Application of biomarker CCL26 in diagnosis of colorectal adenoma

Abstract

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

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to application of biomarker CCL26 in diagnosis of colorectal adenoma. BACKGROUND

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

[0003] The existing colorectal adenoma screening technology mainly relies on colonoscopy. Since endoscopy is an invasive operation, the patient compliance is low, and the endoscopy resources are limited and affected by the technical level of the operator, the examination results are uneven and the missed diagnosis rate is high. At the same time, the sensitivity and specificity of some markers for detecting colorectal adenoma in the existing technology are poor, resulting in low overall detection rate of adenoma. SUMMARY

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

[0005] The first aspect of the present application provides an application 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 comprises a CCL26 marker.

[0006] Further, the sample comprises blood, plasma, serum, saliva, urine, feces, cerebrospinal fluid, semen, vaginal secretion, 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 exhaled breath of a subject.

[0007] Further, the sample comprises blood, plasma, serum, tissue biopsy or epithelial cells.

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

[0009] Further, the sample is plasma.

[0010] Further, the sample is blood.

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

[0012] The term "CCL26" as used herein is intended to encompass 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 publicly available on 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 human are well known.

[0013] The term "colorectal adenoma" as used herein refers to a neoplastic polyp located in the mucosal epithelial tissue derived from the colorectal site. In the present invention, "colorectal adenoma" can include a case where adenoma occurs only in the colon site (which can also be referred to as "colonic adenoma"), only in the rectal site (which can also be referred to as "rectal adenoma"), or in both the colon and rectum.

[0014] The term "marker" as used herein refers to a gene or gene product that is a target for modulating one or more target phenotypes (e.g., target phenotypes in myeloid cells). In this context, the term "marker" is synonymous with "target." However, in some embodiments, the term also encompasses measurable entities that have been determined to be indicative of a target output, e.g., one or more diagnostic, prognostic, and / or therapeutic outputs (e.g., for modulating an inflammatory phenotype, a cancer state, etc.). In other embodiments, the term also encompasses compositions that modulate a gene or gene product.

[0015] The term "test sample," "sample" as used herein can be any biological sample isolated from a subject. The term "sample" includes any biological sample that can be extracted from a subject, untreated, treated, diluted, or concentrated. Samples can include, but are not limited to, biological fluids such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, stool (i.e., fecal), tears, sweat, sebum, nipple aspirate, ductal lavage, tumor exudate, synovial fluid, ascites fluid, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymphatic fluid, fine needle aspirate, amniotic fluid, any other body fluid, cell lysate, cell secretion, inflammatory fluid, semen, and vaginal secretion. Samples can include tissue samples and biopsy samples, tissue homogenates, and the like. Advantageously, samples can include samples that contain in detectable amounts any one or more biomarkers taught herein. Suitably, samples can be readily obtained by minimally invasive procedures, thereby allowing the sample to be removed or isolated from the subject. In certain embodiments, the sample comprises blood, particularly peripheral blood, or a fraction or extract thereof. Typically, the sample comprises blood cells, such as mature, immature, or developing white blood cells, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, hemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells, or natural killer cells, or a fraction (e.g., nucleic acid or protein fraction) of such cells.

[0016] Further, the substance for detecting the marker in the test sample includes a reagent used in western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip assay.

[0017] The term "agent" as used herein includes a compound that induces a desired pharmacological and / or physiological effect. The term also encompasses pharmaceutically acceptable and pharmacologically active components of those compounds specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, and the like. When the above term is used, it is understood that this includes the active agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, and the like. The term "agent" is not to be construed narrowly, but is to be expansively construed to include small molecules, protein molecules such as peptides, polypeptides, and proteins, and compositions containing them, as well as genetic molecules such as RNA, DNA, and mimetics and chemical analogs thereof, and cellular agents.

[0018] The second aspect of the present application provides use of a substance for detecting a marker in a test sample in the manufacture of a product for predicting or assisting in predicting colorectal adenoma, the marker comprising a CCL26 marker.

[0019] Further, the sample comprises blood, plasma, serum, sputum, urine, stool, cerebrospinal fluid, semen, vaginal secretion, sputum, sweat, breast milk, synovial fluid, mucus, tears, bile, gastric juice, interstitial fluid, biopsy of tissue or epithelial cells, oral swab, nasal swab, aqueous humor, amniotic fluid, pleural fluid, or exhaled breath of the subject.

[0020] Further, the sample comprises blood, plasma, serum, biopsy of tissue or epithelial cells.

[0021] Further, the sample comprises biopsy of tissue or epithelial cells.

[0022] Further, the sample is plasma.

[0023] Further, the sample is blood.

[0024] Further, the sample is biopsy of tissue or epithelial cells.

[0025] The third aspect of the present application provides a product comprising a substance for detecting CCL26 marker.

[0026] Further, the product comprises a product for diagnosing or aiding in diagnosing whether the sample to be tested is a colorectal adenoma sample, a product for diagnosing or aiding in diagnosing whether the subject to be tested is a colorectal adenoma patient, a product for predicting or aiding in predicting whether the sample to be tested is a colorectal adenoma sample, a product for predicting or aiding in predicting whether the subject to be tested is a colorectal adenoma patient.

[0027] The term "diagnosing" as used in the present application refers to the identification or classification of a molecular or pathological state, disease, or disorder. The term "aiding in diagnosing" refers to a method that aids a clinician in determining the presence or nature of a symptom or condition that is characteristic of a particular type of disease or illness. For example, an aiding in diagnosing method of a disease or disorder can include measuring certain biomarkers in a biological sample from an individual.

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

[0029] Further, the product comprises a reagent, a kit, a test paper, a nucleic acid membrane strip, a chip, a primer, a probe.

[0030] Further, the subject to be tested comprises a human or a non-human mammal.

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

[0032] Further, the reagent includes a reagent for detecting the presence, absence and / or amount of the gene, fragment and protein of CCL26 in the sample by a PCR reaction, RT-PCR derivation reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, dual hybridization probe, complex probe, ISH, microarray, Southern blotting, Northern blotting, multi-analyte profiling test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemical assay, dot blot assay or slot blot assay.

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

[0034] Further, the gene detection kit includes a primer, reagent or chip for detecting the transcription level of the CCL26 gene.

[0035] Further, the protein detection kit includes a reagent, chip, antibody, ligand for detecting the protein level of CCL26 expression.

[0036] Further, the test paper includes a chromatographic test paper.

[0037] "Expression" used in the present application means transcription of a gene to produce an RNA transcript (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.) and, if appropriate, translation of the resulting mRNA transcript into a protein. Thus, it will be clear from context whether expression of a coding sequence results from transcription and translation of the coding sequence or from transcription of a non-coding sequence.

[0038] The term "level of expression" or "expression level" used in the present application is generally used interchangeably, and generally refers to the amount of biomarker in a sample. "Expression" generally refers to a process by which information (e.g., encoded genes and / or epigenetic) is transformed into structures that exist and are operable in a cell. Thus, as used herein, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even modification of a polynucleotide and / or polypeptide (e.g., post-translational modification of a polypeptide). 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 produced by alternative splicing or degraded transcripts, or from post-translational processing of polypeptides, such as by proteolysis. "Expressed genes" include those that are transcribed into polynucleotides (mRNAs) and then translated into polypeptides, as well as those that are transcribed into RNA but not translated into polypeptides (e.g., transfer RNAs and ribosomal RNAs).

[0039] The fourth aspect of the present application provides the use of a marker in constructing a computer model for diagnosing or assisting in diagnosing, predicting or assisting in predicting colorectal adenoma, the marker comprising a CCL26 marker.

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

[0041] Further, the memory is used to store the executed computer program.

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

[0043] The fifth aspect of the present application provides the use of a marker in screening candidate drugs for treating colorectal adenoma, the marker comprising a CCL26 marker.

[0044] The sixth aspect of the present application provides a colorectal adenoma risk prediction model, the diagnostic model comprising a risk prediction model constructed by model training using a machine learning method, the machine learning method comprising a process of machine training using a CCL26 marker.

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

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

[0047] Further, the diagnostic system comprises a processing module for processing the expression level of the marker-related gene or protein in the sample to obtain a processing value.

[0048] Further, the diagnostic system comprises an output module for outputting the obtained diagnostic result.

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

[0050] Further, the individual comprises a human or a non-human mammal.

[0051] Further, the individual is a human.

[0052] The term "pharmaceutical composition" or "pharmaceutical preparation" used in the present application refers to a preparation in a form that allows the biological activity of the active ingredient to be effective, and does not contain other components that have unacceptable toxicity to the subject to which the composition or preparation will be administered.

[0053] The ninth aspect of the present application provides a scoring device for evaluating the risk of colorectal adenoma in a subject, the scoring device comprising the following units:

[0054] The detection unit detects the expression level of the gene or protein such as CCL26 marker in the sample;

[0055] The analysis unit inputs the detected expression level of the gene or protein of the marker as an input variable into the risk prediction model of the sixth aspect of the present application for analysis;

[0056] The scoring unit outputs the risk value of colorectal adenoma in the subject corresponding to the sample.

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

[0058] 1) Collecting the sample of the subject to be detected;

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

[0060] 3) Comparing the expression level result with the expression level of the healthy control to determine whether the subject has colorectal adenoma or the risk of having colorectal adenoma.

[0061] The term "patient", "subject", "testee" or "individual" used in the present application refers to any subject in need of treatment or prevention, particularly a vertebrate subject, even more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present application include, but are not limited to, any member of the subphylum chordata, including a primate (e.g., humans, monkeys and apes, including monkey species (e.g., macaques (genus Macaca) (e.g., 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 great apes such as chimpanzees (Pan troglodytes)), a rodent (e.g., a mouse, a rat, a guinea pig), a rabbit (e.g., a rabbit, a hare), a bovine (e.g., a cow), an ovine (e.g., a sheep), a caprine (e.g., a goat), a porcine (e.g., a pig), an equine (e.g., a horse), a canine (e.g., a dog), a feline (e.g., a cat), an avian (e.g., a chicken, a turkey, a duck, a goose, a companion bird such as a canary, a parrot, etc.), a marine mammal (e.g., a dolphin, a whale), a reptile (a snake, a frog, a lizard, etc.), and a fish.

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

[0063] Further, the detection performed in step 2) also needs to use a reference gene for data normalization operation.

[0064] In the present application, P value, i.e. probability, reflects the likelihood of the occurrence of an event. According to the P value obtained by the significance test method, P<0.05 is generally considered as significant, and P<0.01 is considered as very significant, which means that the probability of the difference between samples caused by sampling error is less than the P value, i.e. probability, reflecting the likelihood of the occurrence of an event.

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

[0066] The CCL26 biomarker exhibits high sensitivity and specificity for detecting colorectal adenomas, enabling early identification and high diagnostic efficiency. CCL26 biomarkers are detectable in tissues and are also present in the blood, making the procedure convenient and eliminating the discomfort and poor patient compliance associated with invasive procedures. Attached Figure Description

[0067] Figure 1 This is a HE staining image of a collection of colorectal adenoma tissue and adjacent normal tissue; CRA represents colorectal adenoma tissue, and NOR represents adjacent normal tissue.

[0068] Figure 2 This is a graph showing the differential expression results of the test set CCL26 in colorectal adenoma tissue and adjacent normal tissue;

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

[0070] Figure 4 This is a HE staining image of a collection of colorectal adenoma tissue and adjacent normal tissue; CRA represents colorectal adenoma tissue, and NOR represents adjacent normal tissue.

[0071] Figure 5 This is an immunohistochemical staining image of CCL26; CRA represents adenoma tissue, and NOR represents normal tissue adjacent to the tumor; positive expression is indicated by yellowish-brown staining in the cytoplasm and blue staining in the nucleus;

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

[0073] Figure 7 This is the ROC curve for validating the use of CCL26 in the diagnosis of colorectal adenoma. Detailed Implementation

[0074] Example 1: The biomarker CCL26 is associated with the diagnosis of colorectal adenoma.

[0075] 1. Experimental subjects

[0076] The whole transcriptome sequencing (RNA-seq) test set included 12 cases of colorectal adenoma and 12 cases of adjacent normal tissue, for a total of 24 cases. Details are shown in Table 1. All samples were formaldehyde-fixed paraffin-embedded adenoma tissue surgically removed by colonoscopy at Wangjing Hospital between 2019 and 2023. All samples were subsequently confirmed as colorectal adenoma and adjacent normal tissue by two senior pathologists. Figure 1 ).

[0077] Table 1 RNA-seq test set patient clinical information

[0078]

[0079]

[0080] 2、Experimental apparatus

[0081] Agilent 2100 Bioanalyzer (Agilent), Qubit 2.0 Fluorometer (Invitrogen), Veriti 96-Well Multi-Block Thermal Cycler (MJ Research), Peltier Thermal Cycler PTC-225 (MJ Research), DNA Quantification Kit (KAPA Biosystem), NEBNext Multiplex Oligos for Illumina (NEB), High Sensitivity DNA Quantification Kit (Agilent), RNA 6000 Pico chip (Agilent).

[0082] 3、Experimental method

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

[0084] 3.1 Library construction and sequencing

[0085] Total RNA was extracted from colorectal adenoma and normal tissue adjacent to the tumor using Trizol, and the RNA integrity was tested using Agilent 2100 Bioanalyzer, with a quality requirement of RIN≥7 and 28S / 18S≥1.5:1. The starting Total RNA was accurately quantified using QUBIT RNA ASSAY KIT, with a starting amount requirement of 0.1-1 ug. mRNA in Total RNA was purified using Beads with Oligod(T), and mRNA was fragmented to the target length range using NEBNext Reaction Buffer, and then the first complementary DNA (cDNA) strand was synthesized using random primers, followed by synthesis of the second cDNA strand under the action of DNA polymerase I. The purified double-stranded cDNA was subjected to end repair, A tail addition and ligation of sequencing adapters, followed by PCR amplification. The PCR product was quality controlled using 2100 Bioanalyzer chip to obtain the final library, and the constructed cDNA library was sequenced through IILumina Novaseq 6000 sequencing platform.

[0086] 3.2 Data filtering and quality control

[0087] The original files of IILumina sequencing were subjected to base calling and converted into raw sequencing sequences (Sequenced Reads). The collection of raw sequencing sequences is called Raw Data. The Raw Data were filtered to remove reads that do not meet the analysis criteria to obtain Clean Reads for subsequent analysis. The filtering conditions are as follows: (1) remove contaminated samples; (2) if the reads contain adapter sequences, remove the adapter sequences first; (3) remove the bases with an average base quality < 15 in the sliding window of 4 bases at both ends of the reads; (4) remove reads pairs with high N content (if the N content in a read is > 5%, remove the entire pair of reads); (5) remove reads pairs with high low-quality base content (if the low-quality base in a read is > 30%, remove the entire pair of reads); (6) remove the entire pair of reads if the length of the reads after removal is < 100 bp; (7) remove unpaired reads.

[0088] 3.3 Sequence alignment and gene quantification

[0089] The index file of the reference genome was downloaded from the official website, and the Clean Reads were aligned to the reference genome sequence using Hisat2 software. The number of reads in the Clean Reads that align to the reference genome was required to be > 70% of the Clean Reads, and the number of reads that align to multiple positions of the reference genome was required to be < 10% of the number of reads that align to the reference genome. The read count of gene expression was calculated using featureCounts and StringTie software. Considering the length and depth of RNA sequences, the Fragments Per Kilobase of exon model per Million mapped fragments (FPKM) was calculated, and the FPKM value of gene expression was filtered: (1) remove genes with a length < 100 bp; (2) if the FPKM of a gene is < 0.0001, it is considered to be expressed as 0, and genes expressed as 0 in all samples are removed.

[0090] 3.4 Differential gene expression analysis

[0091] The data after quantification of gene expression was statistically analyzed, and the R language limma package and DESeq package were used to screen the differential expression genes (DEGs) of colorectal tissues and paraneoplastic normal tissues. The screening criteria were: |log2FC|>1 and P<0.05, wherein FC represents the fold change (FC), and P value represents the statistical significance of the difference (P-Value). At the same time, according to the FPKM value of gene expression, further screening was carried out to obtain the genes that were expressed in colorectal adenoma tissue but almost not expressed in normal tissue.

[0092] 3.5 Statistical analysis

[0093] Statistical analysis was performed using IBM SPSS Statistics 26.0 software. For measurement data conforming to normal distribution, mean ± standard deviation (x ± s) was used, and independent sample t test was used for comparison between two groups. For non-normal distribution, non-parametric test was used. P<0.05 indicates that the difference is statistically significant.

[0094] 4. Experimental results

[0095] According to the whole transcriptome sequencing result analysis, the expression amount 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), as shown in Table 2 and Table 3. In the evaluation of its diagnostic performance, the ROC curve showed that the AUC was 0.9444, at this time the sensitivity could reach 100%, and the specificity could reach 83.33%, as shown in Figure 2 、 Figure 3 .

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

[0097]

[0098] Note: CRA is colorectal adenoma tissue, NOR is paraneoplastic normal tissue.

[0099] Table 3 Gene expression quantification value (FPKM) of CCL26 in each sample of the test set

[0100] Sample No. Adenoma tissue FPKM value Paraneoplastic tissue FPKM value 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] Immunohistochemistry (IHC) verification set with 45 cases of colorectal adenomas and paraneoplastic tissues, a total of 90 cases (see Table 4). The samples used were adenomas fixed in formaldehyde and embedded in paraffin by endoscopy in Wangjing Hospital from 2019 to 2023. The research samples were diagnosed as colorectal adenomas and paraneoplastic normal tissues by 2 senior pathologists again (see Figure 4 ).

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

[0105]

[0106]

[0107] 2. Experimental apparatus

[0108] 4℃ / -20℃ refrigerator (BCD-196F) (Qingdao Haier Co., Ltd.), RM2235 microtome (Germany LEICA), HI1210 slice spreader (Germany LEICA), HI1210 slice oven (Germany LEICA), high-pressure steam sterilization pot (YX280) (Hefei Huatai Medical Equipment Co., Ltd.), electric heat air dryer (101-0AB) (Linmao Technology Beijing Co., Ltd.), triple constant temperature water bath (SH.W21.600) (Shanghai Shule Instrument Co., Ltd.), BX51 microscope (Japan OLYMPUS), hematoxylin counterstain solution (Beijing Shijiyili Biological Technology Co., Ltd.), DAB color reagent kit (Beijing Zhongshan Biotechnology Co., Ltd.), hydrogen peroxide solution (Beijing Haiderrun Pharmaceutical Group Co., Ltd.), CCL26 antibody reagent (bs-15513R) (Beijing Bioss Biological Technology Co., Ltd.), citric acid repair solution (pH 6.0) (Beijing Zhongsang Jinqiao Biological Technology Co., Ltd.), EDTA antigen repair solution (pH 8.0) (Beijing Zhongsang Jinqiao Biological Technology Co., Ltd.), PBS buffer powder (pH 7.3) (Beijing Zhongsang Jinqiao Biological Technology Co., Ltd.).

[0109] 3. Experimental method

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

[0111] (1) Tissue sectioning:

[0112] 1) The CRA paraffin sample was sectioned at a thickness of 2.5 μm;

[0113] 2) Unfold in a film spreader at 48°C;

[0114] 3) Bake in a film barker at 63°C for 1 h.

[0115] (2) Dewaxing and hydration:

[0116] 1) Soak the prepared sections in dewaxing solution 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 min and then in 80% ethanol for 1 min;

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

[0120] (3) Antigen retrieval:

[0121] 1) Put the sections into the EDTA or citric acid retrieval solution in a pressure cooker and heat to boiling;

[0122] 2) After the pressure cooker sprays, start timing for antigen retrieval;

[0123] 3) Timing for 3 min for EDTA and 2.5 min for citric acid;

[0124] 4) Cool the sections at room temperature for 20 min.

[0125] (4) Immunoreaction:

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

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

[0128] 3) Add the primary antibody and incubate in a humidity box at 37°C for 2 h;

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

[0130] 5) Add the secondary antibody and incubate in a humidity box at 37°C for 30 min;

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

[0132] (5) Chemical staining:

[0133] 1) Develop with DAB, check the staining degree under a microscope, and the positive expression is brownish yellow;

[0134] 2) Re-stain with hematoxylin for 5 min, separate the colors with a color separator, and then wash with an anti-blue solution for 3 times.

[0135] (6) Dehydrating and mounting:

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

[0137] 2) Soak in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, respectively;

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

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

[0140] 4. Statistical analysis

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

[0142] 5. Experimental results

[0143] It is found by analyzing the IHC staining results of CCL26 that the average optical density value of CCL26 in colorectal adenoma tissue is significantly higher than that in normal tissue adjacent to the tumor, and there is a very significant statistical difference (P<0.001). In the evaluation of its diagnostic efficiency, the ROC curve shows that the AUC is 0.8741, at this time the sensitivity can reach 88.89%, and the specificity can reach 77.78%, see Figure 5 、 Figure 6 、 Figure 7 .

[0144] The above description of the examples is only for understanding the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, the present application can be improved and modified in several ways, and these improvements and modifications will also fall within the scope of protection of the claims of the present application.

Claims

1. Use of a substance that detects a marker in a sample to be tested in the manufacture of a product for the diagnosis or the aid to the diagnosis of a colorectal adenoma, characterized in that, The marker comprises a CCL26 marker; and the sample is a tissue.

2. Use of a substance detecting a marker in a sample to be tested for the manufacture of a product for predicting or assisting in predicting colorectal adenomas, characterized in that, The marker comprises a CCL26 marker; and the sample is a tissue.

3. A model for predicting risk of colorectal adenoma, characterized in that, The diagnostic model comprises a risk prediction model constructed by model training using a machine learning method, the machine learning method comprising a process of machine training using a CCL26 marker; and the sample is a tissue.

4. The colorectal adenoma risk prediction model according to claim 3, wherein the machine learning method comprises a decision tree model, a random forest model, a K-neighbor algorithm model, a naive Bayes model, a support vector machine model, or a neural network model.

5. A medical aid diagnostic system for early detection of colorectal adenoma, characterized by, The diagnostic system comprises a result determining module for comparing a processing value of the marker obtained by detecting a gene or protein expression level of the CCL26 marker with a set value. The diagnostic system comprises a processing module for processing the expression level of the gene or protein related to the marker in the sample to obtain a processing value. The diagnostic system comprises an output module for outputting the obtained diagnostic result; and the sample is a tissue.

6. A scoring device for assessing the risk of a subject developing a colorectal adenoma, characterized in that, The scoring device comprises the following units: a detecting unit for detecting a gene or protein expression level of a marker such as a CCL26 marker in a sample; an analyzing unit for inputting the detected gene or protein expression level of the marker as an input variable into the risk prediction model according to claim 3 or 4 for analysis; a scoring unit for outputting a risk value of a subject suffering from colorectal adenoma corresponding to the sample; and the sample is a tissue.

Images