Application of biomarker CCL26 in colorectal adenoma diagnosis

Abstract

The invention discloses an application of a biomarker CCL26 in colorectal adenoma diagnosis, and provides an application of a substance for detecting a marker in a to-be-detected sample in preparation of a product for diagnosis or auxiliary diagnosis and prediction or auxiliary prediction of colorectal adenoma. The invention further provides a related product, a risk prediction model, an auxiliary diagnosis system and a scoring device, the CCL26 marker disclosed by the invention has relatively high sensitivity and specificity on the diagnosis effect of the colorectal adenoma, the colorectal adenoma can be recognized in an early stage, and the diagnosis efficiency is high.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of a biomarker CCL26 in the diagnosis of colorectal adenoma. Background Art

[0002] Colorectal adenoma is a common benign intestinal tumor, accounting for 80%-90% of colorectal polyp types, with an incidence rate of 1.8%-17.2% in the general population and as high as 30%-40% in people over 50 years old. At the same time, colorectal adenoma is currently recognized as the most important precancerous lesion of colorectal cancer. About 70%-90% of benign adenomas can develop into colorectal cancer through the "adenoma-cancer" pathway, and the window period is about 7-10 years. Therefore, early detection of colorectal adenoma and intervention are conducive to reducing the incidence and mortality of colorectal cancer.

[0003] Existing colorectal adenoma screening technology is mainly based on colonoscopy. Since colonoscopy is an invasive procedure, patient compliance is low, and colonoscopy resources are limited and affected by the operator's technical level, the examination results are uneven and the missed diagnosis rate is high. At the same time, some markers used to detect colorectal adenomas in existing technologies have poor sensitivity and specificity, resulting in a low overall adenoma detection rate. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solutions:

[0005] The first aspect of the present invention provides the use of a substance for detecting a marker in a sample to be tested in the preparation of a product for diagnosing or assisting in the diagnosis of colorectal adenoma, wherein the marker includes a CCL26 marker.

[0006] Furthermore, the sample includes blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, semen, vaginal secretions, sputum, sweat, breast milk, synovial fluid, mucus, tears, bile, gastric juice, interstitial fluid, tissue biopsy or epithelial cells, oral swab, nasal swab, aqueous humor, amniotic fluid, pleural fluid or the subject's exhaled breath.

[0007] Furthermore, the sample includes blood, plasma, serum, tissue biopsy or epithelial cells.

[0008] Further, the sample includes a tissue biopsy or epithelial cells.

[0009] Furthermore, the sample is plasma.

[0010] Furthermore, the sample is blood.

[0011] Furthermore, the sample is a tissue biopsy or epithelial cells.

[0012] The term "CCL26" as used herein is intended to include fragments, variants (e.g., allelic variants), and derivatives thereof. Representative human CCL26 cDNA and human CCL26 protein sequences are well known in the art and are publicly available from the NCBI website. For example, at least one human CCL26 isoform is known: human CCL26, Gene ID: 10344. Nucleic acid and polypeptide sequences of CCL26 orthologs in organisms other than humans are well known.

[0013] The term "colorectal adenoma" used in the present invention refers to a neoplastic polyp located in the mucosal glandular epithelial tissue originating from the colorectal region. In the present invention, "colorectal adenoma" may include adenoma occurring only in the colon (also referred to as "colonic adenoma"), adenoma occurring only in the rectum (also referred to as "rectal adenoma"), or adenoma occurring in both the colorectum and rectum.

[0014] The term "marker" used in the present invention refers to a gene or a gene product that is a target for regulating one or more target phenotypes (e.g., a target phenotype in a myeloid cell). In this case, the term "marker" is synonymous with "target". However, in some embodiments, the term also encompasses measurable entities that have been determined to indicate a target output such as one or more diagnostic, prognostic and / or therapeutic outputs (e.g., for regulating an inflammatory phenotype, a cancer state, etc.). In other embodiments, the term also encompasses a composition that regulates a gene or a gene product.

[0015] The terms "sample to be tested" and "sample" used in the present invention can be any biological sample separated from a subject. The term "sample" includes any biological sample that can be extracted from a subject, untreated, treated, diluted or concentrated. Samples may include, but are not limited to, biological fluids, such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, feces (i.e., feces), tears, sweat, sebum, nipple aspirate, duct lavage fluid, tumor exudate, synovial fluid, ascites, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymph fluid, fine needle aspiration fluid, amniotic fluid, any other body fluid, cell lysate, cell secretion, inflammatory fluid, semen and vaginal secretion. Samples may include tissue samples and biopsy samples, tissue homogenates, etc. Advantageous samples may include samples containing any one or more biomarkers taught herein in a detectable amount. Suitably, samples can be easily obtained by minimally invasive methods, thereby allowing samples to be removed or separated from subjects. In certain embodiments, the sample comprises blood, especially peripheral blood, or a portion or extract thereof. Typically, the sample comprises blood cells, such as mature, immature or developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, hemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells or natural killer cells, or a portion (e.g., a nucleic acid or protein portion) of such cells.

[0016] Furthermore, substances for detecting markers in the sample to be tested include reagents used in western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, octoglobulin immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip detection.

[0017] The term "agent" used in the present invention includes compounds that induce the desired pharmacological and / or physiological effects. The term also covers pharmaceutically acceptable and pharmacologically active components of those compounds specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, etc. When the above terms are used, it should be understood that this includes the active agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "agent" should not be interpreted narrowly, but should be extended to small molecules, protein molecules, such as peptides, polypeptides and proteins, and compositions containing them, as well as genetic molecules, such as RNA, DNA and their mimetics and chemical analogs and cellular agents.

[0018] The second aspect of the present invention provides the use of a substance for detecting a marker in a sample to be tested in the preparation of a product for predicting or assisting in predicting colorectal adenoma, wherein the marker includes a CCL26 marker.

[0019] Furthermore, the sample includes blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, semen, vaginal secretions, sputum, sweat, breast milk, synovial fluid, mucus, tears, bile, gastric juice, interstitial fluid, tissue biopsy or epithelial cells, oral swab, nasal swab, aqueous humor, amniotic fluid, pleural fluid or the subject's exhaled breath.

[0020] Furthermore, the sample includes blood, plasma, serum, tissue biopsy or epithelial cells.

[0021] Further, the sample includes a tissue biopsy or epithelial cells.

[0022] Furthermore, the sample is plasma.

[0023] Furthermore, the sample is blood.

[0024] Furthermore, the sample is a tissue biopsy or epithelial cells.

[0025] The third aspect of the present invention provides a product, which includes a substance for detecting a CCL26 marker.

[0026] Furthermore, the products include products for diagnosing or assisting in diagnosing whether a sample to be tested is a colorectal adenoma sample, products for diagnosing or assisting in diagnosing whether a person to be tested is a colorectal adenoma patient, products for predicting or assisting in predicting whether a sample to be tested is a colorectal adenoma sample, and products for predicting or assisting in predicting whether a person to be tested is a colorectal adenoma patient.

[0027] As used herein, the term "diagnosis" refers to the identification or classification of a molecular or pathological state, disease or condition. The term "aided diagnosis" refers to a method that aids in the clinical determination of the presence or nature of a particular type of symptom or condition of a disease or disorder. For example, an aided diagnosis method for a disease or condition may include measuring certain biomarkers in a biological sample from an individual.

[0028] Furthermore, the substance for detecting the CCL26 marker includes a substance for detecting gene expression level or a substance for detecting protein level.

[0029] Furthermore, the products include reagents, test kits, test strips, nucleic acid membrane strips, chips, primers, and probes.

[0030] Furthermore, the subject includes a human or a non-human mammal.

[0031] Furthermore, the subject to be tested is a human.

[0032] Further, the reagents include reagents for detecting the presence, absence and / or amount of CCL26 genes, fragments and proteins in a sample by PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, double hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte spectrum test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay.

[0033] Furthermore, the kit includes a gene detection kit and a protein detection kit.

[0034] Furthermore, the gene detection kit includes primers, reagents or chips for detecting the transcription level of the CCL26 gene.

[0035] Furthermore, the protein detection kit includes reagents, chips, antibodies, and ligands for detecting the protein level of CCL26 expression.

[0036] Furthermore, the test paper includes chromatography test paper.

[0037] As used herein, "expression" refers to the transcription of a gene to produce an RNA transcript (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.), and, if appropriate, the translation of the resulting mRNA transcript into a protein. Thus, it is clear from the context that the expression of a coding sequence is produced by the transcription and translation of the coding sequence. In contrast, the expression of a non-coding sequence is produced by the transcription of the non-coding sequence.

[0038] The terms "level of expression" or "expression level" used in the present invention are generally used interchangeably and generally refer to the amount of a biomarker in a sample. "Expression" generally refers to a process by which information (e.g., coding genes and / or epigenetics) is converted into structures present and operable in a cell. Therefore, as used herein, "expression" may refer to transcription into polynucleotides, translation into polypeptides, or even modification of polynucleotides and / or polypeptides (e.g., post-translational modifications of polypeptides). Transcribed polynucleotides, translated polypeptides, or fragments of polynucleotide and / or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered as expression, whether they are derived from transcripts or degraded transcripts produced by alternative splicing, or from post-translational processing of polypeptides, such as by proteolysis. "Expressed genes" include those that are transcribed into polynucleotides (mRNA) and then translated into polypeptides, as well as those that are transcribed into RNA but not translated into polypeptides (e.g., transfer RNA and ribosomal RNA).

[0039] The fourth aspect of the present invention provides the use of markers in constructing a computer model for diagnosis or auxiliary diagnosis, prediction or auxiliary prediction of colorectal adenoma, wherein the markers include the CCL26 marker.

[0040] Further, the computer model includes a memory.

[0041] Furthermore, the memory is used to store a computer program for execution.

[0042] In some specific embodiments, the computer program is used to control the processor to perform operations of different modules.

[0043] The fifth aspect of the present invention provides the use of markers in screening candidate drugs for treating colorectal adenoma, wherein the markers include CCL26 markers.

[0044] A sixth aspect of the present invention provides a colorectal adenoma risk prediction model, wherein the diagnostic model includes a risk prediction model constructed by model training using a machine learning method, and the machine learning method includes a process of machine training using a CCL26 marker.

[0045] Furthermore, the machine learning method includes a decision tree model, a random forest model, a K-nearest neighbor algorithm model, a naive Bayes model, a support vector machine model, and a neural network model.

[0046] The seventh aspect of the present invention provides an early medical auxiliary diagnosis system for colorectal adenoma, the diagnosis system comprising a result determination module for comparing a processed value of a marker obtained by detecting the gene or protein expression level of a CCL26 marker with a set value.

[0047] Furthermore, the diagnostic system includes a processing module for detecting and processing the expression level of a gene or protein related to a marker in a sample to obtain a processed value.

[0048] Furthermore, the diagnostic system includes an output module for outputting the obtained diagnostic results.

[0049] The eighth aspect of the present invention also provides the use of the CCL26 marker in the preparation of a pharmaceutical composition for treating and / or preventing individual colorectal adenoma.

[0050] Furthermore, the individual includes a human or a non-human mammal.

[0051] Furthermore, the individual is a human.

[0052] The term "pharmaceutical composition" or "pharmaceutical formulation" as used herein refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and does not contain other components that are unacceptably toxic to a subject to which the composition or formulation will be administered.

[0053] A ninth aspect of the present invention provides a scoring device for assessing the risk of a subject suffering from colorectal adenoma, the scoring device comprising the following units:

[0054] Detection unit: detecting the gene or protein expression level of markers such as CCL26 in samples;

[0055] Analysis unit: using the gene or protein expression level of the detected marker as an input variable and inputting it into the risk prediction model described in the sixth aspect of the present invention for analysis;

[0056] Scoring unit: outputs the risk value of colorectal adenoma for the subject corresponding to the sample.

[0057] The present invention also provides a method for diagnosing and predicting colorectal adenoma, which comprises the following steps:

[0058] 1) Collecting samples of the object to be tested;

[0059] 2) Detecting the expression level of CCL26 marker in the sample;

[0060] 3) comparing the expression level results with the expression levels of healthy controls to determine whether the subject suffers from colorectal adenoma or the risk of suffering from colorectal adenoma.

[0061] The terms "patient", "subject", "subject" or "individual" used in the present invention refer to any subject in need of treatment or prevention, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present invention include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys and apes, and including monkey species (e.g., genus Macaca (e.g., macaques, such as cynomolgus monkeys (Macaca fascicularis), and / or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (species from the genus Callithrix), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus), and species of apes, such as chimpanzees (Pantroglodytes), rodents (e.g., mice, rats, guinea pigs), Leporidae (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (e.g., cats), avians (e.g., chickens, turkeys, ducks, geese, companion birds such as canaries, budgies, etc.), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish.

[0062] Further, the detection in step 2) includes detection performed by PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, double hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte spectrum test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay.

[0063] Furthermore, the detection performed in step 2) also requires the use of external reference genes to perform data normalization operations.

[0064] In the present invention, the P value is probability, which reflects the probability of an event. The P value obtained by the statistical significance test method is generally significant when P < 0.05 and very significant when P < 0.01, which means that the probability of the difference between samples caused by sampling error is less than the P value, which reflects the probability of an event.

[0065] Advantages and beneficial effects of the present invention:

[0066] The CCL26 biomarker has high sensitivity and specificity for colorectal adenoma patients, and can identify colorectal adenoma at an early stage with high diagnostic efficiency. CCL26 markers can be detected from tissues, and CCL26 exists in the blood, which is easy to operate and solves the problems of physical discomfort and poor compliance caused by invasive operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is the HE staining image of the test set of colorectal adenoma tissue and adjacent normal tissue; CRA is colorectal adenoma tissue, NOR is adjacent normal tissue;

[0068] Figure 2 This is the differential expression result diagram of CCL26 in the test set in colorectal adenoma tissue and adjacent normal tissue;

[0069] Figure 3 This is the ROC curve of CCL26 in the test set for colorectal adenoma diagnosis;

[0070] Figure 4 It is to verify the HE staining of colorectal adenoma tissue and adjacent normal tissue; CRA is colorectal adenoma tissue, NOR is adjacent normal tissue;

[0071] Figure 5 This is the immunohistochemical staining of CCL26; CRA is adenoma tissue, NOR is normal tissue adjacent to the tumor; positive expression is yellow-brown staining of the cytoplasm and blue staining of the nucleus;

[0072] Figure 6 It is a bar graph of the average optical density values ​​of CCL26 in colorectal adenoma tissues and adjacent normal tissues in the validation set;

[0073] Figure 7 This is the ROC curve of CCL26 for colorectal adenoma diagnosis in the validation set; DETAILED DESCRIPTION

[0074] Example 1: Correlation of marker CCL26 with diagnosis of colorectal adenoma

[0075] 1. Experimental subjects

[0076] The whole transcriptome sequencing (RNA sequencing, RNA-seq) test set used 12 cases of colorectal adenoma and 12 cases of adjacent normal tissues, a total of 24 cases, the specific information is shown in Table 1. The samples used were all formaldehyde-fixed paraffin-embedded tissues of adenomas that were removed by colonoscopy and surgery at Wangjing Hospital between 2019 and 2023. The research samples were again confirmed as colorectal adenomas and adjacent normal tissues by 2 senior pathologists ( Figure 1 ).

[0077] Table 1 Clinical information of patients in the RNA-seq test set

[0078]

[0079]

[0080] 2. Experimental equipment

[0081] Agilent 2100 Bioanalyzer (Agilent), Qubit 2.0 Fluorometer (Invitrogen), Veriti 96-well Multiplex Temperature-controlled Gradient Gene Amplifier (MJ Research, USA), Peltier Thermal Cycler PTC-225 (MJ Research, USA), DNA Quantification Kit (KAPABiosystem), Multi-sample Adapter Primer Kit (NEBNext), High Sensitivity DNA Detection Kit (Agilent), RNA 6000 Pico chip (Agilent).

[0082] 3. Experimental methods

[0083] Whole transcriptome sequencing was performed on 12 cases of colorectal adenoma tissues and 12 cases of adjacent normal tissues. The sequencing process mainly included RNA extraction, library construction, sequencing, data quality control, and data analysis.

[0084] 3.1 Library construction and sequencing

[0085] Total RNA from colorectal adenomas and adjacent normal tissues was extracted using Trizol, and RNA integrity was tested using the Agilent 2100 Bioanalyzer, with quality requirements of RIN ≥ 7, 28S / 18S ≥ 1.5:1. The starting Total RNA was accurately quantified using the QUBIT RNA Assay Kit, with a starting amount requirement of 0.1-1ug. Beads with Oligod (T) were used to purify mRNA in Total RNA, and NEB Next Reaction Buffer was used to fragment the mRNA to the target length range, and then the first complementary DNA chain (cDNA) was synthesized using random primers. The second cDNA chain was then synthesized under the action of DNA polymerase I, and the purified double-stranded cDNA was end-repaired, A-tailed, and connected to the sequencing adapter before PCR amplification. The PCR product was quality controlled using the 2100 Bioanalyzer chip to obtain the final library, and the constructed cDNA library was sequenced using the II Lumina Novasek 6000 sequencing platform.

[0086] 3.2 Data filtering and quality control

[0087] Base calling was performed on the raw files of IILumina sequencing and converted into raw sequencing sequences. The collection of raw sequencing sequences was called Raw Data. Raw Data was filtered to delete reads that did not meet the analysis criteria to obtain Clean Reads for subsequent analysis. The filtering conditions were as follows: (1) Remove contaminated samples; (2) If the reads contained adapter sequences, the adapter sequences were truncated first; (3) With a sliding window of 4 bases in length, the bases with an average base quality of <15 at both ends of the reads were truncated; (4) Read pairs with high N content were removed (if the N content in a read was >5%, the entire pair of reads was removed); (5) Read pairs with high content of low-quality bases (bases with a quality value ≤20) were removed (if the low-quality bases in a read were >30%, the entire pair of reads was removed); (6) If the length of the reads after truncation was <100 bp, the entire pair of reads was removed; (7) Unpaired reads were removed.

[0088] 3.3 Sequence alignment and gene quantification

[0089] The index file of the reference genome was downloaded from the official website. Clean Reads were aligned with the reference genome sequence using Hisat2 software. The number of reads aligned to the reference genome in Clean Reads was required to account for ≥70% of the total number of Clean Reads, and the number of reads aligned to multiple positions of the reference genome accounted for ≤10% of the total number of reads aligned to the reference genome. The readcount of gene expression was calculated using featureCounts and StringTie software. The number of fragments per kilobase of exon model per million mapped fragments (FPKM) was calculated taking into account the length and depth of the RNA sequence, and the FPKM value of gene expression was filtered: (1) genes with a length of less than 100 bp were removed; (2) if the FPKM of a gene was < 0.0001, it was considered to be expressed as 0, and genes with an expression of 0 in all samples were removed.

[0090] 3.4 Analysis of differentially expressed genes

[0091] The data after gene expression quantification were statistically analyzed, and the differentially expressed genes (DEGs) between colorectal tissues and adjacent normal tissues were screened out using the limma package and the DESeq package in R language. The screening criteria were: |log2FC|>1 and P<0.05, where FC represents the fold change (FC), and the P value represents the statistical significance of the difference (P-Value). At the same time, the genes expressed in colorectal adenoma tissues but almost not expressed in normal tissues were further screened out based on the FPKM value of gene expression.

[0092] 3.5 Statistical analysis

[0093] IBM SPSS Statistics 26.0 software was used for statistical analysis. The measured data were expressed as mean ± standard deviation (x ± s) when they were in accordance with normal distribution. The independent sample t test was used for comparison between the two groups. Nonparametric test was used for comparison when they were not in accordance with normal distribution. P < 0.05 indicated statistically significant differences.

[0094] 4. Experimental results

[0095] According to the analysis of the whole transcriptome sequencing results, the expression of CCL26 in colorectal adenoma tissue was significantly higher than that in normal tissue, and there was a very significant statistical difference (P < 0.001), see Table 2 and Table 3. In the evaluation of its diagnostic efficacy, the ROC curve showed that the AUC was 0.9444, at which point its sensitivity could reach 100% and its specificity could reach 83.33%, see Figure 2 , Figure 3 .

[0096] Table 2 Differential expression of CCL26 in adenoma tissue and adjacent normal tissue

[0097]

[0098] Note: CRA is colorectal adenoma tissue, NOR is normal tissue adjacent to the tumor.

[0099] Table 3 Gene expression quantitative values ​​(FPKM) of CCL26 in each sample of the test set

[0100] Sample No. FPKM value of adenoma tissue FPKM value of peritumoral tissue 1 1.10725 0 2 1.21224 0 3 1.08291 0 4 0.87007 0 5 3.61919 0.99962 6 0.09352 0 7 0.17421 0 8 3.24256 0 9 0.21426 0 10 2.28141 0 11 1.46447 0.87314 12 3.01914 0

[0101] Example 2 Immunohistochemical verification of the diagnostic performance of CCL26 for colorectal adenoma

[0102] 1. Clinical samples

[0103] The immunohistochemistry (IHC) validation set used 45 cases of colorectal adenomas and 45 cases of adjacent normal tissues, for a total of 90 cases (see Table 4). All samples used were formaldehyde-fixed paraffin-embedded adenoma tissues that were removed by colonoscopy and surgery at Wangjing Hospital between 2019 and 2023. The research samples were again confirmed as colorectal adenomas and adjacent normal tissues by 2 senior pathologists (see Figure 4 ).

[0104] Table 4 Clinical information of patients in the IHC validation set

[0105]

[0106]

[0107] 2. Experimental equipment

[0108] 4℃ / -20℃ refrigerator (BCD-196F) (Qingdao Haier Co., Ltd.), RM2235 slicer (LEICA, Germany), HI1210 slice spreader (LEICA, Germany), HI1210 slice baking machine (LEICA, Germany), high pressure steam sterilizer (YX280) (Hefei Huatai Medical Co., Ltd.), electric blower drying oven (101-0AB) (Linmao Technology Beijing Co., Ltd.), three-purpose constant temperature water bath (SH.W21.600) (Shanghai Shuli Instrument Co., Ltd.), BX51 microscope (OLYMPUS, Japan) ), hematoxylin anti-blue solution (Beijing Shiji Heli Biotechnology Co., Ltd.), DAB color development kit (Beijing Zhongshan Biotechnology Co., Ltd.), hydrogen peroxide solution (Beijing Haiderun Pharmaceutical Group Co., Ltd.), CCL26 antibody reagent (bs-15513R) (Beijing Bioss Biotechnology Co., Ltd.), citric acid repair solution (pH 6.0) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), EDTA antigen repair solution (pH 8.0) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.), PBS buffer powder (pH 7.3) (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).

[0109] 3. Experimental methods

[0110] The CCL26 gene obtained by whole transcriptome sequencing analysis was validated by immunohistochemistry, and the average optical density value of CCL26 was calculated using Image ProPlus 6.0 software for semi-quantitative analysis, ROC curve drawing, and diagnostic value evaluation. The specific immunohistochemical validation process is as follows:

[0111] (1) Tissue sections:

[0112] 1) CRA paraffin samples were sectioned at 2.5 μm thickness;

[0113] 2) Expand in a 48°C slide spreader;

[0114] 3) Bake the slices in a 63°C oven for 1 hour.

[0115] (2) Dewaxing and hydration:

[0116] 1) Soak the prepared sections in dewaxing solutions I and II for 15 min respectively;

[0117] 2) Soak in anhydrous ethanol I and II for 5 min respectively;

[0118] 3) Soak in 95% ethanol for 2 minutes and then soak in 80% ethanol for 1 minute;

[0119] 4) Rinse with distilled water for 3 times and soak in PBS for 5 min × 3 times.

[0120] (3) Antigen retrieval:

[0121] 1) Heat EDTA or citric acid repair solution in a pressure cooker until boiling and then put the slices in;

[0122] 2) After the pressure cooker is sprayed, start antigen retrieval;

[0123] 3) EDTA timing 3min, citric acid timing 2.5min;

[0124] 4) Cool the slices at room temperature for 20 minutes.

[0125] (4) Immune response:

[0126] 1) Soak in PBS for 5 min × 3 times and then soak in 3% H2O2 for 10 min;

[0127] 2) Soak again in PBS for 5 min × 3 times;

[0128] 3) Add primary antibody and incubate at room temperature at 37℃ for 2h;

[0129] 4) Soak in PBS for 5 min × 3 times;

[0130] 5) Add secondary antibody and incubate at 37℃ moisturizing box for 30min;

[0131] 6) Soak in PBS for 5 min × 3 times.

[0132] (5)Chemical dyeing:

[0133] 1) DAB staining, microscopic examination of staining degree, positive expression appears yellow-brown;

[0134] 2) Re-stain with hematoxylin for 5 minutes, separate with color-separating solution, and de-blue with anti-blue solution, then wash with water three times.

[0135] (6) Dehydration and sealing:

[0136] 1) Soak in 80% ethanol for 1 min, 95% ethanol for 2 min, and 90% ethanol II for 2 min;

[0137] 2) Soak in anhydrous ethanol I for 5 min and anhydrous ethanol II for 5 min in sequence;

[0138] 3) Soak in transparent liquid I for 1 min, transparent liquid II for 5 min, and transparent liquid III for 5 min in sequence;

[0139] 4) Fix the slide with neutral gum.

[0140] 4. Statistical analysis

[0141] The statistical analysis method is the same as in Example 1.

[0142] 5. Experimental results

[0143] The results of IHC staining of CCL26 showed that the average optical density of CCL26 in colorectal adenoma tissue was significantly higher than that in normal tissue adjacent to the tumor, and the difference was statistically significant (P < 0.001). In the evaluation of its diagnostic efficacy, the ROC curve showed that the AUC was 0.8741, at which point its sensitivity could reach 88.89% and its specificity could reach 77.78%. Figure 5 , Figure 6 , Figure 7 .

[0144] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of a substance for detecting a marker in a sample to be tested in the preparation of a product for diagnosing or assisting in the diagnosis of colorectal adenoma, characterized in that: The markers include CCL26 markers; Preferably, the sample comprises blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, semen, vaginal secretions, sputum, sweat, breast milk, synovial fluid, mucus, tears, bile, gastric juice, interstitial fluid, tissue biopsy or epithelial cells, oral swab, nasal swab, aqueous humor, amniotic fluid, pleural fluid or breath of the subject; Preferably, the sample comprises blood, plasma, serum, tissue biopsy or epithelial cells; Preferably, the sample comprises a biopsy of tissue or epithelial cells; Preferably, the sample is plasma; Preferably, the sample is blood.

2. Use of a substance for detecting a marker in a sample to be tested in the preparation of a product for predicting or assisting in predicting colorectal adenoma, characterized in that: The markers include CCL26 markers; Preferably, the sample comprises blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, semen, vaginal secretions, sputum, sweat, breast milk, synovial fluid, mucus, tears, bile, gastric juice, interstitial fluid, tissue biopsy or epithelial cells, oral swab, nasal swab, aqueous humor, amniotic fluid, pleural fluid or breath of the subject; Preferably, the sample comprises blood, plasma, serum, tissue biopsy or epithelial cells; Preferably, the sample comprises a biopsy of tissue or epithelial cells.

3. A product, characterized in that: The product includes a substance for detecting the CCL26 marker; Preferably, the products include products for diagnosing or assisting in diagnosing whether a sample to be tested is a colorectal adenoma sample, products for diagnosing or assisting in diagnosing whether a subject to be tested is a colorectal adenoma patient, products for predicting or assisting in predicting whether a sample to be tested is a colorectal adenoma sample, and products for predicting or assisting in predicting whether a subject to be tested is a colorectal adenoma patient.

4. The product according to claim 3, characterized in that The substance for detecting the CCL26 marker includes a substance for detecting gene expression level or a substance for detecting protein level; Preferably, the product includes reagents, kits, test strips, nucleic acid membrane strips, chips, primers, and probes; Preferably, the subject includes a human or a non-human mammal; Preferably, the reagents include reagents for detecting the presence, absence and / or amount of genes, fragments and proteins of CCL26 in a sample by PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, double hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte spectrum test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay; Preferably, the kit includes a gene detection kit and a protein detection kit.

5. Use of the marker in constructing a computer model for diagnosis or auxiliary diagnosis, prediction or auxiliary prediction of colorectal adenoma, characterized in that: The markers include the CCL26 marker.

6. Use of a marker in screening candidate drugs for treating colorectal adenoma, characterized in that: The markers include the CCL26 marker.

7. A colorectal adenoma risk prediction model, characterized in that: The diagnostic model includes a risk prediction model constructed by model training using a machine learning method, wherein the machine learning method includes a process of machine training using a CCL26 marker; Preferably, the machine learning method includes a decision tree model, a random forest model, a K-nearest neighbor algorithm model, a naive Bayes model, a support vector machine model, and a neural network model.

8. An early medical auxiliary diagnosis system for colorectal adenoma, characterized in that: The diagnostic system includes a result determination module for comparing a processed value of a marker obtained by detecting the gene or protein expression level of a CCL26 marker with a set value; Preferably, the diagnostic system comprises a processing module for detecting and processing the expression level of a gene or protein associated with a marker in a sample to obtain a processed value; Preferably, the diagnostic system comprises an output module for outputting the obtained diagnostic results.

9. Use of the CCL26 marker in the preparation of a pharmaceutical composition for treating and / or preventing individual colorectal adenoma.

10. A scoring device for assessing the risk of a subject suffering from colorectal adenoma, characterized in that: The scoring device comprises the following units: Detection unit: detecting the gene or protein expression level of markers such as CCL26 in samples; Analysis unit: using the gene or protein expression level of the detected marker as an input variable and inputting it into the risk prediction model described in claim 7 for analysis; Scoring unit: outputs the risk value of colorectal adenoma for the subject corresponding to the sample.

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