Application of a biomarker for early screening of colorectal adenoma and colorectal cancer

Abstract

The present invention belongs to the technical field of tumor molecular biology, and specifically relates to the application of a biomarker for early screening of colorectal adenoma and colorectal cancer. The biomarker is clusterin. The present invention analyzes serum samples from normal healthy people, hyperplastic polyps, and adenomatous polyps by weak cation exchange magnetic beads (WCX-MB) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), selects differential polypeptides in significantly differentially expressed peaks, and identifies corresponding differential proteins by high-throughput sequencing, thereby exploring potential biological markers for early carcinogenesis of intestinal polyps. The biomarker provided by the present invention has good diagnostic efficacy for adenoma and colorectal cancer, which is significantly better than traditional tumor markers, and is of great significance for early screening and early diagnosis of colorectal tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor molecular biology, and specifically relates to the application of a biomarker for early screening of colorectal adenoma and colorectal cancer. Background Art

[0002] Colorectal cancer (CRC), also known as colorectal cancer, is a malignant tumor that poses a major threat to human health. Colorectal cancer screening methods are divided into invasive and non-invasive examinations, of which non-invasive examinations include stool examinations and imaging examinations. Currently, the detection tools used for stool include the guaiacum fecal occult blood test (gFOBT), fecal immunochemical test (FIT), and a new type of stool DNA test. Fecal testing has the advantages of being cheap and easy to operate, and can reduce the mortality rate of colorectal cancer. However, studies have found that the specificity and sensitivity of stool testing are low, and it is easily interfered by diet and drugs, resulting in false positive results.

[0003] Imaging examination mainly refers to colon CT imaging technology, namely CT simulated colonoscopy, a non-invasive imaging diagnostic technology that uses computer technology to reconstruct the intestine in three dimensions and provide virtual images of the inside of the colon, thereby diagnosing and screening intestinal tumors. However, this technology has many problems, such as intestinal cleaning preparation, high cost, cumbersome operation, false positives, high radiation hazards, and low acceptance by the population. Therefore, it is not recommended for intestinal tumor screening in the population at present. Some other imaging examinations, including magnetic resonance imaging (MRI) and positron emission tomography (PET) / CT simulated colonoscopy, can also be used for auxiliary diagnosis of colon cancer. These two methods are only important components of preoperative evaluation and can provide valuable information for the TNM staging of patients with colorectal cancer. They can be regarded as a supplement to current examination methods, but there are currently no guidelines recommending them for CRC screening.

[0004] There are two types of invasive examinations: colonoscopy and sigmoidoscopy. Both allow direct observation under the endoscope and can obtain pathological specimens. Colonoscopy, as an efficient and accurate medical method, has become the preferred method for early diagnosis and screening of colorectal cancer. However, colonoscopy is invasive, and due to many factors such as patient discomfort during the operation, sufficient intestinal preparation in the early stage, the high cost of painless colonoscopy, and low public acceptance, it is difficult to popularize colonoscopy as a routine screening method for colorectal cancer at this stage. Therefore, it is necessary to find ideal biological markers.

[0005] Biomarkers are the main tools for cancer diagnosis, patient prognosis and treatment selection. The most widely used biomarkers in clinical practice are serum biomarkers. Carcinoembryonic antigen (CEA) is generally considered to be a serum biomarker for CRC. Similar to CEA, carbohydrate carcinoembryonic antigen 199 (CA19-9) and carbohydrate carcinoembryonic antigen 242 (CA24-2) are also gastrointestinal tumor markers. However, these biomarkers have poor specificity and sensitivity in colorectal cancer screening. For example, elevated CEA is not only manifested in colorectal cancer, but also in many cancers such as breast cancer and lung cancer, and even in some benign diseases. In addition, current serum biomarkers have the problem of poor specificity and sensitivity. Summary of the invention

[0006] In order to solve the problem of poor specificity and sensitivity of existing biomarkers, the present invention proposes to seek specific serum biomarkers with the molecular definition of early carcinogenesis of "adenoma-cancer" from the inflammation-polyp-neoplasia pathway, which can be used in the early screening of colorectal adenomas and even intestinal polyps to stop the possibility of progression to colorectal cancer. The present invention provides an application of a serum biomarker, i.e., a biomarker for early screening of colorectal adenomas and colorectal cancer. The serum biomarker, i.e., a biomarker for early screening of colorectal adenomas and colorectal cancer, has the advantages of being cheap, easy to operate, highly acceptable, highly specific, highly sensitive, and early in the stage of disease detection. To achieve the above purpose, the present invention adopts the following technical scheme:

[0007] The application provided by the present invention is the application of a biomarker for early screening of colorectal adenoma and colorectal cancer in the preparation of a product for screening or diagnosing colorectal adenoma and / or colorectal cancer, wherein the biomarker is any one of clusterin CLU and albumin ALB.

[0008] Preferably, the biomarker for early screening of colorectal adenoma and colorectal cancer is clusterin CLU. Clusterin, apolipoprotein J, sulfated glycoprotein 2, complement lysis inhibitor or testosterone-suppressed prostate information 2 gene. The present invention has confirmed that CLU protein is expressed most highly in healthy control populations, followed by hyperplastic polyp patients, and the lowest serum expression level in adenoma group polyp patients, that is, CLU protein has a decreasing gradient in the process of colorectal polyps progressing to colorectal adenomas, and when the CLU protein concentration is lower than 2895.78pg / mL, the possibility of diagnosing adenomatous polyps is high. Among them, the English name of clusterin is Clusterin, CLU; the English abbreviation of apolipoprotein J is APOJ; the English abbreviation of sulfated glycoprotein 2 is SGP2; the English abbreviation of complement lysis inhibitor is CLI; the English abbreviation of testosterone-suppressed prostate information 2 gene is TRPM-2.

[0009] Preferably, the clusterin CLU is a serum marker or a marker in pathological tissue. The present invention is based on collecting serum samples and polyp tissue samples from patients with hyperplastic polyps and adenomatous polyps to analyze and verify the significance and value of clusterin CLU as a biomarker.

[0010] Preferably, the product includes a kit, a detection reagent or an antibody chip. The CLU-MB-WCX magnetic bead kit is used for protein spectrum analysis, and the CLU antibody is coated on the magnetic beads, eliminating the incubation stage of the antibody and the magnetic beads, which is convenient for mass spectrometry detection and analysis. CLU-related detection reagents can carry corresponding fluorescent labels while carrying CLU antibodies, which is convenient for intuitive analysis of changes in the content of CLU protein under a microscope. CLU-related antibody chips can contain CLU antibodies while containing antibodies for other biomarkers, which is convenient for saving the use of samples during sample detection, and jointly determining the expression of all biomarkers under unified processing at the same time.

[0011] The kit includes a colorectal adenoma and / or colorectal cancer screening or diagnosis kit. The CLU diagnostic concentration analyzed by the present invention is set numerically during sample data analysis, which can further shorten the screening and diagnosis of colorectal adenoma and / or colorectal cancer.

[0012] Preferably, the kit comprises an antibody or antibody fragment that specifically binds to the clusterin CLU. Antibodies are prepared according to the sequence of the CLU protein and / or specific peptide segment to obtain CLU-specific antibodies or antibody fragments.

[0013] The detection reagent includes a reagent for detecting serum biomarkers or a reagent for detecting markers in pathological tissues. The present invention mainly analyzes biomarkers in serum samples and polyp pathological tissues of patients with intestinal polyps.

[0014] Preferably, the reagents for serum biomarkers or reagents for detecting markers in pathological tissues are ELISA reagents, immunoblotting reagents, immunofluorescence reagents, immunohistochemical reagents, protein chips or mass spectrometry detection reagents. ELISA reagents can specifically determine the absolute expression concentration of CLU in serum or tissues, and specific reagents include CLU detection antibodies, HRP-labeled streptavidin, color TMB, etc.; immunoblotting reagents can specifically determine the relative expression concentration of CLU in serum and tissues, and specific reagents include RIPA lysate, 10% APS, TBST eluent, etc.; immunohistochemical reagents can intuitively show the expression and location of CLU with images, and specific reagents include 4% paraformaldehyde, xylene, DAB color development solution, etc.; protein chips or mass spectrometry detection reagents can determine the specific protein types in serum or tissue samples, and specific reagents include monoclonal antibodies, magnetic beads, 50% acetonitrile, etc.

[0015] Preferably, the reagents for detecting biomarkers in serum are PCR detection reagents, in situ hybridization reagents, or sequencing reagents. PCR detection reagents can determine the relative expression of CLU in tissue samples from the RNA level, and specific reagents include SYBR Green, RT Enzyme mix, primers, etc.; in situ hybridization reagents can determine the expression of CLU in situ at a fixed point in a tissue sample, and specific reagents include pepsin, 1% paraformaldehyde / 0.1M PBS, hybridization solution, etc.

[0016] Preferably, the reagent for detecting serum biomarkers is an ELISA reagent. ELISA can quickly and conveniently determine the CLU expression content in a large number of serum or tissue samples at the same time and with a uniform treatment level.

[0017] The antibody chip can fix a variety of antibodies including CLU that specifically bind to different antigens on a carrier at a high density, thereby detecting the antigens specifically bound thereto in serum or tissue samples.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention provides an application of a biomarker for early screening of colorectal adenoma and colorectal cancer. The present invention attempts to find at least one protein biomarker by analyzing differential peptides in serum samples of normal healthy people, patients with hyperplastic polyps, and patients with adenomatous polyps, which can accurately and sensitively reflect the stage of the disease in the process of polyps progressing to adenomas or even colorectal cancer according to its expression concentration, and draws a ROC curve to identify its diagnostic value.

[0020] 2. The present invention uses protein spectrum, high-throughput sequencing, ELISA and other technologies, with normal people, hyperplastic intestinal polyps, adenomatous intestinal polyps patients as the subjects, to detect the polyp tissue samples and serum of patients, and analyze the differences in protein expression in tissue and serum samples of different populations. At the same time, ELISA, Western Blotting and other technologies are used to verify the potential biomarkers of polyp transformation to precancerous lesions. The test results will assist clinicians in more reasonable diagnosis and treatment, so as to achieve the purpose of accurate detection and precise treatment.

[0021] Proteomics-based disease research is considered a dynamic and innovative tool that can provide more detailed information than other high-throughput technologies to confirm, supplement or provide relevant data. In addition, the analysis of proteins and metabolites that are the real effector molecules in biological systems can better reflect the physiological changes of the body. Therefore, the application of proteomics technology can reveal changes in protein expression and / or protein processing in the body, which can often serve as molecular markers and may even become the basis of powerful screening tools for diseases.

[0022] The basic principle of mass spectrometry (MS) is to ionize the substance to be tested, separate it according to the mass-to-charge ratio of the ion, and measure the intensity of various ion spectrum peaks to achieve the purpose of analyzing the disease. The three ionization methods currently used to analyze peptides and proteins mainly include electrospray ionization (ESI), matrix-assisted laser desorption / ionization (MALDI) and surface-enhanced laser desorption / ionization (SELDI). These ionization sources are usually combined with time-of-flight (TOF) or quadrupole analyzers to infer the detailed structural characteristics of the peptide by analyzing the mass of the generated fragments. Among them, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a new technology developed in the 1980s. It has been widely used in the detection of three major molecules such as sugars, nucleic acids, and proteins. The structural analysis and molecular weight determination of biological macromolecules and synthetic polymers have become one of the core goals of current proteomics research. This technology has the advantages of high accuracy in calculating mass-to-charge ratio, low cost, high affinity, and good reproducibility. It can not only analyze cells and tissues, but also analyze samples such as powders, solutions, and membrane tissues. It has the advantages of high sensitivity, high mass accuracy, and fast analysis time. Clinically, the samples used for mass spectrometry analysis mainly include biological fluids such as urine, saliva, serum, and plasma, biological tissues, and cell culture media. Since the collection procedure of biological fluids is simpler than others, such as easy to obtain, low cost, and low invasiveness, general mass spectrometry analysis often uses biological fluids, which is conducive to screening non-invasive biomarkers and ultimately achieving the goal of screening cancer. Mass spectrometry proteomics can easily detect and analyze the proteomic information of hundreds of candidate disease markers. Therefore, it has now become one of the most common discovery and verification tools in the field of life sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the primary mass spectrometry analysis of serum samples from normal healthy people (NC, green, 26 cases), hyperplastic polyp patients (IP, blue, 13 cases), and adenomatous polyp patients (AP, red, 17 cases) in the present invention. (AB) The sum of spectra with a mass range of 1 to 10 kDa obtained from NC, IP and AP samples. (C) The binary graph of the sample in component analysis, with the two most different peaks (m / z: 1866.7Da, 5066.2Da). (D) 3D graph of NC, IP and AP samples in principal component analysis component analysis. (Note that the m / z value on the horizontal axis represents the mass-to-charge ratio, which is the relative molecular weight of the protein or polypeptide, measured in Da (Dalton). The intensity on the vertical axis represents the relative content of the protein or polypeptide, that is, the relative abundance in the sample).

[0024] Figure 2It is a gel chromatographic separation diagram of the serum protein polypeptide mixture of the six differential polypeptides screened out in the present invention; wherein, a is a secondary mass spectrum diagram with a mass-to-charge ratio of 2865.37; b is a secondary mass spectrum diagram with a mass-to-charge ratio of 2191.88; c is a secondary mass spectrum diagram with a mass-to-charge ratio of 1619.15; d is a secondary mass spectrum diagram with a mass-to-charge ratio of 1351.03; e is a secondary mass spectrum diagram with a mass-to-charge ratio of 1194.35; and f is a secondary mass spectrum diagram with a mass-to-charge ratio of 2663.05.

[0025] Figure 3 It is the standard curve of CLU protein in the present invention, wherein the abscissa represents the concentration of CLU protein (unit: pg / mL); the ordinate represents the absorbance value (OD value) at 450 nm.

[0026] Figure 4 The expression levels of CLU protein in the serum of each group of people in the internal validation stage of the present invention, wherein the horizontal axis represents the group, wherein NC represents the healthy control group; IP represents the hyperplasia group; AP represents the adenoma group; the vertical axis represents the concentration of CLU protein (unit: pg / mL). *** represents P≤0.001.

[0027] Figure 5 It is the CLU protein expression level in the serum of the three groups of people in the screening stage of the present invention.

[0028] Figure 6 The Western blotting technology in the present invention verifies the expression level of CLU protein in the control group and adenoma group, wherein NC represents the healthy control group; AP represents the adenoma group; -actin is the internal reference protein.

[0029] Figure 7 The expression levels of CLU protein in the serum of the three groups of people in the screening stage combined with the verification stage in the present invention, wherein the horizontal axis represents the group, wherein NC represents the healthy control group; IP represents the hyperplasia group; AP represents the adenoma group; the vertical axis represents the concentration of CLU protein (unit: pg / mL). *** represents P≤0.001.

[0030] Figure 8 It is the ROC curve diagram of CLU protein in the present invention, wherein the abscissa represents 1-specificity; and the ordinate represents sensitivity. DETAILED DESCRIPTION

[0031] The following will be combined with the specific implementation of the present invention to clearly and completely describe the technical solution in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0033] The instruments and reagents used in the following examples of the present invention are as follows:

[0034] 1. Instrument

[0035] Mass spectrometer: Orbitrap, Thermo Scientific; liquid phase EasynLC: 1200, Thermo Scientific; mass spectrometer column; C181.9 mm 150μm×120mm, Thermo Scientific; mass spectrometer pre-column; C183 mm100μm×20mm, Thermo Scientifi; high-speed centrifuge: Pico 17Centrifuge, Thermo Scientific; hot dry concentrator: SC210A, Thermo Scientific; NanoDrop: 2000C, Thermo Scientific; pipette: 2.5\10\20\200\1000, Eppendorf.

[0036] 2. Reagents

[0037] Formic acid (FA); 500 mL, Sigma, RT; H 2 O: 596mL, Wahaha, RT; ammonia (HPLC grade): 500mL, Sigma, 4℃; acetonitrile (ACN); 4L, Sigma, RT; disposable pipette tips; 0.5-10mL, 2-200mL, 00-1000mL, Axygen; centrifuge tube: 1.5mL, Axygen; loading vial; Thermo Scientific; 96-well plate: Thermo Scientific; MB-WCX magnetic bead kit, Bruker Daltonics, Germany.

[0038] 3. Capillary HPLC parameter setting

[0039] Each sample was separated using an Easy nL C 1200nL flow rate HPLC liquid phase system. Buffer A was 0.1% formic acid in water, and buffer B was 0.1% formic acid in acetonitrile. The column was balanced with 95% A solution. The sample was loaded from the autosampler onto the mass spectrometry pre-column C18trap column (C183mm 0.10×20mm), and then separated by the analytical column C18 column (C181.9mm 0.15×120mm) at a flow rate of 600nL / min. The relevant liquid phase gradient is shown in Table 1:

[0040] Table 1 Liquid chromatography elution gradient parameters

[0041] Time (min) <![CDATA[A(0.1%FA / H 2 O)]]> B(0.1%FA / ACN) Flow rate (nL / min) 0 92％ 8％ 600 8 88％ 12％ 600 48 77％ 23％ 600 68 64％ 36％ 600 69 5％ 95％ 600 78 5％ 95％ 600

[0042] 4. Mass spectrometry identification parameter settings

[0043] Each sample was separated by capillary high performance liquid chromatography and then analyzed by mass spectrometry using an Orbitrap Fusion mass spectrometer (ThermoSceientific). The main parameter settings are shown in Table 2:

[0044] Table 2 Mass spectrometer parameter settings

[0045]

[0046]

[0047] Example 1

[0048] 1. Sample collection

[0049] I. A total of 90 patients with simple colorectal polyps admitted to the Department of Gastroenterology, the First Affiliated Hospital of Xi'an Medical College from January 2021 to December 2023 were selected and divided into two groups according to pathological diagnosis: 28 cases in the hyperplasia group and 62 cases in the adenoma group. At the same time, normal healthy people without colorectal polyps whose endoscopic mucosa was basically normal during the same period were collected as the control group (normal healthy group / control group), and serum and tissue samples were collected from each group of people.

[0050] The general clinical data collected for the above-mentioned simple colorectal polyps include gender, age, BMI, HP infection, smoking history, drinking history, size, location, number of polyps and other indicators.

[0051] Among them, HP infection: HP infection is considered to be present if one of the carbon 13 breath test, carbon 14 breath test, or histopathological examination is positive.

[0052] Smoking history: The smoking index (SI) was used for assessment. SI ≥ 200 indicated a history of smoking. The calculation formula = number of cigarettes smoked per day × number of years of smoking.

[0053] Drinking history: A drinking history was considered to exist if the average daily ethanol intake was ≥40 g for men and ≥20 g for women, and the drinking history was more than 5 years.

[0054] BMI classification: According to the guidelines proposed by the China Obesity Working Group, BMI is divided into four categories: underweight (<18.5), normal (18.5≤BMI<24.0), overweight (24≤BMI≤28.0) and obese (BMI>28).

[0055] Polyp location: According to the location of polyps, it is divided into left colon, right colon and whole colon. The left colon includes the splenic flexure, sigmoid colon, descending colon and rectum; the right colon includes the hepatic flexure, cecum, ascending colon and transverse colon; the whole colon refers to polyps in both the left and right colon.

[0056] Polyp size: According to the maximum diameter of the polyp, it is divided into four groups: ≤5mm, 5-10mm, 10-15mm and >15mm.

[0057] Number of polyps: A single polyp is defined as only one polyp found during colonoscopy; multiple polyps refer to ≥2 polyps found under the microscope, and the pathological type of multiple polyps is based on the type with the lowest degree of differentiation.

[0058] During bowel preparation, use polyethylene glycol electrolyte powder (trade names and Content 68.56g / bag, national medicine standard H20030827, produced by Shenzhen Vanward Pharmaceutical Co., Ltd.). All patients with colorectal polyps take 1 bag of 1L drug solution between 16:00-18:00 in the afternoon on the day before the examination. Patients examined in the morning take 2 bags of 2L drug solution between 4:00-6:00 in the morning on the day of the examination. Patients examined in the afternoon will take 2L solution prepared by 2 bags of drug between 6:00-8:00 in the morning on the day of the examination. The day before the examination, it is recommended that patients eat an easily digestible semi-liquid diet and fast after dinner, but can drink water. On the day of the examination, patients need to fast and do light activities during bowel preparation to promote intestinal peristalsis.

[0059] II. Tissue sample collection: After judgment by experienced endoscopists and pathological examination results, intestinal polyp tissues less than 2 cm in length were obtained from patients with hyperplastic polyps and adenomatous polyps using disposable biopsy forceps under endoscopy and placed in EP tubes. One part was stored at -80°C, and the other part was fixed with 4% paraformaldehyde to prepare paraffin specimens.

[0060] III. Collection of serum specimens: 5 mL of blood was collected from the patients with hyperplastic polyps, adenomatous polyps and normal healthy control group in the early morning on an empty stomach and placed in a collection tube. The blood was centrifuged at 2000 rpm / min for 15 min, and the upper layer of serum was collected and divided into Eppendorf tubes to obtain serum samples, which were stored in a -80°C refrigerator.

[0061] 2. Reagent Preparation

[0062] 1. ELISA reagent preparation

[0063] (1) Washing solution (1×): If crystals are precipitated in the washing solution (20×), heat it at 37°C until all the crystals are dissolved. Dilute it at a dilution ratio of 1:20: For example, take 30 mL of concentrated washing solution (20×) and add 570 mL of ultrapure water or deionized water to obtain washing solution (1×).

[0064] (2) Detection antibody (1×): Centrifuge immediately before opening the lid and dilute at a ratio of 1:100. Prepare the total volume required for the experiment (100 μL / well) according to the pre-calculated amount before dilution. Prepare 0.1-0.2 mL more during actual preparation. For example, add 10 μL of detection antibody concentrate (100×) to 990 μL of antibody diluent and mix gently.

[0065] (3) HRP-labeled streptavidin (1×): Centrifuge immediately before opening the lid and dilute at a ratio of 1:100. Prepare the total amount required for the experiment (100 μL / well) according to the pre-calculated amount before dilution. Prepare 0.1-0.2 mL more during actual preparation. For example, add 990 μL of antibody diluent to 10 μL of HRP-labeled streptavidin concentrate (100×) and mix gently.

[0066] (4) Samples to be tested: Different samples are diluted with corresponding sample diluents. If the sample test value exceeds the highest range of the calibration curve, the sample can be diluted to a certain extent before the experiment is carried out to make the sample test value within the calibration curve range. The dilution multiples of different samples need to be optimized by yourself.

[0067] Serial dilution of standard: for example, use 2mL PT1-ef specimen diluent to reconstitute the standard.

[0068] 2. Preparation of WB experimental reagents

[0069] (1) 1× TBST buffer: weigh 2.4228 g TRIS, 8.184 g NaCl, add ddHO 2 O to 1L, stir magnetically to dissolve, then add 500L of Tween-20 and store at room temperature.

[0070] (2) 1× electrophoresis buffer: weigh 3 g TRIS, 14.4 g Glycine, 1 g SDS, add ddH 2 O to 1 L, stir magnetically to dissolve, and store at room temperature.

[0071] (3) 1× transfer buffer: weigh 3 g TRIS, 14.4 g Glycine, add ddHO 2 O was diluted to 800 mL, stirred magnetically to dissolve, and 200 mL of methanol was added and stored at room temperature.

[0072] (4) 10% ammonium persulfate (APS): weigh 1 g of APS powder and add ddH 2 O to 10 mL, dissolve and dispense into 1.5 mL centrifuge tubes, and store at -20°C.

[0073] (5) 10% SDS (100 mL sodium dodecyl sulfate): weigh 10 g SDS and add ddH 2 O to 100 mL and store at room temperature.

[0074] (6) Solution A (100 mL of 30% acrylamide stock solution): weigh 0.8 g of N,N-methylenebisacrylamide and 29.2 g of acrylamide, take 100 mL of ddHO 2 O, mix well, filter, and store in a 4°C refrigerator away from light for later use.

[0075] (7) Solution B (100 mL of 4× separation gel buffer): weigh 18.17 g of Tris base, 80 mL of ddHO 2 O, dissolve Tris base, add 4 mL 10% SDS, adjust pH to 8.8, make up to 100 mL, and store at 4°C.

[0076] (8) Solution C (100 mL of 4× stacking gel buffer): weigh 12.12 g of Tris base and 80 mL of ddHO. 2 O, dissolve Tris base, add 4 mL 10% SDS, adjust the pH to 6.8, and finally make up to 100 mL and store at 4 °C for future use.

[0077] (9) 5% blocking solution (100 mL): Weigh 5 g of skim milk powder, add 100 mL of 1× TBST, mix by magnetic stirring, and store in a refrigerator at 4°C.

[0078] 3. Sample Testing

[0079] 1. MALDI-TOF mass spectrometry analysis

[0080] 5 μL of serum samples collected from the healthy control group, hyperplasia group, and adenoma group were mixed with 10 μL of MB-WCX and 10 μL of MB-WCX binding buffer in the MB-WCX magnetic bead kit, and then 10 μL of WCX-MB magnetic beads were added and mixed by pipetting up and down. After incubation at room temperature for 5 minutes, the test tube was placed in a magnetic separator and the beads were collected until the supernatant was clear for 1 minute. The supernatant was removed and the magnetic beads were washed three times with 100 μL of MB-WCX wash buffer in the kit. Then, the peptide part was eluted from the magnetic beads with 5 μL of MB-WCX elution buffer and 5 μL of MB-WCX stabilization buffer. Finally, 1 μL of eluent and 1 μL of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile were added to the MALDI-TOF MS target, and 0.5% trifluoroacetic acid was added twice to the MALDI anchor surface. To evaluate reproducibility, all samples were performed in triplicate.

[0081] 2. ClinProTools analysis

[0082] All targets were analyzed using an Autoflex III MALDI-TOF MS (Bruker) and FlexControl software (version 3.0, Bruker). Mass calibration was performed using a peptide and protein standard calibration mixture with a mass range of 1000–10,000 Da. All assays were performed in a blinded manner and included serum analysis of three groups: healthy controls, hyperplasia, and adenoma. All serum data were analyzed using Flex analysis software (version 3.0; Bruker), and peptide pattern recognition and identification were performed using ClinProTools software (version 2.2; Bruker).

[0083] 3. Peptide identification

[0084] The differential peptides identified by ClinProTools software were analyzed by liquid chromatography-mass spectrometry (LC-MS) using a NanoAcquity UPLC (Waters, USA) and an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, USA). A C18 column ( C18, 3 μm, 0.10 × 20 mm, nanoAcquity) to capture 20 μL of sample and flow continuously at 600 nL / min for 3 min. The sample was then loaded onto the analytical column ( C18, 1.9 μm, 0.15 × 120 mm, nanometer). Mobile phase A was a solution composed of 5% acetonitrile and 0.1% formic acid, and mobile phase B was a solution composed of 95% acetonitrile and 0.1% formic acid. The column temperature was maintained at 35 °C. The LTQ OrbitrapXL mass spectrometer was operated in data-dependent mode, which can automatically switch between MS and MS / MS acquisition. Full scan mass spectra were acquired at m / z 300 with 2 microscans (m / z 300–1400) using a Q Exactive mass spectrometer with a mass resolution of 70,000. Ten consecutive LTQ-MS / MS scans were subsequently performed. Dynamic exclusion was set to 2 repeat counts, a repeat time of 18 s, and an exclusion time of 80 s. In MS / MS, the precursor ion was activated at 25% of the normalized collision energy with a default activation q of 0.25. The acquired data will be searched and identified in the Uniprot human protein sequence database (https: / / www.uniprot.org / ).

[0085] 4. WB experiment

[0086] 4.1 Experimental operation of tissue protein extraction

[0087] (1) Take 40 mg of fresh tissue from hyperplastic and adenomatous patients and mince it into a 1.5 mL EP tube.

[0088] (2) Prepare RIPA lysis buffer and place on ice. Based on the ratio of about 200 μL of lysis buffer for every 20 mg of tissue, mix solution A (lysis buffer), solution B (protease), and solution C (phosphatase inhibitor) in a sterile EP tube at a ratio of 1% solution A, 1% solution B, and 1% solution C to make solution D.

[0089] (3) Add the dissolving solution to the EP tube and use a glass homogenizer to grind the tissue blocks until they are completely dissolved. The shaking conditions are: 1200 rpm, 5 cycles, 40 seconds per cycle, with 10 seconds interval between cycles.

[0090] (4) Place the EP tube on ice and continue lysis for 1 hour.

[0091] (5) Centrifuge at 12,000 rpm for 20 min at 4°C.

[0092] (6) Carefully transfer the supernatant to another clean EP tube.

[0093] (7) Protein quantification was performed using the BCA quantification method (protein concentration was determined by Nano Drop).

[0094] (8) Mix 5× Loading Buffer and protein in a ratio of 1:4, boil for 5 minutes, and store at 4°C until use.

[0095] 4.2 Preparation of SDS-PAEG gel

[0096] (1) Clean the glass plate with detergent and dry it in an oven. Then carefully fix the glass plate on the glue rack.

[0097] (2) Prepare the separation gel according to the formula in Table 3. After all components are fully mixed, pour the gel immediately to ensure that no bubbles are generated. When the gel level reaches about 2 cm above the upper edge of the glass plate, stop pouring the gel. Then use a 1 mL pipette to slowly and gently add 1 mL of anhydrous ethanol to the upper edge of the glass plate, and then flatten the separation gel.

[0098] Table 3 Preparation of SDS-PAEG gel

[0099]

[0100] (3) After standing for 30 minutes, tilt the gel-making rack to pour out the anhydrous ethanol and absorb the remaining anhydrous ethanol with clean absorbent paper.

[0101] (4) Prepare the stacking gel according to the recipe in Table 3. Mix the components thoroughly to avoid bubbles, and then pour the mixture into the upper layer of the separation gel until the liquid level exceeds the upper edge of the glass plate. Next, slowly insert the comb and let the gel stand at room temperature for 30 minutes.

[0102] (5) Once the gel has solidified, the sample can be loaded immediately or placed in ultrapure water and stored at 4°C for 3 days.

[0103] 4.3 Electrophoresis

[0104] (1) Assemble the prepared gel into the electrophoresis tank and add electrophoresis buffer to the inner and outer tanks. Slowly pull out the comb to ensure the flatness of the gel.

[0105] (2) Quickly and carefully add the prepared protein sample to the sample well. Add 3 μL of protein marker to the wells on the left and right sides respectively.

[0106] (3) Insert the electrodes and ensure that the positive and negative electrodes are connected correctly. First, set a constant voltage of 80V for electrophoresis for about 30 minutes until the junction of the concentrated gel and the separation gel. Then switch to a constant voltage of 110V and continue electrophoresis until the blue loading buffer runs to the bottom of the gel or the target protein runs to the middle of the gel.

[0107] 4.4 Transfer (wet transfer)

[0108] (1) Cut a piece of PVDF membrane to make it slightly larger than the size of the gel. Soak the PVDF membrane in anhydrous methanol for about 60 seconds to activate it, and then soak it in pre-cooled transfer solution. At the same time, soak the sponge and filter paper in the pre-cooled transfer solution.

[0109] (2) Remove the glass plate from the electrophoresis tank and carefully separate the two plates. Take out the gel and place it in the operating tray containing transfer solution to keep it moist. Use a blade to cut off the concentrated gel.

[0110] (3) Open the transfer clip and first place the black plate (negative electrode) in the transfer solution. Then place the sponge, filter paper, glue, PVDF membrane, filter paper, and sponge in order from bottom to top. Note that when placing the PVDF membrane, you can pour a small amount of transfer solution on the glue to avoid bubbles. Clamp the transfer clip.

[0111] (4) Place the transfer clip into the transfer rack, making sure the black side of the transfer clip is aligned with the black side of the transfer rack. Pour the pre-cooled transfer solution into the transfer clip. Insert the positive and negative electrodes and connect the power supply. Adjust the current to 250mA and transfer the membrane for about 2.5 hours at a constant current. To prevent the transfer solution from overheating, place the electrotransfer chamber in an ice box and cover the area around the electrotransfer chamber with crushed ice.

[0112] 4.5 Blocking and Antibody Incubation

[0113] (1) After the transfer is completed, take out the membrane and soak it in 5% milk blocking solution. Place the membrane on a shaker and shake it slowly. Block it at room temperature for 60 minutes.

[0114] (2) According to the instructions of the antibody manual, add the antibody to the antibody incubation solution in proportion (the antibody incubation solution is a mixture of the above-mentioned 5% milk blocking solution and 1×TBST, with a ratio of 1:9). Soak the PVDF membrane face down in the antibody incubation solution, making sure to avoid bubbles. Shake slowly at room temperature for 30 minutes, and then store the membrane in a refrigerator at 4°C overnight.

[0115] (3) The next day, slowly rewarm the cells on a shaker for about 30 minutes. Then remove the PVDF membrane and wash the membrane three times with 1× TBST, each time for about 10 minutes.

[0116] (4) According to the instructions of the antibody, select an appropriate dilution ratio of the secondary antibody. Soak the PVDF membrane in the secondary antibody incubation solution, face down, and be careful to avoid bubbles. Incubate on a shaker at room temperature for 1.5 hours.

[0117] (5) Remove the PVDF membrane and wash it with 1× TBST for 10 minutes each time, for a total of three times.

[0118] 4.6 Luminescence

[0119] (1) Mix chemiluminescent solution A and solution B in a volume ratio of 1:1 to prepare a working solution (prepare and use immediately). Approximately 0.1 mL of working solution is required for each square centimeter of membrane. When preparing and using the working solution, be sure to avoid light and turn on the operating system.

[0120] (2) Use tweezers to pick up the PVDF membrane and gently absorb the washing solution on it with filter paper. Then completely immerse the membrane in the prepared working solution.

[0121] (3) The soaked PVDF membrane is taken out, exposed and an image is collected.

[0122] 5. ELISA technology

[0123] Customized ELISA kit (Human Clusterin ELISA Kit, 1×96well plate, Cat No. KE00110, sandwich ELISA, Wuhan Sanying Biotechnology Co., Ltd.). Before conducting the ELISA experiment, ensure that the required reagents are equilibrated at room temperature for 20-30 minutes (except for the detection antibody concentrate and HRP-labeled streptavidin concentrate, which can be used immediately); when adding standards, samples and different reagents, change the pipette tip to avoid contact with the inner surface of the microplate, and use different sample slots. The following are the specific experimental steps:

[0124] (1) Take out the required enzyme-labeled strips and place the remaining enzyme-labeled strips in an aluminum foil bag with desiccant. Seal the bag and store it at 4°C. Use it within one week to ensure the stability of the strips.

[0125] (2) Perform sample loading operation and set up zero well, standard wells and sample wells. Add 100 μL of sample diluent to zero well. Then add gradient diluted standards or samples to other wells, adding 100 μL to each well. Be careful to avoid bubbles. It is recommended to perform duplicate processing on both standards and samples to reduce experimental errors and ensure that continuous loading is completed within 10 minutes.

[0126] (3) Cover the ELISA plate and incubate the plate at 37°C for 2 hours.

[0127] (4) Perform the washing step. Gently remove the sealing film, pour off the liquid, and pat dry. Wash the plate strips with 1× washing solution, adding 400 μL of washing solution to each well. After washing, pour off the liquid and pat dry. Repeat this step 4 times to ensure that the washing is thorough, avoid foreign matter from entering the wells, and prevent the plate strips from drying out.

[0128] (5) Add 100 μL of detection antibody (1×) to each well, cover with sealing film, and continue incubation at 37°C for 1 hour.

[0129] (6) Repeat the washing procedure in step 4.

[0130] (7) Add 100 μL of HRP-labeled streptavidin (1×) to each well, cover with a sealing film, and incubate again at 37°C for 40 min.

[0131] (8) Repeat the washing procedure in step 4.

[0132] (9) Perform color development step: Add 100 μL of TMB color development solution to each well and keep it at 37°C in the dark for 15 minutes (if the color is lighter, the color development time can be appropriately extended, but not more than 30 minutes). During the color development process, make sure to always keep it away from light. It should be colorless and transparent before color development. Do not use it if there is any color change.

[0133] (10) Stop reaction: Add 100 μL of stop solution to each well to turn the blue color into yellow. The order of adding the stop solution is the same as that of the TMB colorimetric solution.

[0134] (11) Reading: Using 630 nm as the calibration wavelength, measure the optical density (OD value) of each well on a microplate reader at 450 nm. Read within 5 minutes after adding the stop solution.

[0135] (12) Perform data analysis: Subtract the OD value of the zero well from the OD value of each standard and sample, set up duplicate wells, and take the average value. Use the concentration of the standard as the horizontal axis and the OD value as the vertical axis to perform four-parameter fitting in professional software (such as Origin, ELISACalc, etc.). Calculate the fitting concentration based on the OD value of the sample through the standard curve, and multiply it by the dilution factor to obtain the actual measured concentration of the sample. The operation process is shown in Table 4:

[0136] Table 4 ELISA technical operation process

[0137]

[0138] 6. Data Analysis

[0139] The raw data of mass spectrometry analysis were RAW files, and database detection and protein identification were performed using the software Sequest HT search engine and ThermoProteomeDiscoverer 2.5.0 search engine and Uniprot Human database. GraphPadPrism version 6.0 was used for plotting.

[0140] 7. Database search parameter settings

[0141] Submit the RAW file to the Sequest server through Proteome Discoverer, select the established database, and then search the database. The relevant parameters are as follows in Table 5:

[0142] Table 5 Database search parameters

[0143] Item Value Enzyme NoEnzyme Variable modifications Oxidation(M); Acetyl(ProteinN-term), Deamidated(N) PeptideMassTolerance ±15ppm FragmentMassTolerance 0.02Da peptideconfidence High peptidelength >4

[0144] Note: The result filtering parameter is: Peptide FDR≤0.01.

[0145] 8. Statistical methods

[0146] In the statistical analysis, we used SPSS 25.0 (IBM Corporation, New York, USA) and GraphPad Prism 6.0 (GraphPad, San Diego, USA). For count data, we used the number of cases and percentages (n, %) for description. For quantitative data, we used the Shapiro-Wilk test to test normal distribution and the Levene test to test the homogeneity of variance. For quantitative data that conformed to normal distribution, we used mean ± standard deviation to express them, and used independent sample T test analysis to compare the differences between the two groups. For data that did not conform to normal distribution (i.e., skewed distribution), we used "median (interquartile range)" to express them, and used the rank sum test to compare the two groups. For categorical data, we used the chi-square test to compare the dichotomous data between the two groups. If the conditions for the chi-square test were not met (i.e., the sample size was ≥40, and at least one theoretical frequency 1≤T≤5 was used, the corrected chi-square test was used), the sample size was <40, or at least one theoretical frequency T<5, Fisher's exact probability method was used. Among them, P < 0.05 was considered statistically significant.

[0147] IV. Results

[0148] 1. Comparison of general data between the hyperplasia group and the adenoma group of patients with intestinal polyps

[0149] 1.1 Comparison of gender and age between the two groups of patients with intestinal polyps

[0150] A total of 90 patients with simple colorectal polyps were collected in the present invention, including 28 cases in the hyperplasia group, aged between 24 and 72 years old, with an average age of (49.14±10.87) years old, and 62 cases in the adenoma group, aged between 26 and 77 years old, with an average age of (54.63±12.38) years old. There was a statistically significant difference in age between the two groups.

[0151] There was no statistical difference in gender between the two groups. In the hyperplasia group, there were 17 males, accounting for 60.7%, and 11 females, accounting for 39.3%. In the adenoma group, there were 36 males, accounting for 58.1%, and 26 females, accounting for 41.9%. There were more male patients than female patients in both groups.

[0152] 1.2 Comparison of personal history and HP infection between the two groups of patients with intestinal polyps

[0153] The two groups of polyp patients were compared in terms of smoking history, drinking history, BMI and HP infection. The results are shown in Table 6.

[0154] Table 6 Distribution characteristics of personal history of 90 patients in the hyperplasia group and adenoma group

[0155]

[0156]

[0157] The results in Table 6 showed that there was no statistical difference.

[0158] 1.3 Comparison of the number, location and size of intestinal polyps between the two groups

[0159] The comparison results of clinical characteristics of patients with intestinal polyps in the hyperplasia group and adenoma group are shown in Table 7.

[0160] Table 7 Analysis of polyp characteristics in 290 patients in the hyperplasia group and adenoma group

[0161]

[0162] The results showed that the size of polyps was statistically significant between the two groups, with adenomatous polyps mostly 5-10 mm and hyperplastic polyps mostly ≤5 mm, while the number and location of polyps were not statistically significant.

[0163] 2. Differences in serum protein expression among the normal healthy group, hyperplasia group, and adenoma group

[0164] 2.1 Screening stage

[0165] A total of 26 healthy controls (normal healthy group / control group), 13 patients with hyperplastic intestinal polyps (hyperplasia group), and 17 patients with adenomatous intestinal polyps (adenoma group) were collected. Serum proteins were analyzed by primary mass spectrometry chromatograms to find differentially expressed protein peaks. The purpose was to evaluate the diagnostic value of serum proteomics for early canceration of colorectal polyps. The results are shown in Figure 1 AB. The principal components found that there were significant differences between the groups, see Figure 1 CD.

[0166] The results confirmed that there were indeed significant differential expression peaks between the groups, that is, the groups were comparable.

[0167] According to statistical analysis, a total of 63 peptides were differentially expressed in serum samples of each group (see Table 9 for details). According to the decreasing trend of the ratios of hyperplasia group / control group and adenoma group / control group, a total of 5 peptides were screened, of which the mass-to-charge ratios (m / z) were 2865.37 (hyperplasia group / control group = 0.62; adenoma group / control group = 0.35), 2191.88 (hyperplasia group / control group = 0.81; adenoma group / control group = 0.58), 1619.15 (hyperplasia group / control group = 0.56; adenoma group / control group = 0.40), 1351.03 (hyperplasia group / control group = 0.50; adenoma group / control group = 0.43), and 1194.35 (hyperplasia group / control group = 0.65; adenoma group / control group = 0.45). The peptide with a mass-to-charge ratio (m / z) of 2663.05 (where hyperplasia group / control group = 0.30; adenoma group / control group = 0.30) has the lowest ratio between the hyperplasia group / control group and the adenoma group / control group and the values ​​are similar. In order to explore the differences in the serum of each group, a total of 6 peptides were selected for secondary mass spectrometry analysis to find differentially expressed proteins. After mass spectrometry analysis, a total of 6 differentially expressed proteins were found (specific protein information is shown in Table 9), and the secondary gel chromatography separation diagram of the serum protein polypeptide mixture of the 6 differentially expressed peptides is shown in Figure 2 shown.

[0168] Table 83 Serum peptide composition and statistical analysis of the population

[0169]

[0170]

[0171]

[0172] Note: The red part is the significant serum peptide segment selected according to the screening criteria of decreasing adenoma / control and inflammation / control ratios. * indicates

[0173] P≤0.05; **P≤0.01; ***P≤0.001.

[0174] Table 9 General characteristics of differentially expressed peptide segments in the hyperplasia group and adenoma group compared with the control group

[0175] Molecular weight Peptide sequence Location Protein ID Protein name 2865.37 K.NPKFMETVAEKALQEYRKKHREE. 427-449 P10909 Clusterin 2191.88 K.SSSYSKQFTSSTSYNRGDST.F 575-595 P02671 Fibrinogenalphachain 1619.15 K.DVFLGMFLYEYAR.R 348-360 P02768 Albumin 1351.03 D.SGEGDFLAEGGGVR.G 22-35 P02671 Fibrinogenalphachain 1194.35 D.SGEGDFLAEGGGV.R 22-34 P02671 Fibrinogenalphachain 2663.05 A.DEAGSEADHEGTHSTKRGHAKSRPV.R 605-629 P02671 Fibrinogenalphachain

[0176] 2.2 Internal Verification Phase

[0177] During the internal validation phase, 45 serum samples from the normal healthy control group, 15 serum samples from the hyperplasia group, and 45 serum samples from the adenoma group were collected. Five serum samples from each group were randomly matched to form a group, namely, 9 normal healthy groups, 5 hyperplasia groups, and 9 adenoma groups. ELISA was used to detect the CLU expression concentration of the target protein in the serum samples, the absorbance value of each detection well was measured, the concentration value of each well was calculated, and the curve of the ELISA standard was drawn (see Figure 4 ).

[0178] The results are shown in Table 10 and Figure 4 As shown: The serum concentration of the target protein CLU in the colorectal adenoma group was significantly lower than that in the hyperplastic polyp group and the healthy control group, and the CLU protein concentration showed a gradient decreasing trend in the normal healthy group, hyperplasia group, and adenoma group, with statistical differences among the groups.

[0179] Table 10 Serum CLU protein expression levels in each group of people during the internal validation phase

[0180] Group Number of cases / group CLU protein concentration (pg / mL) F-number P-value Normal healthy group (NC) 45 cases (9 groups) 4069.99±1288.16 13.90 0.000*** Proliferation group (IP) 15 cases (5 groups) 2142.04±233.73 Adenoma group (AP) 45 cases (9 groups) 1890.48±594.97

[0181] Note: *** indicates P ≤ 0.001.

[0182] In the screening phase, 56 serum samples were collected, including 26 normal healthy group, 13 hyperplasia group, and 17 adenoma group (consistent with the experimental grouping in the internal validation phase), namely 5 normal healthy groups, 3 hyperplasia groups, and 4 adenoma groups. ELISA was performed again to detect the concentration of target protein CLU in serum samples of each group, and the difference of CLU protein in each group was analyzed. The results once again confirmed that the serum concentration of CLU protein in the adenoma group was significantly lower than that in the control group, and there was a statistical difference among the three groups (the results are shown in Tables 11 and Figure 5 ).

[0183] Table 11 CLU protein expression levels in serum of each group of people during the screening phase

[0184] Group Number of cases / group CLU protein concentration (pg / mL) F-number P-value Normal healthy group (NC) 26 cases (5 groups) 5232.28±1033.91 17.21 <![CDATA[0.001 *** ]]> Proliferation group (IP) 13 cases (3 groups) 3649.84±495.88 Adenoma group (AP) 17 cases (4 groups) 2060.74±604.49

[0185] Note: *** indicates P ≤ 0.001.

[0186] In the validation phase, 2 tissue samples from the normal healthy control group and 2 tissue samples from the adenoma group were randomly collected for Western Blotting experiments to verify the expression of CLU protein in each group. The results once again confirmed that compared with the normal healthy control group tissue samples, the expression level of CLU protein in the adenoma group decreased (see Figure 6 ).

[0187] 2.3 External Validation Phase

[0188] In order to verify the diagnostic predictive value of the target protein CLU in adenomatous polyps, the CLU protein concentrations detected in each group in the serum samples collected in the screening stage and the validation stage were combined, and the expression levels of CLU protein among the groups were analyzed. The results once again confirmed that the target protein concentration showed a gradient decreasing trend in each group, and there were significant statistical differences in the CLU protein concentrations in the serum of each group (see Table 12 and Figure 7 ).

[0189] Table 12 CLU protein expression levels in serum of three groups of people in the screening phase and validation phase

[0190] Group Number of cases / group CLU protein concentration (pg / mL) F-number P-value Normal healthy group (NC) 71 cases (14 groups) 4485.10±1261.41 23.11 <![CDATA[0.000 *** ]]> Proliferation group (IP) 28 cases (6 groups) 2895.94±895.68 Adenoma group (AP) 62 cases (13 groups) 1942.88±577.90

[0191] Note: *** indicates P ≤ 0.001.

[0192] The ROC curve (Receiveroperating characteristic curve) was drawn using the CLU protein concentrations of each group in the screening and validation stages. The ROC curve analysis was used to evaluate the diagnostic effect of the protein in adenomatous polyps and non-adenomatous polyps, and to determine the best critical value for the diagnostic effect. (See Figure 8 ). The area under the ROC curve AUC = 0.92, where CLU < 2895.78 pg / mL indicates a high possibility of diagnosing adenomatous polyps.

[0193] From the above experimental results, it can be seen that a total of 63 peptides were found to be differentially expressed in the serum protein profiles of the three groups: normal health group, hyperplasia group, and adenoma group (P≤0.05), and the expression of CLU protein in the normal health group, hyperplasia group, and adenoma group showed a gradient decrease trend. According to the trend of gradient expression of biomarkers in the process of polyp development into adenoma, six peptides with mass-to-charge ratios of 2865.37, 2191.88, 1619.15, 1351.03, and 1194.35 were selected for analysis, and three proteins, Clusterin (CLU), Albumin (ALB), and Fibrinogen alpha chain (FGA), were identified.

[0194] In the present invention, the expression of CLU protein in the normal health group, hyperplasia group, and adenoma group shows a gradient decreasing trend. In order to evaluate the diagnostic and prognostic value of CLU protein for adenomatous polyps, ROC curve analysis was performed to evaluate the diagnostic effect of the target protein on subjects with adenomatous polyps and non-adenomatous polyps, wherein the area under the ROC curve (AUC) was 0.92, indicating that the diagnostic accuracy of the target protein was high. The optimal diagnostic critical value was further determined using the coordinates under the ROC curve. It is worth noting that when CLU is less than 2895.78 pg / mL, the Youden index value is the largest, that is, when the concentration is lower than 2895.78 pg / mL, the diagnosis of adenomatous polyps may be high, and colonoscopy is strongly recommended. There is a risk of further development of colorectal cancer, so clinicians and patients should be vigilant.

[0195] In summary, CLU protein is expected to become a screening tool protein for early carcinogenesis of intestinal polyps, and may provide application prospects for targeted treatment of colon cancer.

[0196] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0197] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of a biomarker for early screening of colorectal adenoma and colorectal hyperplasia in the preparation of a product for screening or diagnosis of colorectal adenoma and / or colorectal hyperplasia, characterized in that: The biomarker is clusterin CLU; The clusterin CLU is a marker among serum markers; The product includes a kit, a detection reagent or an antibody chip; The kit includes a colorectal adenoma and / or colorectal hyperplasia screening or diagnosis kit; The kit comprises an antibody or an antibody fragment that specifically binds to the clusterin CLU, and also comprises a reagent for detecting the clusterin CLU.

2. The use according to claim 1, characterized in that: The detection reagents include reagents for detecting markers in the reagents for serum biomarkers.

3. The use according to claim 2, characterized in that: The marker reagent in the reagent for detecting serum biomarkers is an ELISA reagent, an immunoblotting reagent, an immunofluorescence reagent, an immunohistochemistry reagent, a protein chip or mass spectrometry detection reagent, a PCR detection reagent, an in situ hybridization reagent, or a sequencing reagent.

4. The use according to claim 3, characterized in that: The reagent for detecting serum biomarkers is a PCR detection reagent including CLU detection antibody, HRP-labeled streptavidin, and color-developing TMB.

5. The use according to claim 3, characterized in that: The reagent for detecting serum biomarkers is an ELISA reagent including SYBR Green, RT Enzyme mix, and primers.

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