Application of biomarkers based on gmfb function in msh diagnostic

Abstract

The application provides an application of a modified form of GMFB, GMFBS53 phosphorylated protein, as a biomarker in preparation of a MASH early diagnosis reagent. The application also provides a kit for quantitatively or semi-quantitatively detecting a protein, which contains an antibody capable of specifically combining with the GMFBS53 phosphorylated protein. The application can more efficiently and accurately perform early diagnosis on a subject suffering from MASH by detecting the expression level of the GMFBS53 phosphorylated protein, and provides an important basis for early prevention and treatment of potential liver cancer patients. The kit can be applied to early diagnosis of MASH.

Description

Technical Field

[0001] This invention relates to the application of GMFB-based biomarkers in MASH diagnosis and treatment, and belongs to the field of oncology medicine. Background Technology

[0002] The key to cancer treatment lies in early detection and diagnosis, enabling appropriate treatment measures. Currently, only by striving for early treatment can cancer be completely cured. If the cancer progresses to the middle or late stages, with tumor tissue enlargement or metastasis, even with various treatment methods, a complete cure is unlikely. However, MASH (Metabolic Dysfunction-Associated Steatohepatitis), as a precancerous lesion of malignant liver cancer, can significantly delay the progression to cirrhosis or even remain at the MASH stage if diagnosed and treated appropriately in its early or relatively early stages.

[0003] Currently, MASH diagnosis methods are divided into three levels: ① Clinical diagnosis: a presumptive diagnosis based on clinical symptoms, signs, and imaging examinations, with reference to specific MASH diagnostic criteria; ② Surgical diagnosis: a judgment made solely based on visual observations of the liver after surgery or various endoscopic examinations, without pathological confirmation; ③ Histopathological diagnosis: a diagnosis obtained through pathological analysis of tissue obtained via fine-needle aspiration. The reliability of the diagnosis increases sequentially among these three levels, with liver biopsy considered the gold standard. However, these methods cannot achieve early detection and diagnosis of MASH. More often, the disease progresses to a certain stage, leading to cirrhosis or liver cancer, at which point the optimal treatment period is missed, leaving patients in a highly passive position for later treatment. Furthermore, pathological diagnosis, which offers better diagnostic results, requires puncture to obtain cells or tissue sections, causing significant harm and negative impact on the patient's mental and physical well-being. Therefore, finding more effective biomarkers to achieve early diagnosis of MASH with minimal invasiveness is crucial.

[0004] Current research on the mechanisms of MASH development and progression is still insufficient, severely hindering the development of biomarkers for the diagnosis and treatment of this disease. Glial maturation factor (GMF) is a member of the conserved actin depolymerization factor homology family from yeast to mammals. GMFB (glia maturation factor beta) is a subtype of this family, a growth and differentiation factor mainly expressed in the central nervous system (CNS) and testes, primarily involved in cell growth and differentiation as well as the chemotaxis and adhesion of immune cells. Proteomics has confirmed that GMFB is expressed in the liver and can promote liver regeneration through the STAT3 signaling pathway. Some existing studies have mentioned the relationship between GMFB expression and hepatocellular carcinoma (HCC). For example, Chen et al., by detecting GMFB expression in HCC tissues and analyzing the relationship between GMFB expression levels and patient clinicopathological parameters, prognosis, and Ki-67 expression in HCC tissues, found that the relative expression levels of GMFB mRNA and protein in HCC tissues were significantly higher than those in adjacent normal tissues, revealing the role of GMFB expression in the diagnosis of HCC. Furthermore, patent document CN112195244A also mentions that the expression level of GMFB is significantly correlated with the prognosis of HCC patients and can be used as a biomarker for the assessment and diagnosis of HCC. Although these studies have recognized that GMFB can serve as a biomarker for the diagnosis of liver cancer, these studies have focused on the diagnosis and prognostic assessment of patients who have already developed malignant tumors or even those in the middle or late stages or after surgery. For the assessment or early diagnosis of precancerous lesions of liver cancer (especially MASH), there is still a lack of sensitive and targeted biomarkers.

[0005] Given the above background, in order to more accurately assess the occurrence and development of MASH and achieve early prevention, diagnosis and treatment of MASH, a precancerous lesion of liver cancer, it is necessary to identify key biomarkers involved in the key mechanisms of MASH occurrence and development and use them for early diagnosis of MASH. Summary of the Invention

[0006] The technical problem or primary objective of this invention is to address the problem of early diagnosis of MASH (precancerous lesions of liver cancer) by applying novel biomarkers.

[0007] Another object of the present invention is to provide a product for early diagnosis of MASH.

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

[0009] In one aspect, the present invention uses the modified form of GMFB, GMFBS53 phosphorylated protein, as a biomarker for the preparation of MASH early diagnostic reagents.

[0010] In the present invention, the GMFBS53 phosphorylated protein refers to the phosphorylation of amino acid position 53 of the GMFB protein; the NCBI sequence number of GMFB is: NM_004124.3.

[0011] In the present invention, by using an immunoassay reagent targeting the phosphorylated GMFBS53 protein to detect the expression level of GMFBS53 phosphorylated protein in samples from subjects, early diagnosis of liver disease in subjects can be effectively achieved. Furthermore, the sample from the subject can be either peripheral venous blood or liver tissue from the subject.

[0012] In a preferred embodiment of the present invention, the MASH early diagnostic reagent is an immunoassay reagent based on the Western Blot method.

[0013] Secondly, the present invention also provides an antibody that is capable of specifically binding to the phosphorylated GMFBS53 protein.

[0014] Thirdly, the present invention also provides a kit for quantitative or semi-quantitative detection of proteins, containing the antibody described in the second aspect of the present invention.

[0015] In a preferred embodiment of the present invention, the reagent kit for quantitative or semi-quantitative detection of proteins is a reagent kit for detecting proteins by Western blotting; the reagent kit contains a first antibody and a second antibody; the first antibody is the antibody described in the second aspect of the present invention, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

[0016] Fourthly, this invention also provides the application of antibodies that specifically bind to the phosphorylated GMFBS53 protein in the preparation of MASH diagnostic kits.

[0017] In a further preferred embodiment of the present invention, the kit further comprises one or more substances selected from the group consisting of: a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, or a solvent.

[0018] The instruction manual describes how to use the kit for testing, and how to use the test results to assess the development of MASH and select a treatment plan.

[0019] This invention addresses the technical challenges in the early diagnosis of MASH (metastatic inflammatory response) by providing a novel specific biomarker, GMFBS53 phosphorylated protein, for use in the early diagnosis of MASH. By detecting the expression level of this biomarker, early diagnosis of MASH can be achieved. This biomarker provides a powerful tool for the early diagnosis of MASH; by detecting its expression level, it is possible to more effectively conduct preliminary assessments and predictions of a subject's MASH status, providing important evidence for the early prevention and treatment of liver cancer. The detection kit prepared based on the aforementioned biomarker is applicable to the early diagnosis of MASH.

[0020] The GMFBS53 phosphorylated protein described in this invention, as a specific post-translational modification of GMFB, is highly correlated with the occurrence, development, and poor prognosis of MASH. Specifically, our research found that the expression of GMFB S53 phosphorylated protein in hepatocytes of MASH patients is significantly higher than that in normal hepatocytes, and shows a further increasing trend with the progression of MASH, and is associated with poor prognosis in MASH patients. These findings highlight the potential of GMFB S53 phosphorylated protein in the diagnosis and treatment of MASH.

[0021] The above findings were obtained through the following research:

[0022] 1. Materials and Methods

[0023] 1.1 Materials used:

[0024] (1) Cell types:

[0025] Huh7 cells (human liver cancer cell line) and SK-Hep-1 cells (human liver cancer cell line) were obtained from the Cell Bank of the Chinese Academy of Sciences.

[0026] (2) Laboratory animals:

[0027] c57 mice. Detection, comparison, and analysis of p-GMFB(S53) protein expression in liver tissues of normal / high-fat fed c57 mice.

[0028] (3) Patient samples:

[0029] Tissue samples were obtained from the Second Affiliated Hospital of Zhejiang University. Comparative analysis was performed on human MASH tissue specimens and healthy liver tissue.

[0030] 1.2 Test Methods

[0031] (1) Protein level detection:

[0032] First, proteins were extracted from cells used in cell experiments and mouse liver tissue used in animal experiments. Then, corresponding antibodies were used to react with the proteins to form an antigen-antibody reaction, and the expression and function of specific target proteins were determined by substrate color development.

[0033] (2) Acquisition and analysis of human MASH samples

[0034] MASH liver tissue was obtained by surgery / puncture, fixed in 4% formaldehyde, embedded in paraffin, and histologically analyzed by H immunohistochemical staining to determine the MASH phenotype.

[0035] 2. Results and Analysis:

[0036] (1) Under free fatty acid (FFA) stimulation, the expression level of p-GMFB(S53) protein in Huh7 and SK-Hep-1 was upregulated compared with that in the control group (e.g., Figure 1 (As shown).

[0037] (2) Figure 2 As shown, 6-week-old c57 mice were fed a high-fat diet (HFD), and fresh liver samples were obtained after 24 weeks. Compared with the livers of mice fed a normal diet (CD), the p-GMFB (S53) protein level in the livers of mice fed a high-fat diet was significantly increased (e.g., ...). Figure 2 (As shown).

[0038] (3) Analysis results of human MASH samples are as follows: Figure 3 As shown, compared with normal liver, the expression level of p-GMFB(S53) protein was significantly upregulated in MASH liver, consistent with the results of cell experiments.

[0039] (4) Figure 4 As shown, immunohistochemical staining of normal human / MASH liver samples revealed a significant difference in p-GMFB(S53) expression scores between the two groups.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] 1. Based on the mechanism of MASH occurrence and development, this invention has identified new biomarkers for cell metabolism, and early diagnosis of MASH can be achieved by detecting the expression level of the biomarkers.

[0042] 2. The test can be completed by collecting peripheral venous blood samples, which greatly reduces the psychological burden and physical harm to the subjects. Attached Figure Description

[0043] Figure 1 This study reflects the changes in p-GMFB(S53) protein levels in Huh7 and SK-Hep-1 under free fatty acid (FFA) stimulation.

[0044] Figure 2 This study reflects the changes in p-GMFB(S53) protein levels in the livers of c57 mice fed with normal (Fed with CD) / high-fat diet (Fed with HFD).

[0045] Figure 3 This reflects the expression level of p-GMFB(S53) protein in normal (No steatosis) / MASH liver in humans.

[0046] Figure 4 This reflects the difference in p-GMFB(S53) protein expression levels in immunohistochemical samples from normal (No steatosis) / MASH livers. Detailed Implementation

[0047] To make the objectives and technical solutions of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the following tests are conventional methods; for tests where specific techniques or conditions are not specified, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions; unless otherwise specified, the reagents and materials described are commercially available.

[0048] This invention provides the application of GMFBS53 phosphorylated protein (p-GMFB(S53)) as a biomarker in the early diagnosis of MASH.

[0049] This invention also provides the application of p-GMFB(S53) in the preparation of reagents for early diagnosis of MASH.

[0050] The reagents include those for detecting the expression level of p-GMFB(S53) in a sample. The sample is peripheral venous blood or tumor tissue.

[0051] The reagent comprises a first antibody and a second antibody. The first antibody is a p-GMFB(S53) antibody that specifically binds to p-GMFB(S53). The second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase.

[0052] This invention also provides a MASH early diagnostic kit based on the Western Blot method. The kit contains reagents for detecting p-GMFB(S53) expression levels. Each kit includes a first antibody and a second antibody. The first antibody is a p-GMFB(S53) antibody that specifically binds to p-GMFB(S53), and the second antibody is an antibody homologous to the first antibody and labeled with horseradish peroxidase. The kit may also include a container, instructions for use, a positive control, a negative control, a buffer, adjuvants, and a solvent. The instructions for use describe how to use the kit for detection and how to use the test results to assess tumor development and select treatment options. The kit components can be packaged in an aqueous medium.

[0053] The method for early diagnosis of MASH in patients using the kit described in this invention includes the following steps:

[0054] 1) Detect the expression levels of the biomarkers mentioned in the samples;

[0055] When using peripheral venous blood as a sample, the sample processing method includes: after collecting peripheral venous blood, it should be placed on ice for 15 minutes, and then centrifuged at 3500 rpm for 15 minutes to obtain serum or plasma for testing or storage at -80℃. Samples that cannot be tested immediately should be stored at -80℃ and thawed on ice before testing.

[0056] The expression level of GMFBS53 phosphorylated protein in the processed samples was detected by methods such as Western Blot.

[0057] 2) Based on the expression level of GMFBS53 phosphorylated protein in the samples obtained in 1), the subjects were diagnosed with MASH early.

[0058] The Western blotting method mentioned in the diagnostic method described in this invention, also known as protein immunoblotting, is a technique commonly used in research for the isolation and identification of proteins. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample. The separated proteins are then transferred to a nitrocellulose or PVDF membrane, which is subsequently incubated with a specific antibody against the target protein. During membrane washing, unbound antibodies are washed away, leaving only antibodies bound to the target protein. Finally, the bound antibodies are detected by developing film or fluorescence scanning. Because the antibody binds only to the target protein, typically only a single, clear band is observed, with the band thickness corresponding to the protein content. By analyzing the location and intensity of specific reactions, the expression information of the target protein in a given cell or tissue homogenate can be obtained. Due to the high resolution of gel electrophoresis and the high specificity and sensitivity of immunoblotting, Western blotting analysis can detect target proteins as low as 1 ng. This method is widely used in molecular biology fields such as molecular biology, biochemistry, and immunogenetics.

[0059] Based on the above description of specific embodiments, the present invention provides the following examples to further illustrate the technical solution and its technical effects.

[0060] Example 1.

[0061] A Western blot (WB) test kit containing the following reagents and items:

[0062] Primary antibody (p-GMFB(S53) antibody);

[0063] Secondary antibody (antibody homologous to p-GMFB(S53) antibody and labeled with horseradish peroxidase);

[0064] Substrate solution;

[0065] Sealing solution: Milk powder sealing solution;

[0066] Washing buffer: TBS / T;

[0067] Transfer buffer;

[0068] PVDF membrane;

[0069] Electro-hydraulic transfer;

[0070] Membrane staining solution;

[0071] Sample buffer solution;

[0072] Standard protein or marker protein: used to estimate the molecular weight of the target protein;

[0073] Instruction manual.

[0074] Example 2.

[0075] A method for early diagnosis of MASH in subjects includes the following steps:

[0076] I. The expression level of GMFBS53 phosphorylated protein in peripheral venous serum of subjects was detected using the WB detection kit described in Example 1.

[0077] The specific testing steps are as follows:

[0078] 1. Prepare samples

[0079] 1) Sample collection: Peripheral venous blood was collected from the subjects, placed on ice for 15 min, and centrifuged at 3500 rpm for 15 min at 4℃ to obtain serum for testing.

[0080] 2) Make ice and prepare ice boxes;

[0081] Preparation of cell lysis buffer: Determine the required lysis buffer based on the number of cells: 50 μL / well of a six-well plate;

[0082] Lysis buffer formulation: 100 μL Beyotime lysis buffer + 1 μL protease inhibitor mixture + 1 μL PMSF;

[0083] 3) Prepare cells according to experimental requirements: Remove the culture medium, wash 3 times with 1×PBS (to remove serum from the culture medium), and add an appropriate amount of lysis buffer to each well (6-well plate). Quickly scrape cells off with a cell scraper and transfer them to a 1.5 mL tube, place on ice for 20 min, vortex to mix, and then place on ice for another 10 min;

[0084] 4) Centrifuge at 12000g, 4℃ for 15min, and collect the supernatant into another 1.5mL tube;

[0085] 5) Take 2.5 μL of sample and dilute it with 22.5 μL of triple-distilled water for use in the BCA method to determine protein concentration;

[0086] 6) Add 5× Loading Buffer (at a ratio of 2.5 mL Buffer / 10 mL protein) to the remaining samples and boil at 95 °C for 10 min, shaking once during the process;

[0087] 7) Directly load the sample for gel running or dispense it for long-term storage at -80℃.

[0088] 2. SDS-PAGE polyacrylamide gel electrophoresis

[0089] 1) Prepare the separating gel (5 mL / gel);

[0090] 2) Carefully inject the separating gel, leaving about 2cm of space (below the red border of the gel casting frame) for the concentrating gel, cover the top with deionized water, and let stand for about 30 minutes;

[0091] 3) Prepare the stacking gel (2 mL / gel);

[0092] 4) Pour the stacking gel into the top of the separating gel, being careful to avoid air bubbles;

[0093] 5) Insert the comb and wait for the concentrated gel to solidify (there is a clear boundary between the gel and the comb, plus the solidification time of the separating gel).

[0094] (For more than 2 hours), clean the pores with double-distilled water to remove gel fragments, and then blot dry with filter paper;

[0095] 6) Place the gel into the electrophoresis tank, and add 1× electrophoresis buffer to both the upper and lower tanks (do not reuse more than 3 times);

[0096] 7) Sample loading: Take 5 μL of prestained marker into the marker well and add an appropriate amount of 1× loading buffer to make the total volume the same as the sample well. The sample loading volume is generally 15-25 μL. First, boil it in a heating block at 95℃ for 5-10 minutes, shaking once during the process. Then, centrifuge quickly and load the sample for gel running. Add an equal volume of 1× loading buffer to the wells without sample loading.

[0097] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the stacking gel, increase the current to 100-120V. The electrophoresis time is determined according to the size of the target protein and the position of the marker. Generally, the target protein is ready when it reaches two-thirds of the separating gel.

[0098] 3. Membrane transfer

[0099] 1) Cut the gel according to the marker indication and the position of the target band (note to mark the cut corner of the gel). Immerse the eluted gel in the transfer buffer for 15 minutes.

[0100] 2) After marking the PVDF membrane, immerse it in methanol for 1 minute, then immerse it together with 4 sheets of 3mm filter paper and a sponge in the transfer buffer for 15 minutes.

[0101] 3) Preparing the "sandwich cookie":

[0102] The order is as follows: fiber mat—filter paper—PVDF membrane—gel—filter paper—fiber mat;

[0103] Note: Align each item as you add it, ensuring there are no air bubbles;

[0104] 4) Transfer: The transfer time is determined according to 1).

[0105] One side of the PVDF membrane is connected to the positive electrode (red), and the other side of the gel is connected to the negative electrode (black).

[0106] 4. Membrane blocking and antibody incubation

[0107] 1) After the transfer, wash the membrane with 10 mL of 1×TBS at room temperature for 10 min;

[0108] 2) Incubate 5 mL of the milk powder blocking solution at room temperature for 2 hours or at 4°C with gentle shaking overnight. Since milk powder is relatively difficult to dissolve, it should be prepared at least 1 hour in advance.

[0109] 3) Wash the membrane three times with 10 mL of TBS / T solution, 5 min each time;

[0110] 4) Add 5 mL of primary antibody dilution buffer (dilute the antibody according to the instructions), incubate at room temperature for 2 hours or at 4°C with gentle shaking overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μL / mL of primary antibody solution, and store at 4°C (less frequently used antibodies can be stored at -20°C for a long time). It can be reused.

[0111] 5) Wash the membrane three times with 10 mL of TBS / T solution, 5 min each time;

[0112] 6) Add the secondary antibody (usually diluted 1:2000) and gently shake at room temperature for 1 hour;

[0113] 7) Wash the membrane three times with 10 mL of TBS / T solution, each time for 5 min.

[0114] 5. Development and fixing (or direct fluorescence scanning after incubation with fluorescently labeled secondary antibody)

[0115] 1) Development steps: First, lay down plastic wrap, then place another layer of plastic wrap on top of absorbent paper. Pour water, developer (if the color darkens, do not use), and fixer into their respective trays. Mix 2.5mL of ECL-A and 2.5mL of ECL-B, and keep away from light. Take the ECL mixture, film, etc., into the darkroom. Close and lock the door, and pull back the curtain. Pour the ECL mixture into a small box. Slightly pat the film dry with absorbent paper and place it in the ECL mixture, shaking at room temperature for 5 minutes (to ensure the ECL is evenly spread over the film). After patting dry with absorbent paper, place it on plastic wrap, film side down, and place it on a clip. Cut an X-film (note that you can only hold the edge of the X-film) and place it on the film, cutting one corner as a marker (note: when handling the film, turn the light to minimum and do not face the light; keep it away from the light). Adjust the exposure time according to the brightness of the strips. Generally, you can expose for 2 minutes first, observe the depth of the strips, and then determine the optimal exposure time.

[0116] 2) Development and fixing: After taking out the X-film and placing it in the developer for a certain period of time (depending on the intensity of the target band and the background), wash it once with water and then place it in the fixer for at least 5 minutes.

[0117] Finally, the target protein band on the PVDF membrane is obtained. By comparing the band with the protein marker, the size and location of the target protein can be determined, and the expression result of the target protein can be obtained.

[0118] II. Early diagnosis of MASH in patients based on the protein expression results detected in step one.

[0119] If a protein band of p-GMFB(S53) appears on the PVDF membrane, it suggests that the subject may have MASH disease.

[0120] We collected serum samples from 130 individuals at the Second Affiliated Hospital of Zhejiang University, including 72 MASH patients (diagnosed according to current clinical diagnostic criteria) and 58 healthy individuals.

[0121] Following the early assessment or prediction method of this embodiment, the expression level of p-GMFB(S53) in the serum of 130 patients was detected using the aforementioned WB detection kit, and assessment and prediction were performed based on the obtained protein bands. The results showed that 68 out of 72 MASH patients were positive, and 6 out of 58 healthy individuals were positive. This indicates that the method has a sensitivity of 94.4% and a specificity of 89.7%.

[0122] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Application of the modified form of GMFB, GMFBS53 phosphorylated protein, as a biomarker in the preparation of MASH early diagnostic reagents.

2. The application as described in claim 1, characterized in that: The MASH early diagnostic reagent is used to detect the expression level of GMFBS53 phosphorylated protein in samples from subjects.

3. The application as described in claim 2, characterized in that: The expression level of phosphorylated GMFBS53 protein in samples from subjects was detected by Western blotting.

4. The application as described in any one of claims 2 or 3, characterized in that: The detection of the expression level of GMFBS53 phosphorylated protein in samples from the subject refers to the detection of the expression level of GMFBS53 phosphorylated protein in peripheral venous blood or liver tissue from the subject.

5. A kit for quantitative or semi-quantitative detection of proteins, containing an antibody that specifically binds to GMFBS53 phosphorylated protein.

6. The kit as described in claim 5, characterized in that: The aforementioned reagent kit for quantitative or semi-quantitative detection of proteins is a reagent kit for detecting proteins using the Western blotting method; the reagent kit contains a first antibody and a second antibody; the first antibody is an antibody that specifically binds to GMFBS53 phosphorylated protein, and the second antibody is an enzyme-labeled antibody homologous to the first antibody.

7. The kit according to any one of claims 5-6, characterized in that: The antibody that specifically binds to the phosphorylated GMFBS53 protein is a monoclonal antibody against the phosphorylated GMFBS53 protein.

8. Application of antibodies against GMFBS53 phosphorylated protein in the preparation of MASH diagnostic kits.

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