A myeloma biomarker SERPINF2 and its application

Abstract

The present invention relates to the field of molecular biology and biomedicine, and more particularly to a myeloma biomarker and its application. According to the above-mentioned application of the present invention, by detecting the expression level of the SERPINF2 gene or SERPINF2 protein in a subject, it is possible to determine whether the subject has myeloma, whether the subject is at risk of developing myeloma, or whether the subject has a good prognosis, thereby guiding clinicians to provide effective preventive measures or treatment plans for the subject, which is conducive to improving the subject's survival rate.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology and biomedicine technology, and in particular to a myeloma biomarker (SERPINF2) and applications thereof. Background Art

[0002] Myeloma (also known as plasmacytoma) is a malignant tumor originating from plasma cells in the bone marrow and is a relatively common malignant tumor. It can be either single or multiple. Multiple myeloma, also known as MM, is caused by the malignant transformation of plasma cells that synthesize and secrete immunoglobulins. The proliferation of large numbers of monoclonal malignant plasma cells easily involves soft tissues and can metastasize extensively in late stages, though lung metastasis is rare. It is more common in the spine, accounting for 10% of primary spinal tumors, with a higher incidence in the lumbar region. It is more common in men over 40 years old, with a male to female ratio of approximately 2:1. Common sites of MM include the spine, ribs, skull, and sternum. In recent years, the incidence of MM has been increasing, and the age of onset has also been decreasing.

[0003] Multiple myeloma has a gradual onset, with no obvious symptoms in the early stages. Its diverse clinical manifestations often lead to misdiagnosis and missed diagnosis, ultimately delaying treatment. The diagnosis of multiple myeloma generally requires bone marrow biopsy, imaging studies, and blood tests, combined with clinical manifestations for differential diagnosis. The Revised International Staging System (R-ISS) for multiple myeloma incorporates plasma biomarkers (lactate dehydrogenase, β2-microglobulin, and albumin) and cytogenetic abnormalities of known prognostic significance to predict disease behavior, but there is considerable heterogeneity among patients. Hematopoietic stem cell transplantation is the most effective treatment for multiple myeloma, but its clinical application is limited by factors such as bone marrow source, HLA matching, and high medical costs. Chemotherapy remains the main treatment strategy, but patients are prone to developing drug resistance and intolerance over the course of drug treatment. To address these issues, it is imperative to develop simple, non-invasive, sensitive, rapid, and highly specific diagnostic, therapeutic, and prognostic strategies. Summary of the Invention

[0004] In a first aspect of the present invention, there is provided a use of a product for detecting the SERPINF2 gene or SERPINF2 protein in the preparation of a tool for the diagnosis and / or prognosis of myeloma.

[0005] Specifically, products for detecting the SERPINF2 gene or SERPINF2 protein include products for detecting the expression level of the SERPINF2 gene or SERPINF2 protein.

[0006] Specifically, the product for detecting the SERPINF2 gene may include a nucleic acid capable of binding to the SERPINF2 gene.

[0007] Specifically, products for detecting the SERPINF2 gene can function based on known methods using nucleic acid molecules: for example, polymerase chain reaction (PCR), Southern blot hybridization, Northern blot hybridization, dot hybridization, fluorescence in situ hybridization (FISH), DNA microarrays, high-throughput sequencing platforms, and the like, particularly PCR methods, such as real-time fluorescence quantitative PCR. This product can be used to perform qualitative, quantitative, or semi-quantitative analysis.

[0008] Specifically, the nucleic acid contained in the product for detecting the SERPINF2 gene can be obtained by chemical synthesis, or by preparing a gene containing the desired nucleic acid from biological materials and then amplifying it using primers designed to amplify the desired nucleic acid.

[0009] Specifically, the nucleic acid may include primers that specifically amplify the SERPINF2 gene.

[0010] Specifically, the nucleic acid may also include a probe that specifically recognizes the SERPINF2 gene.

[0011] Specifically, the product for detecting the SERPINF2 gene can be a reagent, a kit, a test paper, a gene chip, etc., which can contain a nucleic acid that can bind to the SERPINF2 gene (for example, a primer that specifically amplifies the SERPINF2 gene and / or a probe that specifically identifies the SERPINF2 gene); the product for detecting the SERPINF2 gene can also be a high-throughput sequencing platform, which can use a nucleic acid that can bind to the SERPINF2 gene (for example, a primer that specifically amplifies the SERPINF2 gene and / or a probe that specifically identifies the SERPINF2 gene) to detect the SERPINF2 gene.

[0012] Specifically, the product for detecting the SERPINF2 protein may include a substance capable of binding to the SERPINF2 protein (eg, an antibody or a fragment thereof).

[0013] Specifically, products for detecting SERPINF2 protein can function based on known methods using proteins: for example, ELISA, radioimmunoassay, immunohistochemistry, Western blotting, proteomics (such as antibody chips, mass spectrometry (such as Data Independent Acquision (DIA) mass spectrometry), etc.) can be used.

[0014] Specifically, products for detecting SERPINF2 protein may include antibodies or fragments thereof that specifically bind to SERPINF2 protein. Antibodies or fragments thereof of any structure, size, immunoglobulin class, origin, etc. may be used, as long as they bind to the target protein. The antibodies or fragments included in products for detecting SERPINF2 protein may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody (partial fragment) or a peptide containing a portion of an antibody that retains the antibody's antigen-binding activity. Antibody fragments may include F(ab')2, Fab', Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv) or polymers thereof, dimerized V regions (diabodies), or peptides containing CDRs. Products for detecting SERPINF2 protein may include isolated nucleic acids encoding the amino acid sequence of the antibody or antibody fragment, vectors containing the nucleic acid, or cells carrying the vector. Antibodies may be obtained by methods well known to those skilled in the art, or available commercial products may be used.

[0015] Specifically, the product for detecting SERPINF2 protein can be a reagent, a kit, a test paper, a gene chip, etc., which can contain a substance that can bind to the SERPINF2 protein (such as an antibody or a fragment thereof); the product for detecting SERPINF2 protein can also be an instrument platform, which can include a measurement module (for measuring the content of SERPINF2 protein in the sample to be tested) and an analysis module (for analyzing the difference in the content of SERPINF2 protein in the sample to be tested and the reference sample).

[0016] Specifically, the measurement module can be based on mass spectrometry, such as DIA-MS, where the DIA acquisition scheme consists of 32 fixed windows with an acquisition range of 400-1200 mass-to-charge ratio (m / z).

[0017] Specifically, when the product for detecting SERPINF2 protein is an instrument platform, the sample to be tested is pretreated before testing, and the pretreatment may include: diluting the sample to be tested with a lysis buffer, disulfide reduction, alkylation treatment, enzymatic hydrolysis, acidification, and desalting; specifically, the pretreatment may include: diluting the sample to be tested with a urea solution, performing disulfide reduction with dithiothreitol (DTT) in a 37°C water bath, then alkylating with 500 mmol / L iodoacetamide (IAA) at 25°C in the dark, enzymatic hydrolysis with trypsin at 37°C, acidifying the enzymatically hydrolyzed peptide with trifluoroacetic acid solution (TFA, pH = 2-3), and then desalting with a C18 desalting column. The desalted peptide is then dried under vacuum and dissolved in a buffer containing 0.1% formic acid and 2% acetonitrile, and separated with an analytical column to obtain quantitative peptides for DIA-MS analysis.

[0018] Specifically, myeloma includes single myeloma and multiple myeloma, especially multiple myeloma.

[0019] Specifically, samples for the detection of SERPINF2 gene or SERPINF2 protein can be, for example, tissue samples or fluids obtained from biopsy subjects, such as tissue, blood, plasma, serum, lymph, urine, serous cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, etc., or their fractions or processed materials.

[0020] In one embodiment of the present invention, the sample used for the detection of SERPINF2 gene or SERPINF2 protein is blood (particularly peripheral blood) or a fraction thereof (such as serum), particularly serum, of a subject.

[0021] In a second aspect of the present invention, a tool for diagnosis and / or prognosis of myeloma is provided, which comprises a product for detecting the SERPINF2 gene or SERPINF2 protein.

[0022] Specifically, the product for detecting the SERPINF2 gene or SERPINF2 protein has the above corresponding definition of the present invention.

[0023] Specifically, the above-mentioned tools can be reagents, kits, test strips, gene chips, high-throughput sequencing platforms, proteomics analysis products (such as antibody chips, DIA-MS), etc.

[0024] In a third aspect of the present invention, there is provided a use of an inhibitor of the SERPINF2 gene or SERPINF2 protein in the preparation of a medicament for treating myeloma.

[0025] Specifically, the inhibitor can inhibit the expression or activity of substances in the upstream or downstream pathways of SERPINF2.

[0026] In a fourth aspect of the present invention, a drug for treating myeloma is provided, comprising an inhibitor of the SERPINF2 gene or SERPINF2 protein.

[0027] In a fifth aspect of the present invention, a method for diagnosing or prognosing myeloma is provided, comprising the step of detecting the SERPINF2 gene or SERPINF2 protein.

[0028] Specifically, the above method may include the following steps:

[0029] (1) Obtaining subject samples;

[0030] (2) detecting the expression level of SERPINF2 gene or SERPINF2 protein in the subject's sample;

[0031] (3) Correlating the measured expression level of the SERPINF2 gene or SERPINF2 protein with whether the subject is ill or not.

[0032] In particular, if the expression level of the SERPINF2 gene or SERPINF2 protein is elevated compared to a normal control, the subject may be diagnosed with myeloma or at high risk of developing myeloma, or the subject may be determined to have a poor prognosis. It should be noted that the specific disease risk, severity, and prognosis require a comprehensive assessment by the clinician in conjunction with other test indicators of the subject.

[0033] Specifically, myeloma includes single myeloma and multiple myeloma, especially multiple myeloma.

[0034] Specifically, the sample can be, for example, a tissue sample or fluid obtained from a biopsy subject, such as tissue, blood, plasma, serum, lymph, urine, serous cavity fluid, spinal fluid, synovial fluid, aqueous humor, tears, saliva, etc., or fractions thereof or processed materials. In one embodiment of the present invention, the sample is blood (particularly peripheral blood) or a fraction thereof (e.g., serum), particularly serum, obtained from a biopsy subject.

[0035] In particular, the subject is a mammal, especially a human.

[0036] In a sixth aspect of the present invention, a method for treating myeloma is provided, comprising inhibiting the SERPINF2 gene or SERPINF2 protein.

[0037] Specifically, the method includes inhibiting the expression of the SERPINF2 gene and / or inhibiting the activity of the SERPINF2 protein.

[0038] In the seventh aspect of the present invention, a method for screening tumor drugs is provided, which may include detecting the expression level of SERPINF2 gene or SERPINF2 protein at a certain period after administering the test drug to tumor cells or administering the test drug to tumor model animals to evaluate the effect of the test drug on improving tumor prognosis.

[0039] Specifically, when the expression level of the SERPINF2 gene or SERPINF2 protein decreases or returns to normal level after administration of the test drug, the drug can be selected as a therapeutic drug for improving tumor prognosis.

[0040] Specifically, the tumor is myeloma, especially multiple myeloma.

[0041] The present invention uses DIA-MS to analyze serum samples to obtain differential proteins and select potential diagnostic markers, prepares an antibody chip based on the selected diagnostic markers, and evaluates the stability and specificity of the antibody chip. The myeloma markers screened by the present invention can detect and screen myeloma patients with high sensitivity and high specificity. The antibody chip prepared by the present invention has high stability and high sensitivity and can be used for clinical testing. The myeloma serum markers screened by the present invention and the antibody chip prepared can greatly improve the detection efficiency of myeloma, achieve early diagnosis, and effectively reduce the detection cost under the high-throughput chip detection platform. According to the above-mentioned application of the present invention, by detecting the expression level of the SERPINF2 gene or SERPINF2 protein in a subject, it can be determined whether the subject has myeloma, whether the subject is at risk of developing myeloma, or whether the subject has a good prognosis, thereby guiding clinicians to provide effective preventive measures or treatment plans for the subject, which is conducive to improving the subject's survival rate and can also provide new drug targets for the development of myeloma treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Shown are the detection results of targets on the SERPINF2 protein by DIA-MS mass spectrometry.

[0043] Figure 2 Shown are the results of antibody microarray validation of myeloma diagnostic markers.

[0044] Figure 3 Shown is a volcano plot of mass spectrometry and antibody array detection results.

[0045] Figure 4 Shown are representative results of antibody microarray assays. DETAILED DESCRIPTION

[0046] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0047] Alpha-2 antiplasmin (serpin peptidase inhibitor clade F member 2, SERPINF2), referred to herein as SERPINF2.

[0048] In the present invention, "expression level" refers to the measurable amount of a SERPINF2 gene product in a sample, wherein the gene product can be a transcription product or a translation product. Thus, expression level is related to a nucleic acid gene product (such as mRNA or cDNA) or a polypeptide gene product (such as a SERPINF2 protein).

[0049] In the present invention, "SERPINF2 gene" includes the SERPINF2 gene itself and any functional equivalent polynucleotide of the SERPINF2 gene, such as a DNA sequence that has more than 70% (e.g., more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, more than 99.5%) homology with the SERPINF2 gene DNA sequence in the current international public nucleic acid sequence database GeneBank, and encodes the same functional protein.

[0050] In the present invention, "diagnosing myeloma" includes determining whether a subject already has myeloma, and also includes determining whether a subject has a risk of developing myeloma.

[0051] In the present invention, "prognosis" refers to the process or outcome of a myeloma patient after myeloma is suppressed or alleviated by surgical treatment or the like. In this specification, prognosis can be the vital status 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or more after myeloma is suppressed or alleviated by surgical treatment. Prognosis can be predicted by examining the biomarker, i.e., the SERPINF2 gene or SERPINF2 protein. Prognosis prediction can be performed by determining whether the patient's prognosis is good or poor, or determining the probability of a good or poor prognosis, based on the presence or absence, or increase or decrease, of the biomarker.

[0052] In the present invention, "good prognosis" means that after the patient's myeloma is suppressed or alleviated by surgical treatment, the patient has no critical condition for a long period of time (e.g., 3, 5, 6, 7, 8, 9, 10, 15, 20 years or longer). Alternatively, "good prognosis" can mean survival, no metastasis, no recurrence for such a long period of time. For example, "good prognosis" can mean at least 3 years or particularly at least 5 years of existence, preferably without metastasis or recurrence. The most preferred state of good prognosis is long-term disease-free survival. In the present invention, "good prognosis" can also include such a state: the disease such as metastasis can be found, but the nausea is low and does not seriously affect the ability to survive.

[0053] In the present invention, "poor prognosis" refers to a patient developing a fatal condition within a short period (e.g., 1, 2, 3, 4, 5 years or less) after myeloma is suppressed or remitted by surgical treatment or the like. Alternatively, "poor prognosis" refers to death, metastasis, or relapse within such a short period. For example, "poor prognosis" may mean relapse, metastasis, or death within at least 3 years, and particularly at least 5 years.

[0054] Predicting prognosis means predicting the course or outcome of a patient's condition, and does not mean that the course or outcome of a patient's condition can be predicted with 100% accuracy. Predicting prognosis means determining whether the likelihood of a certain course or outcome has increased, and does not mean determining the likelihood of a certain course or outcome by comparing it to a situation where the certain course or outcome does not occur. As in the present invention, in patients with elevated or decreased levels of the SERPINF2 gene or SERPINF2 protein, a particular course or outcome is more likely to be observed than in patients who do not exhibit this characteristic.

[0055] The disclosures of various publications, patents, and published patent specifications cited herein are incorporated by reference in their entirety.

[0056] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0057] Example 1: Screening of Myeloma Diagnostic Markers

[0058] 1. Sample collection

[0059] Samples were collected at Beijing Chaoyang Hospital affiliated to Capital Medical University. Twenty-two healthy volunteers (N) were recruited from Chaoyang Hospital with an average age of 57.95±3.40 years and a male-to-female ratio of 8:3 as the control group. Patients with clinically diagnosed myeloma were selected. The average age of myeloma patients (MM) was 58.16±9.14 years and the male-to-female ratio was 8:3. Informed consent was obtained from all patients.

[0060] 2. Myeloma diagnostic marker screening

[0061] The above samples were subjected to mass spectrometry detection using DIA-MS technology to obtain differential proteins between the N and MM groups of samples and select potential diagnostic markers.

[0062] The DIA-MS analysis steps are as follows:

[0063] 2 μL of serum sample was added to a 1.5 mL centrifuge tube and diluted with 6 mol / L urea (Sigma, USA) lysis buffer. Disulfide reduction was performed with 10 mmol / mL dithiothreitol (DTT) in a 37°C water bath for 60 min. The sample was then alkylated with 500 mmol / L iodoacetamide (IAA) at 25°C in the dark for 45 min. The sample was then enzymatically digested with 0.04 mg / mL trypsin at 37°C for 16 h. The enzymatically digested peptides were then eluted with 1% trifluoroacetic acid. The peptides were acidified with 1% tantalum phosphate (TFA, pH 2-3) and then desalted using a C18 desalting column. The desalted peptides were dried under vacuum and dissolved in 20 μL of a buffer containing 0.1% formic acid and 2% acetonitrile. Peptide concentrations were determined using a Nanodrop scanner under ultraviolet light at an absorbance of A280 nm. 1.5 μg of peptides were separated using an analytical column (150 μm × 250 mm) and injected into a QE-HF (Q Exactive HF Hybrid Quadrupole Orbitrap™, Thermo Fisher) mass spectrometer. The DIA acquisition protocol consisted of 32 fixed windows over a mass-to-charge ratio (m / z) range of 400-1200, with MS1 and MS2 resolution profiles of 60,000 and 30,000, respectively. The raw files were imported into Spectronaut Pulsar for analysis using the default software parameters.

[0064] The results of mass spectrometry screening of myeloma diagnostic markers are as follows Figure 1 shown.

[0065] Example 2: Antibody Chip Analysis of Myeloma Diagnostic Markers

[0066] The potential diagnostic markers obtained in Example 1 were verified using a specially prepared antibody chip.

[0067] 1. Preparation of Antibody Chip

[0068] (1) Experimental materials

[0069] SERPINF2 antibody was purchased from R&D Company, catalog number: MAB1470-SP.

[0070] Negative control: 100 μg / mL BSA and 1× PBS.

[0071] Positive control: 10 μg / mL Alexx-55-goat anti-human IgG; 100 μg / mL Biotin-IgG.

[0072] (2) Preparation steps:

[0073] Using the Ultra Marathon II spotter from Arrayjet (UK), various antibodies, negative controls, and positive controls were spotted on a Jingxin polymer three-dimensional substrate D (Biobio Group Co., Ltd., Beijing) by spraying. The spotted chips were placed in a closed environment with a humidity of 60% and a temperature of 25°C for 2 hours and then stored in a -20°C refrigerator for future use.

[0074] 2. Antibody chip detection process

[0075] (1) Experimental materials

[0076] Sample diluent: 1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4);

[0077] The washing solution was 0.05% PBST (0.05% Tween, 1× PBS). Tween was purchased from Amresco, USA.

[0078] 5% milk blocking solution (5% milk, 1× PBST) and skim milk powder were purchased from BD Biosciences, USA;

[0079] Biotin labeling reagent (NHS-PEG4-Biotin) and fluorescent dye (Streptavidin, R-PhycoerythrinConjugate (SAPE)) were purchased from Thermo Fisher Scientific, USA;

[0080] BSA (Albumin from bovine serum) was purchased from Sigma-Aldrich, USA;

[0081] Biochip scanner (4300A) was purchased from Molecular Devices, USA;

[0082] Incubation plates (3 / 5) were purchased from PEPperPRINT, Germany;

[0083] The temperature-controlled mixer (MixMate) and benchtop centrifuge (Centrifuge 5810R) were purchased from Eppendorf AG, Germany;

[0084] The micro separation column Bio-Spin6 was purchased from Bio-Rad, USA;

[0085] PCR-384M2-C microplate was purchased from Axygen, USA.

[0086] (2) Experimental steps:

[0087] Labeling of serum samples:

[0088] All serum protein molecules were labeled with biotin using the method described in Xu, M., Deng, J., Xu, K. et al. In-depth serum proteomics reveals biomarkers of psoriasis severity and response to traditional Chinese medicine. Theranostics. 2019, 9, 2475-2488. Figure 2 As shown, 10 μL of serum was diluted with 90 μL of filtered 1× PBS, and then the serum was labeled with 1 μL of biotin labeling reagent. After incubation at room temperature for 1 h, the serum was centrifuged at 1000×g for 2 min using a Bio-Spin6 separation column (Bio-Rad, USA) to remove excess biotin molecules. The collected biotinylated proteins were dissolved in 500 μL of 1× PBS containing 5% skim milk and stored at -20°C.

[0089] Testing of serum samples:

[0090] The antibody array was taken out of the −20°C freezer, placed at room temperature for 30 min, and then fixed on an incubation plate (PEPperPRINT, Germany), with each array corresponding to one fence;

[0091] (1) Blocking: After centrifugation, 500 μL of 5% skim milk (containing 0.05% PBST) was slowly added to each enclosure of the incubation plate and incubated at room temperature for 1 h.

[0092] (2) Sample addition: vacuum pump aspirates the milk in the incubation plate, and the treated serum marker is added to each fence of the incubation plate, and the reaction is carried out overnight at 4°C on a shaker;

[0093] (3) Washing: Use a vacuum pump to remove the solution in the incubation plate, wash three times with 0.05% PBST (1× PBS containing 0.05% Tween), each for 10 min, and then wash three times with deionized water, each for 5 min;

[0094] (4) Add fluorescent dye: Add 2 mg / L fluorescent dye (solvent is 5% skim milk) to the chip array and incubate at room temperature in the dark for 1 h;

[0095] (5) Washing in the dark: the same operation as step (3);

[0096] (6) Chip detection: Dry the chip, scan it with a fluorescence chip scanner and extract the fluorescence signal data.

[0097] Antibody chip test results Figure 2 shown.

[0098] The volcano plot of mass spectrometry and antibody chip test results is as follows Figure 3 shown.

[0099] Example 3: Clinical detection accuracy of antibody chip

[0100] Figure 4 These are representative results from antibody chip testing. Panel A shows the results from a healthy individual, while panel B shows the results from a myeloma patient. The inventors found that the signals in panel B are generally stronger than those in panel A. This preliminarily suggests that myeloma patients have more upregulated proteins in their serum than healthy individuals.

[0101] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0102] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience, and preference of those skilled in the art.

[0103] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. Use of a product for detecting SERPINF2 protein in the preparation of a tool for diagnosing myeloma.

2. The use according to claim 1, characterized in that The products include products for detecting the expression level of SERPINF2 protein.

3. The use according to claim 1, characterized in that The product comprises a substance capable of binding to the SERPINF2 protein.

4. The use according to claim 1, wherein The product is selected from the group consisting of: reagents, test kits, test strips, antibody chips, and instrument platforms.

5. The use according to claim 4, characterized in that The instrument platform comprises a measuring module for measuring the content of SERPINF2 protein in a sample to be tested.

6. The use according to claim 1, wherein The myeloma includes single myeloma and multiple myeloma.

7. The use according to claim 1, wherein The sample used for the detection of SERPINF2 protein is the subject's blood or a fraction thereof.

8. The use according to claim 1, wherein The sample used for the detection of SERPINF2 protein is serum.

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