Plasma exosome protein marker VCAM1 for diagnosis of hepatocellular carcinoma, kit and application thereof

Abstract

The present invention discloses a plasma exosome protein marker VCAM1, a kit and its application for the diagnosis of hepatocellular carcinoma, which belongs to the field of molecular marker technology. The technical problem to be solved is how to improve the sensitivity and / or specificity of hepatocellular carcinoma diagnosis. The present invention provides the use of biomarkers and / or substances for detecting the biomarkers in the diagnosis of hepatocellular carcinoma or in the preparation of products for the diagnosis of hepatocellular carcinoma; the biomarker is a plasma exosome VCAM1 protein or a combination of plasma exosome VCAM1 protein and plasma AFP. The biomarker VCAM1 protein and the detection method of the present invention have the characteristics of high sensitivity and high specificity. Their application in the diagnosis of clinical hepatocellular carcinoma patients can improve the diagnosis rate of early liver cancer, screen out subclinical liver cancer or occult liver cancer from the population, and help improve the cure rate of liver cancer and reduce the mortality rate.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular markers for testing molecular materials by means of the chemical or physical properties of the test materials, and in particular to a plasma exosome protein marker VCAM1 for diagnosing hepatocellular carcinoma, a kit and applications thereof. Background Art

[0002] Primary liver cancer (PLC) is the sixth most common digestive system malignancy and the third most common cause of death worldwide. Hepatocellular carcinoma (HCC) accounts for approximately 75%-85% of PLC cases. Hepatitis B virus (HBV) infection is the most significant risk factor for the development and progression of HCC, with over 50% of individuals infected with HBV eventually developing HCC. HCC is insidious and difficult to diagnose, with over 70%-80% of patients diagnosed in the advanced stages. Furthermore, the 5-year recurrence and metastasis rate after resection of advanced HCC is as high as 40%-70%, and the 5-year survival rate is only 14.1%. Recent studies have shown that the diagnosis of HCC can increase the 5-year survival rate to 50%-75%. Therefore, developing more effective diagnostic methods or strategies for HCC and increasing the rate of early diagnosis of HCC in high-risk populations will hopefully improve patient outcomes.

[0003] Serum / plasma biomarkers are highly effective and noninvasive diagnostic methods for the disease. Currently, the main serum / plasma biomarkers associated with hepatocellular carcinoma used in clinical practice include alpha-fetoprotein (AFP), alpha-fetoprotein isoform L3 (AFP-L3), and des-γ-decarboxy prothrombin (DCP). However, existing data show that the ability of these biomarkers to detect hepatocellular carcinoma remains limited. In the diagnosis of clinical cases of hepatocellular carcinoma, the sensitivity and specificity of AFP, AFP-L3, and DCP are only 18%-60% and 85%-90%, 28%-56% and 92%-97%, and 56%-77% and 82%-87.2%, respectively. Therefore, the development of new diagnostic biomarkers for hepatocellular carcinoma with higher sensitivity and specificity is urgently needed.

[0004] Exosomes are small, intraluminal vesicles with a lipid bilayer, approximately 40 to 200 nanometers in diameter, formed by inward budding of the membrane of multivesicular endosomes (MEVs) during the maturation process of intracellular endosomes. In recent years, researchers have discovered that proteins, nucleic acids, and lipids contained in exosomes can serve as diagnostic and / or prognostic markers for cancer, with potential clinical application. Studies have reported that proteins in extracellular vesicles derived from tumor tissue and plasma from certain cancer patients can distinguish cancer patients with a sensitivity and specificity exceeding 90%. In patients with hepatocellular carcinoma, galectin-3-binding protein (LGALS3BP), which is highly expressed in serum exosomes, may serve as a candidate biomarker for distinguishing HCC patients from controls. However, to date, no exosome protein markers or molecular classifiers derived from HCC plasma have been reported. Therefore, the discovery of plasma exosome protein markers derived from HCC plasma and the corresponding molecular classifiers will help to further establish non-invasive, easy-to-operate, highly sensitive and highly specific HCC diagnosis or diagnostic detection methods, which will have great practical significance for the early detection, diagnosis and treatment of HCC. Summary of the Invention

[0005] The present invention aims to provide a biomarker for the diagnosis of hepatocellular carcinoma. The technical problem to be solved is how to improve the sensitivity and / or specificity of hepatocellular carcinoma diagnosis. The technical problem to be solved is not limited to the technical subject matter described herein. Other technical subjects not described herein will be readily apparent to those skilled in the art through the following description.

[0006] To solve the above technical problems, the present invention first provides any of the following applications of a biomarker and / or a substance for detecting the biomarker:

[0007] A1) Use in the diagnosis of hepatocellular carcinoma or in the preparation of a product for the diagnosis of hepatocellular carcinoma;

[0008] A2) use in screening for hepatocellular carcinoma or in the preparation of a product for screening for hepatocellular carcinoma;

[0009] A3) Use in assessing the risk of hepatocellular carcinoma or in the preparation of a product for assessing the risk of hepatocellular carcinoma;

[0010] A4) Use in the prognosis assessment of hepatocellular carcinoma or in the preparation of a product for the prognosis assessment of hepatocellular carcinoma;

[0011] A5) Use in identifying and differentiating hepatocellular carcinoma from cirrhosis or in preparing a product for identifying and differentiating hepatocellular carcinoma from cirrhosis;

[0012] A6) Use in identifying and differentiating hepatocellular carcinoma from chronic hepatitis B or in preparing products for identifying and differentiating hepatocellular carcinoma from chronic hepatitis B;

[0013] The biomarker may be any of the following:

[0014] B1) VCAM1 protein in plasma exosomes and AFP in plasma;

[0015] B2) VCAM1 protein in plasma exosomes.

[0016] Furthermore, the expression level of VCAM1 protein in the plasma exosomes in hepatocellular carcinoma samples (plasma samples from liver cancer patients) and control samples (plasma samples from healthy people or patients with cirrhosis or chronic hepatitis B) was significantly different.

[0017] The VCAM1 protein is vascular cell adhesion molecule 1 (VCAM1).

[0018] Furthermore, the expression level of VCAM1 protein in the plasma exosomes is upregulated in liver cancer samples.

[0019] For healthy controls (HC), patients with liver cirrhosis (LC), patients with chronic hepatitis B (CHB), and patients with hepatocellular carcinoma (HCC), VCAM1 protein in plasma exosomes can be used as a biomarker to distinguish HCC from LC, CHB, and HC.

[0020] The expression level of VCAM1 in plasma exosomes of healthy subjects, patients with cirrhosis and chronic hepatitis B was lower than that in patients with hepatocellular carcinoma.

[0021] In the above applications, the substance may be a reagent for detecting the VCAM1 protein content (i.e., the concentration of VCAM1 protein in plasma exosomes) (such as a reagent for detecting the VCAM1 protein content in plasma exosomes) and a reagent or kit for detecting the AFP content (such as a reagent or kit for detecting the AFP content in plasma); or the substance may be a reagent for detecting the VCAM1 protein content (such as a reagent for detecting the VCAM1 protein content in plasma exosomes).

[0022] In the above applications, the substance for detecting the biomarker may include a substance for detecting the biomarker by enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR or biotin-avidin technology.

[0023] In the above application, the reagent for detecting the VCAM1 protein content may include VCAM1 protein (such as Ag1993, Proteintech, USA) and an antibody binding to VCAM1 protein (such as ab216035, Abcam, UK); the reagent for detecting the AFP content includes AFP and an antibody binding to AFP.

[0024] The kit for detecting the content of AFP comprises AFP and an antibody that binds to AFP.

[0025] The reagent for detecting the content of VCAM1 protein includes VCAM1 protein as a standard.

[0026] The reagent for detecting the AFP content includes AFP protein as a standard.

[0027] Furthermore, the substance used to detect the expression level of VCAM1 protein can be an antibody that specifically binds to VCAM1 protein (such as ab216035, Abcam, UK).

[0028] The present invention also provides a hepatocellular carcinoma diagnostic kit, which includes a reagent for quantitatively detecting the marker of the present invention (VCAM1 protein in plasma exosomes). Furthermore, the above-mentioned hepatocellular carcinoma diagnostic kit also includes a control sample, which can be plasma exosomes from healthy people, plasma exosomes from patients with cirrhosis, or plasma exosomes from patients with chronic hepatitis B.

[0029] The present invention also provides a composition comprising the reagent for detecting the VCAM1 protein content and the reagent for detecting the AFP content, and the composition has at least one of the following uses:

[0030] U1) Diagnosis of hepatocellular carcinoma or preparation of products for diagnosis of hepatocellular carcinoma;

[0031] U2) Hepatocellular carcinoma screening or preparation of products for hepatocellular carcinoma screening;

[0032] U3) assessing the risk of hepatocellular carcinoma or preparing a product for assessing the risk of hepatocellular carcinoma;

[0033] U4) Hepatocellular carcinoma prognosis assessment or preparation of a product for use in hepatocellular carcinoma prognosis assessment;

[0034] U5) Identifying and differentiating hepatocellular carcinoma from cirrhosis or preparing products for identifying and differentiating hepatocellular carcinoma from cirrhosis;

[0035] U6) Identifying and differentiating hepatocellular carcinoma from chronic hepatitis B or preparing products for identifying and differentiating hepatocellular carcinoma from chronic hepatitis B.

[0036] The reagent for detecting the content of VCAM1 protein and the reagent for detecting the content of AFP in the composition are both packaged independently.

[0037] The product can use plasma as the sample to be tested.

[0038] The present invention also provides a kit comprising the composition or the reagent for detecting the content of VCAM1 protein, wherein the kit has at least one of the following uses:

[0039] F1) Diagnosis of hepatocellular carcinoma or preparation of products for diagnosis of hepatocellular carcinoma;

[0040] F2) Hepatocellular carcinoma screening or preparation of products for hepatocellular carcinoma screening;

[0041] F3) assessing the risk of hepatocellular carcinoma or preparing products for use in assessing the risk of hepatocellular carcinoma;

[0042] F4) Hepatocellular carcinoma prognosis assessment or preparation of a product for use in hepatocellular carcinoma prognosis assessment;

[0043] F5) Identifying and differentiating hepatocellular carcinoma from cirrhosis or preparing products for identifying and differentiating hepatocellular carcinoma from cirrhosis;

[0044] F6) Differentiating hepatocellular carcinoma from chronic hepatitis B or preparing products for differentiating hepatocellular carcinoma from chronic hepatitis B.

[0045] In the above kit, the test sample of the kit may be plasma.

[0046] Furthermore, the kit includes a reagent for quantitatively detecting VCAM1 protein in plasma exosomes.

[0047] Furthermore, the kit further comprises a standard substance, which may be a VCAM1 protein standard substance.

[0048] Furthermore, the kit may also include exosome enrichment and purification reagents.

[0049] Furthermore, the kit may be a hepatocellular carcinoma diagnostic kit, which may also include a control sample, and the control sample may be plasma exosomes from healthy subjects and / or plasma exosomes from patients with cirrhosis and / or plasma exosomes from patients with chronic hepatitis B.

[0050] The present invention also provides any of the following applications of the kit:

[0051] C1) Use in the diagnosis of hepatocellular carcinoma or in the preparation of a product for the diagnosis of hepatocellular carcinoma;

[0052] C2) use in screening for hepatocellular carcinoma or in the preparation of a product for screening for hepatocellular carcinoma;

[0053] C3) Use in assessing the risk of hepatocellular carcinoma or in the preparation of a product for assessing the risk of hepatocellular carcinoma;

[0054] C4) Use in the prognosis assessment of hepatocellular carcinoma or in the preparation of a product for the prognosis assessment of hepatocellular carcinoma;

[0055] C5) Use in identifying and differentiating hepatocellular carcinoma from cirrhosis or in preparing a product for identifying and differentiating hepatocellular carcinoma from cirrhosis;

[0056] C6) Use in identifying and differentiating hepatocellular carcinoma from chronic hepatitis B or in preparing a product for identifying and differentiating hepatocellular carcinoma from chronic hepatitis B.

[0057] Herein, the product may be a reagent, a kit or a chip.

[0058] The present invention also provides the use of the biomarker (VCAM1 protein in plasma exosomes) as a target in the preparation of a drug for treating or preventing hepatocellular carcinoma.

[0059] The present invention also provides a method for screening or assisting in the diagnosis of hepatocellular carcinoma, which may be any of the following:

[0060] D1) The method comprises using the composition to detect the VCAM1 protein level and the AFP level in the plasma of the patient to be tested, and determining whether the patient to be tested is a hepatocellular carcinoma patient based on the VCAM1 protein level and the AFP level;

[0061] D2) The method includes using the reagent for detecting the VCAM1 protein content to detect the VCAM1 protein content in the plasma of the patient to be tested, and determining whether the patient to be tested is a hepatocellular carcinoma patient based on the VCAM1 protein content.

[0062] Furthermore, detecting the VCAM1 protein content in the plasma of the patient to be tested may include detecting the VCAM1 protein content in exosomes in the plasma of the patient to be tested.

[0063] The diagnosis of hepatocellular carcinoma according to the present invention may include the following steps:

[0064] E1) collecting hepatocellular carcinoma samples;

[0065] E2) detecting the protein expression level of plasma exosomal VCAM1 in the hepatocellular carcinoma sample and the control sample using the hepatocellular carcinoma diagnostic reagent (a substance that detects the biomarker);

[0066] E3) If the absolute expression level of VCAM1 protein in the hepatocellular carcinoma sample is higher than that in the control sample, the patient has a risk of developing hepatocellular carcinoma.

[0067] The hepatocellular carcinoma sample may be a plasma sample.

[0068] Furthermore, the detection can be performed by detecting the expression level of VCAM1 protein in plasma exosomes through an enzyme-linked immunosorbent assay (ELISA) method.

[0069] The biomarkers of the present invention can be applied to conventional ELISA protein detection experiments.

[0070] The present invention also provides an ELISA method for detecting VCAM1 protein in plasma exosomes.

[0071] In one embodiment of the present invention, the ELISA method comprises the following steps:

[0072] The expression level of VCAM1 protein in exosomes was detected by ELISA method 1, and the steps were as follows: (1) 100 μL rabbit anti-human CD63 monoclonal antibody (1:1000; ab68418, Abcam, UK) was coated in a 96-well ELISA plate (95029180, Thermo Fisher Scientific, USA), incubated at 4°C overnight, washed, and patted dry; (2) 200 μL of 5% skim milk powder solution containing 0.05% Tween-20 was added to each well, incubated at room temperature for 2 h, washed, and patted dry; (3) 100 μL of plasma sample and exosomes were added to each well, incubated at 37°C for 2 h, washed, and patted dry. Pat dry the water droplets; (4) Add 100 μL of mouse anti-human VCAM1 protein monoclonal antibody (1:500; ab216035, Abcam, UK) to each well, incubate at 37°C for 2 h, wash, and pat dry the water droplets; (5) Add 100 μL of HRP-conjugated goat anti-mouse IgG polyclonal antibody (1:1000; ab205719, Abcam, UK) to each well, incubate at 37°C for 1 h, wash, and pat dry the water droplets; (6) Add 90 μL of TMB to each well, incubate at room temperature for 15 min, and then add 50 μL of stop solution (2M H2SO4) to each well; (7) Use a SunriseTM plate reader to read the reaction signal at 450 nm, and quantify the VCAM1 protein content in the plasma sample based on the standard curve.

[0073] The purpose of the above-mentioned applications and methods may be disease diagnosis, disease prognosis and / or disease treatment, or their purpose may be non-disease diagnosis, non-disease prognosis and non-disease treatment; their direct purpose may be to obtain information on intermediate results of disease diagnosis, disease prognosis and / or disease treatment, or their direct purpose may be non-disease diagnosis, non-disease prognosis and / or non-disease treatment.

[0074] After extensive and in-depth research, the inventors unexpectedly discovered that VCAM1 protein is highly expressed in the plasma exosomes of patients with hepatocellular carcinoma (HCC). Based on this, they developed a biomarker for the diagnosis and detection of HCC. VCAM1 protein as a biomarker can be used for non-invasive diagnosis, screening, and prognostic assessment of HCC. Furthermore, VCAM1 protein can be used as a drug target in the screening and development of HCC drugs. Specifically, the presence of HCC can be determined by measuring the expression level of VCAM1 protein in a patient's plasma exosomes. When the expression level of VCAM1 is significantly higher than that of a control individual, this can be used as a diagnostic indicator for HCC, guiding clinicians in early intervention and treatment, thus promising promising applications.

[0075] This invention proposes plasma exosome protein markers for the diagnosis of hepatocellular carcinoma and their applications. The invention screens and further identifies a specific hepatocellular carcinoma-associated protein (VCAM1 in plasma exosomes) and a molecular classifier composed of it and plasma AFP. By detecting its protein expression, the diagnosis of hepatocellular carcinoma is achieved. This method is suitable for the development of ELISA hepatocellular carcinoma-related detection kits, and thus for clinical diagnosis of hepatocellular carcinoma patients.

[0076] The VCAM1 protein in plasma exosomes, a biomarker for the diagnosis and detection of hepatocellular carcinoma, and the substances, kits, and detection methods for detecting the expression level of the VCAM1 protein disclosed herein are characterized by high sensitivity and specificity; the higher the sensitivity, the lower the missed diagnosis rate (false negative rate). In the diagnosis and screening of liver cancer, since liver cancer is often discovered late and almost incurable once discovered, increased sensitivity is particularly important. This increased sensitivity can improve the diagnosis rate of early-stage liver cancer, screen out subclinical or occult liver cancer from the population, and help improve the cure rate and reduce the mortality rate of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 To evaluate the quality of extracted plasma exosomes; A: Nanoparticle tracking analysis showed that the enriched exosomes were between 30 and 150 nm in size, with a peak size of approximately 74.49 nm; B: The size and structure of exosomes observed by transmission electron microscopy; C: The expression levels of marker molecules FLOT1 and TSG101 proteins in the enriched exosomes.

[0078] Figure 2 Figure 3: Comparison results of plasma exosome proteins in the present invention with the exosome databases ExoCarta and Vesiclepedia; A: Normalized mass spectrometry data of each group of samples; B: The abundance of the identified plasma exosome markers ranked top among all proteins; C: Venn diagram comparing plasma exosome proteins in HCC, LC, CHB, and HC groups; D: Venn diagram comparing plasma exosome proteins in each group with the exosome databases ExoCarta and Vesiclepedia.

[0079] Figure 3 Figure 2: Analysis of differential expression of plasma exosome proteins between HCC and LC, CHB, and HC groups; A: Venn diagram of high-quality proteins retained for analysis in HCC and LC, CHB, and HC groups after quality control; B: Volcano plot of differentially expressed proteins between HCC and LC groups; C: Volcano plot of differentially expressed proteins between HCC and CHB groups; D: Volcano plot of differentially expressed proteins between HCC and HC groups.

[0080] Figure 4Results of Hallmark pathway enrichment analysis of differentially expressed exosomal proteins; A: Hallmark pathway enrichment analysis of differentially expressed exosomal proteins between HCC and HC groups; B: Hallmark pathway enrichment analysis of differentially expressed exosomal proteins between HCC and CHB groups; C: Hallmark pathway enrichment analysis of differentially expressed exosomal proteins between HCC and LC groups.

[0081] Figure 5 Figure 3: Identification of exosomal proteins associated with hepatocellular carcinoma based on the population; A: Venn diagram of differentially expressed proteins between HCC and LC, CHB, and HC groups; B: Heat map of 10 differentially expressed proteins; C: Box plot of expression levels of differentially expressed proteins among the four groups.

[0082] Figure 6 Figure 3. Establishment and evaluation of the ELISA method for detecting VCAM1 protein levels in plasma exosomes; A: Detection of VCAM1 in intact exosomes by immunofluorescence staining; B: Schematic diagram of the self-established ELISA method for detecting VCAM1 protein in plasma exosomes; C: Standard curve of ELISA method 1 in purified exosomes; D: Standard curve of ELISA method 1 in plasma; E: Correlation diagram of the detection results of ELISA method 1 in purified exosomes and plasma; F: Standard curve of ELISA method 2 in VCAM1 protein standard; G: Expression levels of VCAM1 protein in plasma exosomes detected by ELISA method 1 in each group of the validation cohort; H, Expression levels of plasma VCAM1 detected by ELISA method 2 in each group of the validation cohort.

[0083] Figure 7 Figure 3: Evaluation of the diagnostic value of plasma exosome VCAM1 protein for hepatocellular carcinoma; A: Expression levels of plasma exosome VCAM1 protein in each group of the validation cohort; B: Expression levels of plasma AFP in each group of the validation cohort; C: ROC of plasma exosome VCAM1 protein alone and in combination with AFP for differentiating HCC. DETAILED DESCRIPTION

[0084] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0085] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0086] Protein extraction

[0087] The plasma exosomes of healthy subjects and patients diagnosed with hepatocellular carcinoma were extracted using conventional exosome protein extraction methods in the art as the experimental materials of the present invention. The number of healthy subjects and patients diagnosed with hepatocellular carcinoma met the minimum statistical sample collection requirements and was therefore statistically representative.

[0088] Sample inclusion criteria:

[0089] Healthy people (HC): ① No tumor; ② No hyperlipidemia or hypertension; ③ No systemic inflammatory response; ④ No cysts, suspicious nodules, etc.

[0090] Chronic hepatitis B (CHB) patients: patients who have been HBsAg positive for more than 6 months.

[0091] Patients with liver cirrhosis (LC): confirmed by pathological biopsy or two imaging examinations (ultrasound combined with CT or MRI).

[0092] Hepatocellular carcinoma (HCC) patients: HCC patients with American Association for the Study of Liver Diseases (AASLD) clinical stages I, II, III, and IV.

[0093] The expression of plasma AFP (alpha-fetoprotein) was detected using a kit

[0094] The expression level of AFP in plasma was detected using an enzyme-linked immunosorbent assay kit (KA0202, Abnova, China) according to the manufacturer's instructions. The procedure was briefly as follows: (1) Plasma sample diluent and protein standard (AFP, KA0202, Abnova) were added to the reaction wells in sequence, incubated, and then washed; (2) Biotinylated detection antibody was added to each well, incubated, and then washed; (3) Avidin-horseradish peroxidase (HRP) was added to each well, incubated, and then washed; (4) 3,3′,5,5′-tetramethylbenzidine (TMB) was added to each well. Under the catalysis of HRP, 3,3′,5,5′-tetramethylbenzidine (TMB) produced a blue product, which turned yellow after the addition of acidic stop solution; (5) The reaction signal was read at 450 nm using a Sunrise™ plate reader (Tecan, Switzerland), and the AFP content in the plasma sample was quantified according to the standard curve.

[0095] Establishment of an ELISA method for detecting VCAM1 protein levels in plasma exosomes

[0096] The expression level of VCAM1 protein in exosomes was detected by ELISA method 1, as follows: (1) 100 μL rabbit anti-human CD63 monoclonal antibody (1:1000; ab68418, Abcam, UK) was coated in a 96-well ELISA plate (95029180, Thermo Fisher Scientific, USA), incubated at 4°C overnight, washed, and patted dry; (2) 200 μL of 5% skim milk powder solution containing 0.05% Tween-20 was added to each well, incubated at room temperature for 2 h, washed, and patted dry; (3) 100 μL of plasma sample and exosome standard were added to each well, incubated at 37°C for 2 h, and then washed and patted dry. Wash and pat dry the water droplets; (4) Add 100 μL of mouse anti-human VCAM1 monoclonal antibody (1:500; ab216035, Abcam, UK) to each well, incubate at 37°C for 2 h, wash and pat dry the water droplets; (5) Add 100 μL of HRP-conjugated goat anti-mouse IgG polyclonal antibody (1:1000; ab205719, Abcam, UK) to each well, incubate at 37°C for 1 h, wash and pat dry the water droplets; (6) Add 90 μL of TMB to each well, incubate at room temperature for 15 min, and then add 50 μL of stop solution (2M H2SO4) to each well; (7) Use a SunriseTM plate reader to read the reaction signal at 450 nm and quantify the VCAM1 protein content in the plasma sample according to the standard curve.

[0097] The expression level of VCAM1 protein in plasma was detected by ELISA method 2, as follows: (1) 100 μL rabbit anti-human VCAM1 polyclonal antibody (1:1000; 11444-1-AP, Proteintech, USA) was coated in a 96-well ELISA plate (95029180, Thermo Fisher Scientific, USA), incubated at 4°C overnight, washed, and patted dry; (2) 200 μL of 5% skim milk powder solution containing 0.05% Tween-20 was added to each well, incubated at room temperature for 2 h, washed, and patted dry; (3) 100 μL of plasma sample and VCAM1 protein standard (Ag1993, Proteintech, USA) were added to each well. tech, USA), incubated at 37°C for 2 h, washed, and patted dry; (4) 100 μL of mouse anti-human VCAM1 monoclonal antibody (1:500; ab216035, Abcam, UK) was added to each well, incubated at 37°C for 2 h, washed, and patted dry; (5) 100 μL of HRP-conjugated goat anti-mouse IgG polyclonal antibody (1:1000; ab205719, Abcam, UK) was added to each well, incubated at 37°C for 1 h, washed, and patted dry; (6) 90 μL of TMB was added to each well, incubated at room temperature for 15 min, and then 50 μL of stop solution (2 M H2SO4) was added to each well; (7) The reaction signal was read at 450 nm using a SunriseTM plate reader, and the VCAM1 protein content in the plasma samples was quantified according to the standard curve.

[0098] Immunofluorescence (IF) was used to identify the localization of VCAM1 protein on exosomes. The steps were as follows: (1) 20 μL of isolated and purified exosomes were mixed with 500 μL of 2% paraformaldehyde (80096618, Sinopharm, China) and dropped onto a coverslip for fixation for 20 min, followed by washing twice with PBS; (2) 5% normal horse serum (16050122, Gibco, USA) was added to block the exosomes for 30 min, followed by washing twice with PBS; (3) 400 mL of rabbit anti-human VCAM1 monoclonal antibody (1:250; ab134047, Abcam, UK) was added to the coverslip and incubated for 20 min, followed by washing twice with PBS; (4) 400 μL of goat anti-rabbit IgG (1:200, A27034, Thermo Fisher Scientific) was added to the coverslip. The cells were incubated for 20 min and washed twice with PBS. (5) 400 μL of anti-fluorescence quenching mounting solution (P10144, Thermo Fisher Scientific, USA) was added to the coverslips. The exosomes were observed and photographed under a laser scanning confocal microscope (GE Healthcare, USA). Exosomes treated with goat anti-mouse IgG alone were used as a control.

[0099] Example 1. Extraction and identification of plasma exosomes

[0100] The inventors first used size exclusion chromatography to extract plasma exosomes from 11 HCC, 9 LC, 10 CHB, and 10 HC individuals in the excavation population (Table 1). The quality of the extracted exosomes was assessed by nanoparticle size analysis, transmission electron microscopy, and immunoblotting. Nanoparticle tracking analysis showed that the enriched exosomes were approximately 30 to 150 nm in diameter, with a peak size of approximately 74.49 nm and a concentration of approximately 2.19 × 10 9 particles / mL( Figure 1 A in the middle), which is similar to the size of exosomes reported in the literature. Transmission electron microscopy analysis clearly shows that the size of plasma exosomes is between 40 and 150 nm ( Figure 1 The shape of the enriched exosomes is similar to that of the saucer or oval, which is consistent with the morphological characteristics of exosomes. Immunoblotting analysis showed that the enriched exosomes highly expressed specific markers FLOT1 and TSG101 ( Figure 1 In summary, these results indicate that high-quality plasma exosome particles were isolated and enriched.

[0101] Table 1 Clinical characteristics of the excavation cohort

[0102]

[0103]

[0104] Note: TNM, Tumor-Node-Metastasis; HBsAg, Hepatitis B virus surface antigen; AFP, Alpha-fetoprotein; HCC, Hepatocellular carcinoma; LC, Live cirrhosis; CHB, Chronic hepatitis B; HC, Healthy control.

[0105] Example 2: Proteomic Analysis of Plasma Exosomes

[0106] To map the proteomic landscape of hepatocellular carcinoma plasma exosomes, and to perform high-throughput mass spectrometry detection of the whole proteome using a shotgun strategy and data-independent acquisition (DIA) data acquisition method.

[0107] After quality control filtering, data normalization and protein qualitative and quantitative analysis ( Figure 2 A total of 1463 proteins were identified. Among them, 20 proteins are known characteristic markers of the exosome surface, and their expression abundance ranks first among all identified proteins ( Figure 2 In the HCC, LC, CHB, and HC groups, 1357, 1381, 1426, and 1386 proteins were identified, respectively ( Figure 2 There were 1296 common proteins among the four groups, and the 1463 identified proteins were compared with the exosome database ExoCarta and Vesiclepedia ( Figure 2 A comparison was performed in (D) and the results showed that 87.3% of the proteins were exosomal proteins, further suggesting that high-quality plasma exosomes were enriched.

[0108] Example 3: Identification of candidate protein markers associated with hepatocellular carcinoma in plasma exosomes

[0109] To ensure the reliability of the analysis results, only proteins detected in at least 7 samples were retained, and the KNN (k-Nearest Neighbor) method was used to fill in missing values. 885, 882, 1021, and 897 proteins were retained in the HCC, LC, CHB, and HC groups, respectively. Figure 3A), further, differential expression analysis was performed by retaining plasma exosome proteins ( Figure 3 (B, C, D).

[0110] To explore the changes in metabolic pathways associated with hepatocellular carcinoma, Hallmark was used to compare the signaling pathways enriched in differentially expressed proteins between HCC and HC, CHB, and LC groups. The results showed that the differentially expressed proteins between HCC and HC groups were mainly enriched in complement, epithelial-mesenchymal transition (EMT), heme metabolism, KRAS signaling, and PI3K / AKT / mTOR signaling pathways ( Figure 4 Middle A); The proteins that were differentially expressed between HCC and CHB groups were mainly enriched in complement, reactive oxygen species pathway, EMT, inflammatory response and bile acid metabolism pathways ( Figure 4 Middle B); The proteins that differed between HCC and LC groups were mainly enriched in pathways such as coagulation, complement, EMT, angiogenesis and inflammatory response ( Figure 4 (C) Hallmark pathway enrichment analysis revealed that complement and EMT pathways were enriched in all three groups. Recent studies have found that the complement system is associated with liver inflammation, fibrosis, carcinogenesis, and the development of HCC, but there are no reports on the interaction between exosomes and complement affecting HCC progression. Studies have shown that the EMT pathway also plays an important role in the progression of HCC. For example, EMT can lead to increased malignancy in hepatocytes associated with tumor cell invasion and metastasis. Among them, the EMT pathway enriched in the Hallmark pathway mainly involves vascular cell adhesion molecule 1 (VCAM1) and versican (VCAN). Studies have reported that exosomes secreted by HCC cells can promote HCC progression by mediating EMT through the MAPK / ERK signaling pathway; miR92a-3p in HCC-derived exosomes promotes HCC metastasis by mediating EMT by inhibiting PTEN and activating the Akt / Snail signaling pathway. These clues suggest that exosome proteomics-based research is of great significance in revealing the progression of HCC.

[0111] Finally, through the overlap analysis of the three groups of differentially expressed proteins, 10 common differentially expressed proteins (P < 0.05, fold change > 2 or < 0.5) were found ( Figure 5 A and B), the expression levels of VCAM1 protein in HCC, LC and CHB groups were significantly increased compared with those in HC group ( Figure 5 C), suggesting that it may have potential value as a diagnostic marker for hepatocellular carcinoma.

[0112] Example 4. Establishment and evaluation of an ELISA method for detecting VCAM1 protein levels in plasma exosomes

[0113] To evaluate the diagnostic value of VCAM1 protein in plasma exosomes as a candidate HCC marker, an independent HCC validation cohort was first recruited ( Table 2 ), including 22 HCC patients, 22 LC patients, 22 CHB patients, and 22 HC individuals.

[0114] Table 2 Clinical characteristics of the validation cohort

[0115]

[0116] Note: TNM, Tumor-Node-Metastasis; HBsAg, Hepatitis B virus surface antigen; AFP, Alpha-fetoprotein; HCC, Hepatocellular carcinoma; LC, Live cirrhosis; CHB, Chronic hepatitis B; HC, Healthy control.

[0117] Studies have reported that VCAM1 is a cell membrane-localized protein. Therefore, to evaluate the feasibility of ELISA as a method for detecting VCAM1 protein expression levels in plasma exosomes, immunofluorescence (IF) experiments were performed to identify its localization on exosomes. The results showed that VCAM1 protein labeled with VCAM1 antibody had a high abundance on the exosome membrane without disrupting the exosome membrane ( Figure 6 A in the middle, suggesting that its expression level can be detected by ELISA. Subsequently, an ELISA-based VCAM1 protein detection method was established, and its detection capability and scope of application were systematically evaluated ( Figure 6 First, an ELISA method was established using an antibody against the exosome-specific protein marker CD63 as a capture antibody and an antibody against VCAM1 as a detection antibody. Figure 6 (B) We evaluated its detection ability in exosomes purified from plasma and plasma respectively. The results showed that the R value of the standard curve of ELISA method 1 in plasma purified exosomes was 2 The value reached 0.985( Figure 6 C); R of the standard curve directly detected in plasma 2 The value also reached 0.984 ( Figure 6 More importantly, the results obtained by the two detection strategies were significantly positively correlated (R = 0.999, P < 0.001; Figure 6 E), and the OD value (450 nm) corresponding to the concentration gradient of exosomes purified from plasma was no more than 0.03 higher than that of the plasma sample, indicating that ELISA method 1 can detect exosome proteins directly from plasma samples without having to detect them from pre-purified exosomes.

[0118] In order to exclude the interference of free non-exosome VCAM1 protein in plasma, the capture antibody was replaced with VCAM1 antibody and an ELISA method was established2( Figure 6 The results showed that this method showed high specificity (R 2 =0.999; Figure 6 F). Subsequently, 8 HCC patients and 8 HC individuals were randomly selected from the validation population, and the expression levels of VCAM1 protein in plasma were detected by ELISA method 1 and ELISA method 2. The results of ELISA method 1 showed that the expression level of VCAM1 protein in plasma exosomes was significantly higher in the HCC group than in the HC group ( Figure 6 The results of ELISA method 2 showed that there was no statistical difference in the expression level of plasma VCAM1 protein between HCC and HC samples ( Figure 6 These results not only preliminarily confirmed that the ELISA method can be used for the quantitative detection of plasma exosome proteins, but also suggested that VCAM1 protein in plasma exosomes may serve as a potential diagnostic marker for hepatocellular carcinoma.

[0119] Example 5. Evaluation of the diagnostic value of VCAM1 protein in plasma exosomes for hepatocellular carcinoma

[0120] The receiver operating characteristic (ROC) reflects the balance between sensitivity and specificity. The area under the ROC curve is an important indicator of test accuracy. The larger the area under the ROC curve, the greater the diagnostic value of the test.

[0121] Sensitivity (true positive rate): The percentage of actual HCC patients who are correctly diagnosed as HCC patients according to the experimental criteria. The higher the sensitivity, the better, and the ideal sensitivity is 100%.

[0122] Specificity (true negative rate): The percentage of patients who are actually non-HCC but are correctly diagnosed as non-HCC patients according to the experimental criteria. The higher the specificity, the better, and the ideal specificity is 100%.

[0123] To further evaluate the diagnostic value of VCAM1 protein in plasma exosomes for hepatocellular carcinoma, the established ELISA method 1 was used to detect the expression level of VCAM1 protein in plasma exosomes in samples from the validation population (the hepatocellular carcinoma validation population in Table 2 included 22 HCC patients, 22 LC patients, 22 CHB patients, and 22 HC individuals). Simultaneously, the expression level of plasma AFP was also measured in parallel using an ELISA method (enzyme-linked immunosorbent assay kit, Abnova, Cat. No. KA0202). The results showed that the expression level of VCAM1 protein in plasma exosomes in the HCC group was significantly higher than that in the LC, CHB, and HC groups (P < 0.05; Table 2). Figure 7 Meanwhile, the expression level of plasma AFP in HCC group was significantly higher than that in LC, CHB and HC groups (P<0.05; Figure 7 Middle B).

[0124] Receiver operating characteristic (ROC) curves were constructed to further evaluate the ability of plasma exosome VCAM1 and plasma AFP to distinguish HCC patients from controls (LC patients, CHB patients, and HC individuals). The results showed that plasma exosome VCAM1 alone had an AUC of 0.944 for distinguishing HCC, with an optimal cutoff of 16.591 ng / mL and sensitivity and specificity of 0.864 and 0.985, respectively. Plasma AFP alone had an AUC of 0.885 for distinguishing HCC, with an optimal cutoff of 4.923 ng / mL and sensitivity and specificity of 0.955 and 0.742, respectively. These results preliminarily suggest that plasma exosome VCAM1 has high specificity, while plasma AFP has high sensitivity, demonstrating significant complementarity between the two. When plasma AFP and plasma exosome VCAM1 were combined, the AUC for distinguishing HCC reached 0.979, with sensitivity increased to 0.955 and specificity to 0.924, both exceeding the diagnostic capabilities of either assay alone. The above results suggest that VCAM1 protein in plasma exosomes can be used as a candidate plasma marker for the diagnosis of HCC, and it may have a higher diagnostic value when used in combination with AFP (Table 3, Figure 7 Middle C).

[0125] Table 3: Diagnostic ability of plasma AFP and plasma exosome VCAM1 detection alone and in combination for HCC

[0126]

[0127] Note: AFP, Alpha-fetoprotein; HCC, Hepatocellular carcinoma; AUC, Area under the curve; CI, Confidence interval.

[0128] Similarly, based on the marker combination of the present invention, the detection reagents and detection kits further developed can also accurately indicate hepatocellular carcinoma with mild clinical symptoms that is difficult to detect, and have extremely high medical and practical value.

[0129] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present invention. Throughout this specification, the schematic representations of these terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0130] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Any of the following uses of a biomarker or a substance for detecting the biomarker: A1) Use in the preparation of a product for the diagnosis of hepatocellular carcinoma; A2) Use in the preparation of a product for screening for hepatocellular carcinoma; A3) Use in the preparation of a product for assessing the risk of hepatocellular carcinoma; A4) Use in the preparation of a product for the prognosis assessment of hepatocellular carcinoma; A5) Use in the preparation of a product for identifying and distinguishing hepatocellular carcinoma from cirrhosis; A6) Use in the preparation of products for identifying and distinguishing hepatocellular carcinoma from chronic hepatitis B; The biomarker is any of the following: B1) VCAM1 protein in plasma exosomes and AFP in plasma; B2) VCAM1 protein in plasma exosomes.

2. The use according to claim 1, characterized in that The substance is a reagent for detecting the content of VCAM1 protein and a reagent for detecting the content of AFP; or the substance is a reagent for detecting the content of VCAM1 protein.

3. The use according to claim 1 or 2, characterized in that The substances for detecting the biomarkers include substances for detecting the biomarkers by enzyme-linked immunosorbent assay, radioimmunoassay, immunoprecipitation, immunoblotting, high performance liquid chromatography, capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence, colloidal gold immunoassay, fluorescent immunochromatography, surface plasmon resonance or immuno-PCR.

4. The use according to claim 2, characterized in that The reagent for detecting the content of VCAM1 protein includes VCAM1 protein and an antibody that binds to VCAM1 protein; the reagent for detecting the content of AFP includes AFP and an antibody that binds to AFP.

5. Use of a kit for detecting the content of VCAM1 protein in plasma exosomes and the content of AFP in plasma in any of the following: C1) Use in the preparation of products for diagnosis of hepatocellular carcinoma; C2) Use in the preparation of a product for hepatocellular carcinoma screening; C3) Use in the preparation of a product for assessing the risk of hepatocellular carcinoma; C4) Use in the preparation of a product for the prognosis assessment of hepatocellular carcinoma; C5) Use in the preparation of a product for identifying and distinguishing hepatocellular carcinoma from liver cirrhosis; C6) Use in the preparation of products for identifying and distinguishing hepatocellular carcinoma and chronic hepatitis B.

Images