Application of biomarker based on GMFB function in MASH diagnosis

Abstract

The invention provides an application of a GMFBS53 phosphorylated protein in a modified form of GMFBB as a biomarker in preparation of an MASH early diagnosis reagent. The invention further provides a kit capable of quantitatively or semi-quantitatively detecting the protein, and the kit contains an antibody capable of being specifically combined with the GMFBS53 phosphorylated protein. By detecting the expression level of the GMFBS53 phosphorylated protein, early diagnosis can be more efficiently and accurately carried out on the MASH condition of a subject, and an important basis is provided for early prevention and treatment of potential liver cancer patients. The kit provided by the invention can be suitable for early diagnosis of MASH.

Description

Technical Field

[0001] The present invention relates to the application of biomarkers based on GMFB function in the diagnosis and treatment of MASH, and belongs to the field of tumor medicine. Background Art

[0002] The key to cancer treatment is whether it can be detected and diagnosed early so that appropriate treatment measures can be taken. At present, cancer can only be completely cured if it is treated early. If the tumor develops to the middle or late stage, the tumor tissue expands or has metastasized, it is difficult to cure it even if various treatment methods are taken. MASH (Metabolic Dysfunction-Associated Steatohepatitis) is a precancerous lesion of liver malignancy. If it can be diagnosed and treated reasonably at an early or relatively early stage, a considerable number of patients can delay the progression of MASH to cirrhosis, or even stay in the MASH stage.

[0003] At present, the diagnosis of MASH is divided into three levels: ① Clinical diagnosis: based on clinical symptoms, signs and imaging examinations, the presumptive diagnosis is made with reference to the MASH disease special diagnosis; ② Surgical diagnosis: after surgery or various endoscopic examinations, the judgment is made only based on the liver performance seen by the naked eye, without pathological confirmation; ③ Histopathological diagnosis: the diagnosis is obtained after pathological analysis of the tissue obtained after fine needle puncture. Among these three levels, the reliability of the diagnosis increases successively, with liver tissue biopsy as the gold standard. However, the above methods cannot achieve early detection and early diagnosis of MASH. More often, after the disease develops to a certain extent, patients develop cirrhosis or liver malignant tumors. At this time, the best treatment period is often missed, making the later treatment in a very passive state. In addition, the pathological diagnosis with better diagnostic effect requires puncture to obtain cells or tissue sections, which has great damage and negative effects on the patient's mental and physical health. Therefore, it is very important to find more effective biomarkers to achieve early diagnosis of MSAH under less traumatic conditions.

[0004] At present, there are still many deficiencies in the study of the mechanism of occurrence and development of MASH, which seriously hinders the development of biomarkers for the diagnosis and treatment of this disease. Glial maturation factor (GMF) is a member of the homologous family of actin depolymerization factors that are conserved from yeast to mammals. GMFB (glia maturation factor beta) is a subtype of this family. It is a growth and differentiation factor mainly expressed in the central nervous system (CNS) and testis, and is mainly involved in cell growth and differentiation and chemotaxis and adhesion of immune cells; proteomics has confirmed that GMFB is expressed in the liver, and it can promote liver regeneration through the STAT3 signaling pathway. Some existing studies have mentioned the relationship between GMFB expression and liver cancer. For example, Chen Xin et al. detected the expression of GMFB in hepatocellular carcinoma tissues, and analyzed the relationship between GMFB expression levels and clinical pathological parameters, prognosis, and Ki-67 expression in hepatocellular carcinoma tissues of patients. It was found that the relative expression levels of GMFB mRNA and protein in HCC tissues were significantly higher than those in adjacent tissues, revealing the role of GMFB expression in the diagnosis of hepatocellular carcinoma. In addition, patent document CN112195244A also mentioned that the expression of GMFB is significantly correlated with the prognosis of HCC patients, and can be used as a biomarker to evaluate and diagnose HCC. Although people have realized through these studies that GMFB can be used as a biomarker for liver cancer diagnosis, these studies are all aimed at the diagnosis and prognosis evaluation of patients who have developed malignant tumors or even in the middle and late stages or after surgery. There is still a lack of sensitive and targeted biomarkers for the evaluation or early diagnosis of liver cancer precancerous lesions (especially MASH).

[0005] In view of the above background, in order to more accurately evaluate the occurrence and development of MASH and achieve early prevention, diagnosis and treatment of MASH in liver cancer precancerous lesions, it is necessary to find key markers involved in the key mechanisms of the occurrence and development of MASH and use them for the early diagnosis of MASH. Summary of the invention

[0006] The technical problem to be solved or the primary purpose of the present invention is to solve the problem of early diagnosis of MASH, a precancerous lesion of liver cancer, by using new biomarkers.

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

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention uses the modified form of GMFB, GMFBS53 phosphorylated protein, as a biomarker for preparing an early diagnostic reagent for MASH.

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

[0011] In the scheme described in the present invention, by using an immunoassay reagent for GMFBS53 phosphorylated protein to detect the expression level of GMFBS53 phosphorylated protein in a sample from a subject, early diagnosis of liver disease in the subject can be effectively performed. Furthermore, the sample from the subject is any one of the subject's peripheral venous blood or liver tissue.

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

[0013] In a second aspect, the present invention further provides an antibody, wherein the antibody is an antibody capable of specifically binding to the GMFBS53 phosphorylated protein.

[0014] In a third aspect, the present invention further provides a kit for quantitative or semi-quantitative detection of proteins, comprising the antibody described in the second aspect of the present invention.

[0015] In a preferred embodiment of the present invention, the kit capable of quantitatively or semi-quantitatively detecting proteins is a kit capable of detecting proteins by Western Blot method; the 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, the present invention also provides the use of an antibody that specifically binds to the GMFBS53 phosphorylated protein in the preparation of a MASH diagnostic kit.

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

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

[0019] The present invention is committed to solving the technical challenges in the field of early diagnosis of MASH, and provides a new specific biomarker GMFBS53 phosphorylated protein, and uses it in the early diagnosis of MASH. By detecting the expression level of the 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 can more effectively conduct a preliminary assessment and prediction of the subject's MASH condition, and provide an important basis for the early prevention and treatment of liver cancer. The detection kit prepared based on the biomarker of the present invention can be applied to the early diagnosis of MASH.

[0020] The GMFBS53 phosphorylated protein described in the present invention, as a specific post-translational modification form of GMFB, is highly correlated with the occurrence, development and poor prognosis of MASH. Specifically, we found through research that the expression of GMFB S53 phosphorylated protein in hepatocytes in MASH disease is significantly higher than that in normal hepatocytes, and shows a further upward trend with the progression of MASH, and is associated with the poor prognosis of MASH patients. These findings emphasize 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 type:

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

[0026] (2) Experimental animals:

[0027] c57 mice. The expression of p-GMFB (S53) protein in liver tissues of normal / high-fat fed c57 mice was detected, compared and analyzed.

[0028] (3) Patient samples:

[0029] The sample tissues were obtained from the Second Affiliated Hospital of Zhejiang University. Human MASH tissue specimens and healthy liver tissues were compared and analyzed.

[0030] 1.2 Test methods

[0031] (1) Protein level detection:

[0032] First, proteins are extracted from cells in cell experiments and mouse liver tissues in animal experiments. Then, corresponding antibodies are used to react with proteins to form antigen-antibody reactions, and the expression and action of specific target proteins are determined through substrate color development.

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

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

[0035] 2 Results and Analysis:

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

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

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

[0039] (4) Figure 4 As shown, immunohistochemical staining was performed on human normal / MASH liver samples, and the p-GMFB (S53) expression scores were significantly different between the two.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

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

[0042] 2. The test can be completed by collecting samples from peripheral venous blood, which greatly reduces the psychological burden and physical damage of the subjects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0044] Figure 2 The results show the changes of p-GMFB(S53) protein levels in liver of normal (Fed with CD) / high-fat fed (Fed with HFD) c57 mice.

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

[0046] Figure 4 It reflects the difference score of p-GMFB (S53) protein expression level in human normal (No steatosis) / MASH liver immunohistochemistry. DETAILED DESCRIPTION

[0047] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings. The experimental methods described in the following experiments are all conventional methods unless otherwise specified; if no specific technology or conditions are specified in the experiment, the technology or conditions described in the literature in the field or the product instructions are used; the reagents and materials described can be obtained from commercial channels unless otherwise specified.

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

[0049] The present invention also provides the use of p-GMFB (S53) in preparing a reagent for early diagnosis of MASH.

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

[0051] The reagents include a first antibody and a second antibody. The first antibody is a p-GMFB (S53) antibody that can specifically bind to p-GMFB (S53). The second antibody is an antibody that is homologous to the first antibody and is labeled with horseradish peroxidase.

[0052] The present invention also provides a MASH early diagnosis kit based on the Western Blot method, wherein the kit contains reagents for detecting the expression level of p-GMFB (S53), and the reagents in each kit include a first antibody and a second antibody, wherein the first antibody is a p-GMFB (S53) antibody that can specifically bind to p-GMFB (S53), and the second antibody is an antibody that is homologous to the first antibody and is labeled with horseradish peroxidase. The kit may also include a container, instructions for use, a positive control, a negative control, a buffer, an adjuvant, and a solvent. The instructions for use record how to use the kit for detection, and how to use the detection results to judge tumor development and select a treatment plan. The components of the kit may be packaged in the form of an aqueous medium.

[0053] The method for using the kit of the present invention to diagnose early MASH in patients comprises the following steps:

[0054] 1) detecting the expression level of the biomarker in the sample;

[0055] When peripheral venous blood is used as a sample, the sample processing method includes: after collecting the peripheral venous blood, it needs to be placed on ice for 15 minutes, and then centrifuged at 3500rpm for 15 minutes to obtain serum or plasma, which is then tested or stored at -80°C. Samples that cannot be tested immediately need to be stored at -80°C and thawed on ice during testing.

[0056] The treated samples are used to detect the expression level of GMFBS53 phosphorylated protein therein by Western Blot or other methods;

[0057] 2) Performing early diagnosis of MASH on subjects based on the GMFBS53 phosphorylated protein expression level in the samples detected in 1).

[0058] The Western Blot method mentioned in the diagnostic method of the present invention, i.e., protein immunoblotting experiment, is a technique commonly used to separate and identify proteins in research. It utilizes SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate various proteins contained in a specified sample, and then transfers the separated proteins to a nitrocellulose or PVDF membrane, and then incubates the membrane with a specific antibody to the target protein. During the membrane washing process, unbound antibodies are washed away, leaving only antibodies bound to the target protein, and finally the bound antibodies are detected by developing film or fluorescence scanning. Since the antibody is only bound to the target protein, generally only a clear band can be seen, and the thickness of the band corresponds to the amount of protein. By analyzing the position and intensity of a specific reaction, 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 strong specificity and high sensitivity of immunity, Western blot analysis can detect target proteins as low as 1ng. This method is widely used in molecular biology, biochemistry, immunogenetics and other molecular biology fields.

[0059] Based on the description of the above specific implementation methods, the present invention specifically lists the following embodiments to further illustrate the technical solution and technical effects of the present invention in detail.

[0060] Example 1.

[0061] A WB detection kit containing the following reagents and items:

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

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

[0064] Substrate solution;

[0065] Blocking solution: milk powder blocking solution;

[0066] Wash buffer: TBS / T;

[0067] Transfer buffer;

[0068] PVDF membrane;

[0069] Electroporation fluid;

[0070] Membrane staining solution;

[0071] Sample buffer;

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

[0073] Instructions for use.

[0074] Example 2.

[0075] A method for early diagnosis of MASH in a subject, comprising the following steps:

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

[0077] The specific detection steps are as follows:

[0078] 1. Sample Preparation

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

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

[0081] Prepare cell lysis solution: Determine the required lysis solution according to the number of cells: 50 μL / well of a six-well plate;

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

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

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

[0085] 5) Take 2.5 μL of sample and dilute with 22.5 μL of triple distilled water for BCA method to measure protein concentration;

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

[0087] 7) Load the sample directly onto the gel or aliquot and store at -80℃ for long term storage.

[0088] 2. SDS-PAGE polyacrylamide gel electrophoresis

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

[0090] 2) Carefully inject the separation gel, leaving about 2 cm of space (below the red frame of the gel rack) for the concentrated gel, cover the top layer with deionized water, and let it stand for about 30 minutes;

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

[0092] 4) Inject the concentrated gel into the upper end of the separation gel, taking care to avoid 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 separation gel

[0094] The wells were washed with double distilled water to remove the gel fragments, and then the water was absorbed 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 5μL prestained marker from the marker well and add an appropriate amount of 1× sample loading buffer to make the total volume the same as that of the sample well. The sample loading volume is generally 15-25μL. First, cook it at 95℃ for 5-10min in a heating block, shake it once during the process, then quickly centrifuge it and load the sample on the gel. Add an equal volume of 1× sample loading buffer to the well without the loaded sample.

[0097] 8) Electrophoresis: Start with a constant voltage of 60-80V. After running through the concentrated 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 runs to two-thirds of the separation gel.

[0098] 3. Membrane Transfer

[0099] 1) Cut the gel according to the marker and the position of the target band (make sure to mark the gel cutting angle), and immerse the eluted gel in transfer buffer for 15 minutes;

[0100] 2) After the PVDF membrane is marked, immerse it in methanol for 1 min, and then immerse it in transfer buffer together with 4 pieces of 3 mm filter paper and sponge for 15 min;

[0101] 3) Preparation of "Sandwich Cookies":

[0102] In the following order: fiber pad - filter paper - PVDF membrane - gel - filter paper - fiber pad;

[0103] Note: Align each item you add to ensure there are no 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 transfer, wash the membrane with 10 mL of 1× TBS at room temperature for 10 min;

[0108] 2) Incubate 5 mL of milk powder blocking solution at room temperature for 2 hours or gently shake at 4°C overnight. Since milk powder is 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, 5 min each time;

[0110] 4) Add 5 mL of primary antibody dilution buffer (antibodies are diluted according to the instructions), shake gently at room temperature for 2 hours or at 4°C overnight, recover the primary antibody, add sodium azide (which can inhibit bacterial growth) at 5 μL / mL of primary antibody solution, store at 4°C (uncommon antibodies can be stored at -20°C for a long time), and can be reused;

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

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

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

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

[0115] 1) Color development step: first lay out plastic wrap, then lay out plastic wrap on absorbent paper; pour water, developer (cannot be used if the color becomes darker) and fixer into their respective plates. Mix 2.5mL ECL-A and 2.5mL ECL-B and protect from light. Take the ECL mixed solution, membrane, etc. into the darkroom. Close the door, lock it, and pull back the cloth. Pour the ECL mixed solution into a small box, slightly dry the membrane with absorbent paper, put it into the ECL mixed solution and shake it at room temperature for 5 minutes (so that the ECL is evenly spread on the membrane), dry it with absorbent paper and place it on the plastic wrap, wrap it face down and place it on the clip, cut the X-film (note that you can only hold the edge of the X-film with your hands) and place it on the membrane, and the cut corner side is the marker (note: when taking the film, adjust the light to the minimum, and do not face the light, but face away from the light). Adjust the exposure time according to the brightness of the strip. Generally, you can expose it for 2 minutes first, observe the depth of the strip, and then determine the best exposure time;

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

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

[0118] 2. Perform early diagnosis of MASH in patients based on the protein expression results detected in step 1.

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

[0120] We collected 130 sera from Zhejiang University Second Hospital, including 72 MASH patients (diagnosed according to current clinical diagnostic criteria) and 58 healthy people.

[0121] According to the early evaluation or prediction method of this embodiment, the expression level of p-GMFB (S53) in 130 serum samples was detected by the WB detection kit, and then the evaluation and prediction were performed based on the obtained protein bands. The results showed that 68 of the 72 MASH patients were positive and 6 of the 58 healthy people were positive. The sensitivity of this method was 94.4% and the specificity was 89.7%.

[0122] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

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

2. The use according to claim 1, characterized in that: The MASH early diagnosis reagent is a reagent used to detect the expression level of GMFBS53 phosphorylated protein in a sample from a subject.

3. The use according to claim 2, characterized in that: The expression level of GMFBS53 phosphorylated protein in samples from subjects was detected by Western Blot method.

4. The use according to any one of claims 2 or 3, characterized in that: The detection of the expression level of GMFBS53 phosphorylated protein in the sample from the subject is to detect the expression level of GMFBS53 phosphorylated protein in the peripheral venous blood or liver tissue from the subject.

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

6. The kit according to claim 5, characterized in that: The kit capable of quantitatively or semi-quantitatively detecting proteins is a kit for detecting proteins by Western Blot method; the kit contains a first antibody and a second antibody; the first antibody is an antibody capable of specifically binding 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 to 6, characterized in that: The antibody that can specifically bind to the phosphorylated GMFBS53 protein is a monoclonal antibody of the phosphorylated GMFBS53 protein.

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

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