μL of the standard respectively, and use ddH2O to make up to 20μL for wells less than 20μL. Add 18μL ddH2O to each sample well, mix the protein sample to be tested, and pipette 2μL into the sample well. Add premixed AB solution to the standard protein well and sample well, mix thoroughly, place in a 37°C incubator for 30 minutes, and then use a microplate reader to detect the absorbance (OD) value at 562nm. Use the concentration and absorbance value of the standard as the X-axis and Y-axis, respectively, to make a standard curve and obtain the relevant function formula. Calculate the protein concentration of the sample using the function formula obtained from the standard curve and the OD value of the sample. Add 6× Loading Buffer at 1 / 5 of the sample volume, mix well, and centrifuge instantly.

[0094] A3: Protein denaturation: Boil the quantified protein sample in a 100℃ water bath for 5 minutes, quickly place it on ice, and store it at -80℃ for later use.

[0095] A4: Protein electrophoresis: Select 10% SDS-page precast gel, add 1X electrophoresis buffer to the vertical electrophoresis tank, carefully remove the tooth comb, add prestained protein molecular weight standard markers at both ends, and add protein samples in order. With positive-positive / negative-negative opposite, turn on the power. 140v constant voltage allows the protein sample to be compressed and separated in the gel until the electrophoresis stops about 1cm from the bottom edge of the gel plate. Remove the gel plate, cover the gel with an NC membrane that matches the gel size, form a "sandwich" sandwich between the filter paper and the sponge pad, close the transfer clip, and start the transfer when the electrodes are aligned. After 200mA constant current transfer for 1h, take out the cellulose membrane and shake it in 5% skim milk pre-prepared in PBST for 1h, then incubate it with the corresponding ENO1 antibody at 4℃ overnight according to the dilution ratio in the instructions, and then take out the strips and wash them 3 times with PBST, each time for 10min. Dilute the strips to the corresponding concentration according to the instructions, incubate at room temperature for 1 hour, and wash three times with PBST, 10 minutes each time. Development: ECL developer and AB solution are mixed in a 1:1 ratio, and the strips are immersed in the developer, incubated for 2-5 minutes, and placed in the Bio-Rad chemiluminescence imaging system for development and image acquisition.

[0096] The results are as follows Figure 1 As shown, bioinformatics analysis showed that the RNA level of ENO1 was associated with disease progression ( Figure 1 The expression of ENO1 in the collected MM patients was evaluated using IHC analysis, qRT-PCR and western blot. The IHC results showed that ENO1 was significantly upregulated in MM bone marrow ( Figure 1 ).

[0097] Example 2 ENO1-related mRNA expression levels in MM

[0098] The specific experimental process is:

[0099] (1) Collect 3 mL of serum samples from 20 clinically diagnosed multiple myeloma patients and 20 healthy controls. All participants' blood samples were drawn in the morning on an empty stomach. 3 mL of peripheral blood was placed in a vacuum EDTA anticoagulant blood collection tube and mixed by inversion;

[0100] (2) Add 0.5 mL of Trizol to the cell pellet, pipette it into a cell suspension, and place it at room temperature for 5 min;

[0101] (3) Add 200 μL of chloroform to each sample, shake vigorously for 15 seconds, and place at room temperature for 15 minutes;

[0102] (4) Place the sample in a low-temperature centrifuge and centrifuge at 12,000 g for 15 min at 4°C. Carefully aspirate the upper aqueous phase containing RNA and transfer it to another high-temperature sterilized 1.5 mL centrifuge tube, taking care to avoid aspirating the precipitate.

[0103] (5) Add 500 mL of isopropanol, mix by inverting several times, and leave at room temperature for 10 min;

[0104] (6) Centrifuge the sample at 4°C, 12000g for 8 min

[0105] (7) Discard the supernatant, add 1 mL of 75% ethanol, and vortex to mix.

[0106] (8) Centrifuge the sample at 7500g for 8 min at 4°C

[0107] (9) Discard ethanol and air dry for 5 minutes

[0108] (10) Add 30 μL of DEPC water to each tube to dissolve RNA.

[0109] (11) Take 1.5 μL RNA and use an ELISA reader to detect the RNA concentration and quality of each sample. The remaining RNA is stored in a -80 °C refrigerator until use.

[0110] RNA is reverse transcribed into cDNA,

[0111] 1) Reverse transcription reaction system (20 μL):

[0112]

[0113] (2) Reverse transcription reaction conditions: 37°C, 15 min; 42°C, 30 min; 98°C, 5 min; 4°C, and store in a -20°C refrigerator after removal.

[0114] qReal-time PCR was used to detect the expression level of ENO1. The upstream and downstream primers of ENO1 were designed by ourselves and synthesized by Shanghai Liuhe BGI. The sequences are as follows:

[0115]

[0116] (2) qReal-time PCR reaction system:

[0117]

[0118] (3) Real-time PCR reaction conditions:

[0119]

[0120] Repeat (a) to (d) 40 times

[0121] (e)Melt Curve 65℃-95℃ increment 0.5℃for 0:05

[0122] (4) Data statistics: Real-time PCR reactions were performed using the CFX96 Real-Time PCR Detection System, and the Ct values ​​of genes were calculated using the software.

[0123] Results: Total RNA was extracted from the bone marrow of MM patients and healthy donors, and the relative ENO1 mRNA levels were detected. qRT-PCR results confirmed that ENO1 was highly expressed in MM bone marrow tissue ( Figure 2 (top middle picture).

[0124] Example 3 ENO1 inhibitors have significant synergistic effects with bortezomib (BTZ) both in vitro and in vivo

[0125] To further evaluate the potential efficacy of BTZ combined with ENO1 inhibitors in the treatment of multiple myeloma, we examined whether ENO1 inhibitors (ENO1i) have synergistic effects with BTZ. AP-III-a4 is a known ENO1 inhibitor. Compared with ENO1i or BTZ alone, the simultaneous administration of ENO1i and BTZ produced a stronger cytotoxic effect on MM cells ( Figure 4 A, left column) and significantly higher levels of apoptosis ( Figure 4In addition, dose-effect analysis revealed a synergistic cytotoxic effect of ENO1i and BTZ, with a ZIP synergy score >10 in MM cells ( Figure 4 A, right column). Two of the four MM patients were newly diagnosed and untreated, and the other two were relapsed patients with resistance to BTZ and dexamethasone; the inventors found that ENO1i had synergistic effects with bortezomib in these four patient samples ( Figure 4 In addition, a MM xenograft model was constructed, and it was observed in vivo that the combination of ENO1i and BTZ resulted in a more significant growth inhibitory effect than the administration of ENO1i or BTZ alone ( Figure 4 D and Figure 4 The combined administration also had significantly lower tumor weight ( Figure 4 In addition, no significant differences were observed in the body weight of mice in each group during the treatment period ( Figure 4 G). The expression of Ki-67 in tumor xenografts was evaluated by IHC, and the synergistic effect of the two drugs was also evaluated by TUNEL assay ( Figure 4 The results showed that the combined administration of ENOi and BTZ resulted in decreased Ki-67 and increased apoptosis compared with each drug alone ( Figure 4 In summary, the combination of ENO1 inhibitors and BTZ improved the anti-tumor activity and may provide a new idea for the development of targeted drugs.

[0126] Example 4 ENO1 interacts with YWHAZ and regulates the stability of YWHAZ protein

[0127] The YWHAZ protein information used in this patent was retrieved from the UniProt database, and its entry number is P63104. In order to identify proteins that may interact with ENO1 molecules, the inventors performed co-IP mass spectrometry (co-IP-MS) analysis ( Figure 5 A). This analysis identified 125 and 295 proteins in the IP-IgG and IP-ENO1 groups, respectively. The Venn diagram showed that there were 195 specific proteins in the IP-ENO1 group ( Figure 5 B). Mass spectrometry results showed that YHWAZ can interact with ENO1 ( Figure 5 C), a positive correlation between ENO1 and YWHAZ was confirmed by database analysis ( Figure 8 A). Next, co-IP was performed to verify the interaction between ENO1 and YWHAZ. In MM cells, YWHAZ was precipitated by ENO1 antibody but not by control IgG antibody ( Figure 5 D). This was further confirmed by reverse verification of the interaction between endogenous ENO1 and YWHAZ ( Figure 8 In addition, exogenous co-IP assays were performed in HEK293T cells transfected with HA-tagged ENO1 (HA-ENO1) and Flag-YWHAZ alone or simultaneously, and the results confirmed the interaction between YWHAZ and ENO1 ( Figure 5 In addition, confocal laser microscopy was used to observe the intracellular distribution of ENO1 and YWHAZ, indicating that YWHAZ and ENO1 were co-localized in MM cells ( Figure 5 F). In order to delineate the specific interaction domain between YWHAZ and ENO1, ENO1 was truncated into three fragments based on the functional structure of ENO1 for co-IP analysis. The results showed that only the C-terminal deletion mutant (HA-ENO1-237-434) could not bind to Flag-YWHAZ, while the N-terminal deletion mutant (HA-ENO1-1-97) and the DNA binding domain deletion mutant (HA-ENO1-97-237) could ( Figure 5 G). We then investigated the effect of ENO1 knockout on YWHAZ expression and found that ENO1 had no effect on the RNA level of YWHAZ ( Figure 8 C), but significantly reduced the protein level of YWHAZ ( Figure 5 To confirm the effect of ENO1 on the stability of YWHAZ protein, cycloheximide (CHX) tracking analysis was performed, which showed that the stability of YWHAZ protein was significantly reduced after ENO1 knockout ( Figure 5 I).

[0128] Example 5 HDAC6 is a deacetylase of YWHAZ, and the acetylation level of YWHAZ is required for mitochondrial autophagy

[0129] Studies have shown that ENO1 has the function of HDAC binding protein. The inventors conducted co-IP mass spectrometry (co-IP-MS) analysis and determined that HDAC6 is the binding ligand of ENO1 ( Figure 6 The inventors subsequently focused on HDAC6 and found that HDAC6 interacted with YWHAZ in MM by co-IP analysis ( Figure 6 B). To confirm whether YWHAZ undergoes acetylation, H929 cells were treated with Tubastatin A HCl, a selective HDAC6 deacetylase inhibitor. Under Tubastatin AHCl treatment, the endogenous YWHAZ acetylation level increased ( Figure 6 C). To further explore the effect of YWHAZ acetylation level on mitophagy, the results showed that the mutation of YWHAZ acetylation site did not significantly change the mitophagy markers compared with the WT cell line ( Figure 6In addition, transmission electron microscopy results showed that the presence of double membrane-bound autophagosomes increased in MM cells with high YWHAZ expression, but not in YWHAZ mutants, confirming that the K138 site is specifically deacetylated, mediating YWHAZ expression and mitophagy ( Figure 6 E).

[0130] Example 6 ENO1 promotes mitochondrial autophagy and drug resistance in MM through YWHAZ

[0131] The inventors verified that YWHAZ was involved in ENO1-mediated tumor protective mitophagy and drug resistance. Western blot analysis and immunofluorescence results showed that overexpression of YWHAZ reversed the inhibitory effect of ENO1 knockout on mitophagy ( Figure 7 In addition, the effect of YWHAZ overexpression on ENO1-mediated drug resistance was investigated. YWHAZ overexpression reversed the sensitivity of MM cells to BTZ caused by ENO1 knockout ( Figure 7 C and D). We further explored whether ENO1-mediated resistance via YWHAZ stems from activation of mitophagy. BTZ treatment induced apoptosis and inhibited cytoprotective mitophagy. Our findings revealed that YWHAZ overexpression reduced ENO1 knockout-mediated resensitization to BTZ, such as that observed after treatment with a mitophagy inducer (CCCP) ( Figure 7 E and F), and downregulation of mitochondrial autophagy and apoptosis-related markers ( Figure 7 G and H). Taken together, these results suggest that YWHAZ acts as a downstream target of ENO1 to promote mitophagy and drug resistance.

[0132] Summary of Examples 3-6

[0133] By analyzing public databases, the inventors found that MM patients with high expression of ENO1 had stronger malignant potential and worse prognosis. In the current study, ENO1 knockout inhibited tumorigenesis and induced MM cell cycle arrest. In addition, ENO1 knockout increased MM sensitivity to BTZ and increased cell apoptosis. In order to further explore the value and significance of inhibiting ENO1 in clinical transformation, the inventors studied whether ENO1 inhibitors (AP-III-a4) can synergize with MM first-line chemotherapy drugs BTZ, and found that co-administration has synergistic effects both in vitro and in vivo. And this synergistic effect is effective in both newly diagnosed MM patients and relapsed MM patients.

[0134] The inventors found that after ENO1 was knocked out, the mitochondrial homeostasis in the cells was disturbed and the mitochondrial autophagy function was impaired, making the cells unable to clear damaged mitochondria, ultimately leading to apoptosis.

[0135] In addition, the inventors found that in MM cells overexpressing ENO1, the increase in mitophagy levels was dependent on the expression of ENO1, which may affect the resistance of MM to BTZ. At the same time, this may explain that the ENO1-promoted MM cell apoptosis escape may be due to the subsequent mitochondrial adaptation caused by the increased clearance of damaged organelles.

[0136] In the current study, the inventors found that ENO1 promotes the stability of YWHAZ protein and triggers cytoprotective mitophagy by interacting with YWHAZ. It has been reported that acetylation and ubiquitination can compete for the same lysine sites. The inventors' data showed that increased acetylation inhibited the ubiquitination degradation of YWHAZ. HDAC6 is a key member of the class IIa histone deacetylase and is involved in the pathogenesis of various cancers. A large number of studies have shown that HDAC6 plays an important role in various biological processes by regulating post-translational modifications. In this study, the inventors found that HDAC6 can bind to YWHAZ and ENO1, and inhibiting the activity of HDAC6 can promote the acetylation of YWHAZ and enhance its protein stability.

[0137] ENO1 increases the acetylation level of YWHAZ, which leads to a decrease in its ubiquitination level and prevents degradation, thereby increasing the stability of YWHAZ and further enhancing its binding to Parkin, which inhibits Parkin degradation and increases the ability of MM cells to clear damaged mitochondria. This promotes mitochondrial homeostasis in MM cells, leading to apoptosis escape and insensitivity to drug stress such as BTZ. It was found that overexpression of YWHAZ in ENO1-KD cells reduced the sensitivity of cells to BTZ.

[0138] In summary, it is proved that ENO1 is a key factor in promoting MM's resistance to apoptosis and drug resistance by regulating mitochondrial autophagy through YWHAZ. ENO1 regulates the acetylation level of YWHAZ at the K138 site through HDAC6, thereby enhancing the protein stability of YWHAZ. In addition, the inventors found that ENO1 inhibitors have synergistic effects with first-line chemotherapeutic drugs for MM, such as BTZ, providing a theoretical basis for the development of new MM therapies.

[0139] Example 7 Detection of mRNA expression level of ENO1-related regulatory gene YWHAZ The specific experimental process is as follows:

[0140] (1) Collect 3 mL of serum samples from 20 clinically diagnosed multiple myeloma patients and 20 healthy controls. All participants' blood samples were drawn in the morning on an empty stomach. 3 mL of peripheral blood was placed in a vacuum EDTA anticoagulant blood collection tube and mixed by inversion;

[0141] (2) Add 0.5 mL of Trizol to the cell pellet, pipette it into a cell suspension, and place it at room temperature for 5 min;

[0142] (3) Add 200 μL of chloroform to each sample, shake vigorously for 15 seconds, and place at room temperature for 15 minutes;

[0143] (4) Place the sample in a low-temperature centrifuge and centrifuge at 12,000 g for 15 min at 4°C. Carefully aspirate the upper aqueous phase containing RNA and transfer it to another high-temperature sterilized 1.5 mL centrifuge tube, taking care to avoid aspirating the precipitate.

[0144] (5) Add 500 mL of isopropanol, mix by inverting several times, and leave at room temperature for 10 min;

[0145] (6) Centrifuge the sample at 4°C, 12000g for 8 min

[0146] (7) Discard the supernatant, add 1 mL of 75% ethanol, and vortex to mix.

[0147] (8) Centrifuge the sample at 7500g for 8 min at 4°C

[0148] (9) Discard ethanol and air dry for 5 minutes

[0149] (10) Add 30 μL of DEPC water to each tube to dissolve RNA.

[0150] (11) Take 1.5 μL RNA and use an ELISA reader to detect the RNA concentration and quality of each sample. The remaining RNA is stored in a -80 °C refrigerator until use.

[0151] RNA is reverse transcribed into cDNA,

[0152] 1) Reverse transcription reaction system (20 μL):

[0153]

[0154] (2) Reverse transcription reaction conditions: 37°C, 15 min; 42°C, 30 min; 98°C, 5 min; 4°C, and store in a -20°C refrigerator after removal.

[0155] qReal-time PCR was used to detect the expression level of YWHAZ. The upstream and downstream primers of YWHAZ were designed by ourselves and synthesized by Shanghai Liuhe BGI. The sequences are as follows:

[0156]

[0157]

[0158] (2) qReal-time PCR reaction system:

[0159]

[0160] (3) Real-time PCR reaction conditions:

[0161]

[0162] Repeat (a) to (d) 40 times

[0163] (e)Melt Curve 65℃-95℃ increment 0.5℃for 0:05

[0164] (5) Data statistics: Real-time PCR reactions were performed using the CFX96 Real-Time PCR Detection System, and the Ct values ​​of genes were calculated using the software.

[0165] Results: Total RNA was extracted from the bone marrow of MM patients and healthy donors, and the relative YWHAZ mRNA levels were detected. qRT-PCR results confirmed that YWHAZ was highly expressed in MM bone marrow tissue ( Figure 2 (lower middle picture).

[0166] Example 8 Prognostic prediction effect of combining ENO1 and YWHAZ on multiple myeloma

[0167] Data of multiple myeloma patients were obtained from the GSE57317 dataset for subsequent survival curve analysis. According to the quartile distribution of ENO1 and YWHAZ expression counts, the samples were divided into high ENO1 high YWHAZ expression group, high ENO1 low YWHAZ expression group, and low ENO1 expression low YWHAZ expression group. The R software package (https: / / www.r-project.org / ) was used to evaluate the prognostic differences between the three groups, and P < 0.05 was identified as statistically significant. All analyses were performed using R software (version 4.2.1).

[0168] The MM patients were grouped according to the expression levels of ENO1 and YWHAZ, and survival curve analysis was performed ( Figure 3 ), and found that MM patients with high expression of both ENO1 and YWHAZ had the worst prognosis, patients with high expression of ENO1 alone had a poor prognosis, and patients with low expression of both ENO1 and YWHAZ had the best prognosis. This result indicates that the detection of ENO1 and YWHAZ can be used as a new indicator to predict the prognosis and survival of MM patients.

Claims

1. Use of an ENO1 inhibitor and bortezomib in the preparation of a drug for treating and / or preventing multiple myeloma.

2. The use according to claim 1, characterized in that The ENO1 inhibitor is AP-III-a4, or an RNA interference reagent for ENO1, and preferably the shRNA primers in the RNA interference reagent are as shown in SEQ ID NOs: 3 and 4, or as shown in SEQ ID NOs: 5 and 6.

3. The use according to claim 1 or 2, characterized in that: The drug has one or more of the following effects: It increases the sensitivity of multiple myeloma to BTZ, produces toxic effects on multiple myeloma cells, increases the apoptosis level of multiple myeloma cells, and reduces the level of Ki-67.

4. Use of ENO1 inhibitor, YWHAZ inhibitor and bortezomib in the preparation of drugs for treating and / or preventing multiple myeloma.

5. The use according to claim 4, characterized in that The ENO1 inhibitor is as defined in the use of claim 2; and / or, the YWHAZ inhibitor is an RNA interference agent of YWHAZ.

6. Use of ENO1 inhibitors and YWHAZ inhibitors in the preparation of drugs for reducing drug resistance of multiple myeloma cells or reducing the mitochondrial autophagy ability of multiple myeloma cells; Preferably, the drug resistance is caused by the administration of bortezomib; More preferably, the ENO1 inhibitor and YWHAZ inhibitor are as defined in the use of claim 4 or 5.

7. Use of a marker combination in the preparation of a drug for predicting and / or diagnosing multiple myeloma, wherein: The marker combination includes ENO1 protein and YWHAZ protein.

8. Use of a reagent for determining a marker combination in the preparation of a kit for determining the prognosis of a subject with multiple myeloma, wherein: The marker combination includes ENO1 protein and YWHAZ protein; Preferably, the reagent is used to detect the expression level of the marker combination, such as protein expression level and / or mRNA transcription level; More preferably, the reagent is a biomolecular reagent that specifically binds to the biomarker or specifically hybridizes with the nucleic acid encoding the biomarker, and the biomolecular reagent is, for example, a primer, a probe and / or an antibody.

9. The use according to claim 8, characterized in that By obtaining the expression levels of ENO1 protein and YWHAZ protein from the biological sample of the subject, Preferably, the biological sample is selected from one or more of peripheral venous blood, lymph node, and bone marrow fluid; More preferably, the biological sample is at least 1-10 mL, for example, 3 mL of peripheral venous blood or 1 mL of bone marrow fluid.

10. A reagent for detecting a marker combination, characterized in that: The marker combination includes ENO1 protein and YWHAZ protein, and the reagent is defined as in the use of claim 8.

11. A prognosis prediction model for multiple myeloma, characterized in that: The prognosis prediction model includes a detection module and an analysis and judgment module; the detection module detects the level of the marker combination in the biological sample to be tested, and transmits the level data to the analysis and judgment module; the analysis and judgment module judges the level data of the marker combination, and outputs a prediction or diagnosis result: the sample has a good prognosis or a poor prognosis; wherein the marker combination includes ENO1 protein and YWHAZ protein; Preferably, the biological sample is as defined in the application of claim 9.

12. A computer-readable storage medium for implementing the function of the prognosis prediction model according to claim 11, wherein the computer-readable storage medium stores a computer program.

13. An electronic device comprising a memory and a processor, wherein the memory stores a computer program, wherein: The processor is configured to execute the computer program to implement the function of the prognosis prediction model according to claim 11.

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