Application of methylation level of specific region of ITGA4 gene in diagnosis and prediction of colorectal cancer

Abstract

The invention discloses application of a detection reagent for the methylation level of a specific region of an ITGA4 gene in preparation of a kit for diagnosing colorectal cancer or predicting the risk of the colorectal cancer, and belongs to the technical field of cancer molecular diagnosis. Wherein the specific region comprises at least one part of chr < 2 >: 181457330 to 181457528, and at least one part of chr < 2 >: 181457528. The methylation level of a specific region of the ITGA4 gene is provided as a marker for early diagnosis or prediction of the colorectal cancer, and choices of technicians in the field are enriched. The methylation level of a specific region of the ITGA4 gene is detected by adopting a methylated DNA co-immunoprecipitation technology, the sensitivity is high, the specificity is strong, and the kit has very important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer molecular diagnosis, and in particular, relates to the application of the methylation level of a specific region of the ITGA4 gene in the diagnosis and prediction of colorectal cancer. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors in the world, and its incidence and mortality rates are increasing year by year. In China, data from the National Cancer Center in 2023 showed that colorectal cancer surpassed gastric cancer to become the second most common cancer, and nearly 80% of patients were diagnosed in the middle and late stages, and nearly half of the patients survived for less than 5 years. Therefore, reducing the incidence and mortality of colorectal cancer has become a major public health issue that needs to be urgently addressed in China and even the world.

[0003] According to the multi-stage theory of carcinogenesis, the occurrence of colorectal cancer is morphologically manifested as a staged evolution from normal mucosal hyperplasia, adenoma formation, adenoma carcinomatosis to invasive metastasis. It takes 10-15 years for adenoma to evolve into colorectal cancer. The cure rate of colorectal cancer detected in the early stage can reach more than 90%, while that of advanced cancer is less than 10%. Intervention through screening is an effective measure to reduce the incidence and mortality of colorectal cancer.

[0004] Colonoscopy is the gold standard for colorectal cancer screening, but due to its high invasiveness and cumbersome intestinal preparation, the compliance of Chinese people with colonoscopy screening is relatively low. In addition, there is a large demand for colonoscopy. With the aging of the population, the number of elderly people and people over 40 years old is increasing, and the number of people undergoing colonoscopy has shown a blowout growth. The large-scale use of colonoscopy for screening will also cause a huge waste of resources. The traditional screening program adopts a two-step screening model combining a questionnaire survey with two fecal occult blood tests (FIT). Anyone who is positive in any one of the items is judged as a positive in the initial screening, indicating that they are at high risk and need to undergo a colonoscopy. This screening has problems such as too high false positives and low colorectal cancer detection rate. In addition, this screening model results in insufficient manpower investment in the hospital, so the project is progressing slowly.

[0005] The current marketed products of colorectal cancer auxiliary diagnosis technology based on blood DNA detection generally have low sensitivity, less than 85%, which cannot meet clinical needs. Clinical practice shows that the main limitation of blood Septin9 methylation detection is that its sensitivity for identifying colorectal cancer and precancerous lesions (adenomas) is relatively low, and the sensitivity for advanced adenomas is only 7.9% to 38.7%.

[0006] The colorectal cancer screening technology based on fecal DNA detection mainly targets the gene mutations and / or methylation and other characteristics of colorectal exfoliated cells, overcomes the main defect of detecting trace bleeding, has single-target and multi-target schemes, and can also be combined with FIT for detection, with the advantages of no need for special equipment, no dietary restrictions, and non-invasive. Although there is a significant improvement in the sensitivity and specificity of colorectal cancer, the detection rate for stage 0-II early cancers is still below 90%, and the detection rate for precancerous lesions and advanced adenomas such as high-grade intraepithelial neoplasia is even lower, all less than 65% or even lower. Although the detection of fecal DNA can achieve home sampling, fecal sampling is relatively private and inconvenient, which is different from the public's medical treatment habits. The public still needs a very long period of health publicity and education to accept fecal DNA detection, so there are still certain problems with detection compliance in the promotion of screening.

[0007] Therefore, it has become an urgent problem to develop new colorectal cancer markers with higher sensitivity based on blood samples with the highest user compliance in clinical practice. Summary of the Invention

[0008] In order to solve the above technical problems, the inventors of the present invention have made a lot of efforts to develop colorectal cancer methylation markers effective in blood based on non-bisulfite technology, and these markers make it possible to diagnose cancer and the risk of canceration at an early stage. Unexpectedly, it was found that a specific region of the ITGA4 gene is methylated in colorectal cancer cells. Using this gene as a biomarker and detecting the methylation level of this gene through a non-bisulfite-treated methylation detection system has high sensitivity and can diagnose colorectal cancer, thus completing the present invention.

[0009] The first aspect of the present invention provides the use of a detection reagent for the methylation level of a specific region of the ITGA4 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer.

[0010] The ITGA4 gene encodes a member of the integrin alpha chain protein family. Integrins are heterodimeric integral membrane proteins composed of an alpha chain and a beta chain, which play a role in cell surface adhesion and signal transduction. The encoded preproprotein undergoes proteolytic processing to generate a light chain and a heavy chain containing the alpha4 subunit. This subunit binds to the beta1 or beta7 subunit to form an integrin that may play a role in cell movement and migration. This integrin is a therapeutic target for the treatment of multiple sclerosis, Crohn's disease, and inflammatory bowel disease. Alternative splicing results in multiple transcript variants.

[0011] Almost all tumors are caused and promoted by genetic alterations and epigenetic variations together. By comparing tumor cells and normal cells, a large number of epigenetic abnormalities have been reported, among which DNA methylation is the most common epigenetic effect.

[0012] Methylation of cytosine on DNA is a covalent "epigenetic" modification of DNA. DNA methylation is carried out by DNA cytosine methyltransferases (DNMTs). DNMTs can transfer a methyl group from an S-adenosylmethionine to the C-5 position of cytosine. DNA methylation almost specifically occurs at CpG dinucleotide positions. CpG dinucleotides are unevenly distributed in the human genome, and regions of concentrated enrichment are generally called CpG islands. Such CpGs exist in repetitive sequences of the human genome and regulatory regions at the 5' ends of many genes. Abnormal DNA methylation in tumors includes two types: hypomethylation (or demethylation) and hypermethylation. Hypermethylation occurs in, but is not limited to, tumor suppressor genes, and hypomethylation occurs in, but is not limited to, proto-oncogenes.

[0013] All aspects of tumorigenesis and development may be related to changes in DNA methylation, involving cell cycle regulation, DNA damage repair, biochemical metabolism of carcinogenic compounds, apoptosis, and angiogenesis. Different types of tumors may have a specific group of hypermethylated tumor suppressor genes and hypomethylated proto-oncogenes, that is, there is a specific methylation map for a cancer type, and the type of cancer may be confirmed based on the methylation map.

[0014] In the present invention, through long-term exploration and verification with a large number of clinical samples, the inventors unexpectedly found that the methylation level in a specific region of the ITGA4 gene is significantly different between colorectal cancer and non-colorectal cancer.

[0015] In some embodiments of the present invention, the methylation region of the specific region of the ITGA4 gene is obtained in colorectal cancer population samples and normal samples using a probe combination. The coverage information of each probe in the probe combination is as follows:

[0016]

[0017] In some specific embodiments of the present invention, the specific region includes at least a part of chr2:181457330-181457528. There are 25 methylation sites in chr2:181457330-181457528, which are chr2:181457331-181457332, chr2:181457339-181457340, chr2:181457345-181457346, chr2:181457352-181457353, chr2:181457356-181457357, chr2:181457371-181457372, chr2:181457376-181457377, chr2:181457382-181457383, chr2:181457393-181457394, chr2:181457395-181457396, chr2:181457403-181457404, chr2:181457412-181457413, chr2:181457419-181457420, chr2:181457429-181457430, chr2:181457441-181457442, chr2:181457445-181457446, chr2:181457451-181457452, chr2:181457453-181457454, chr2:181457466-181457467, chr2:181457471-181457472, chr2:181457475-181457476, chr2:181457480-181457481, chr2:181457484-181457485, chr2:181457486-181457487, chr2:181457494-181457495.

[0018] In some preferred embodiments of the present invention, the specific region may include one or more of the above methylation sites, which may be continuous or spaced apart. In some more preferred embodiments of the present invention, the specific region may include a total of 8 methylation sites in the region of chr2:181457371-181457420. In some most preferred embodiments of the present invention, the specific region includes chr2:181457371-181457420. Further, the specific region may be extended by several bases on this basis. In some specific embodiments of the present invention, the specific region includes chr2:181457370-181457425.

[0019] In some embodiments of the present invention, the detection reagent includes a methylation region enrichment or methylation treatment reagent, and also includes a qPCR detection reagent.

[0020] Methylation enrichment technology is an analytical method for studying methylation modifications on DNA. DNA methylation is an important epigenetic modification that involves the addition of a methyl group to the cytosine ring in the DNA molecule. This modification plays a key role in regulating biological processes such as gene expression, cell differentiation, and genomic stability. Therefore, understanding the status of DNA methylation is very important for understanding biological processes and the occurrence and development of diseases.

[0021] Common methylation enrichment techniques include methylation-specific PCR (MSP), methylation-sensitive restriction enzyme digestion, MeDIP-Seq (Methylated DNA Immunoprecipitation sequencing), and MBD-Seq (Methyl-CpG Binding Domain sequencing), etc., among which:

[0022] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments by PCR. It is simple, fast, and suitable for the analysis of specific CpG sites, but it cannot provide genome-wide methylation information and is only applicable to pre-determined target regions.

[0023] Methylation-sensitive restriction enzyme digestion utilizes the sensitivity difference of restriction enzymes to DNA sequences to distinguish methylated and non-methylated DNA regions. This method is based on the principle that DNA methylation affects the sensitivity of base pairs on the cytosine ring to restriction enzymes. This technique does not require the use of expensive sequencing technologies and can be analyzed by methods such as gel electrophoresis. However, it cannot provide high-resolution information on individual CpG sites and usually provides the methylation status of the overall region. At the same time, it is limited by the specificity of the selected restriction enzyme, and some methylation sites may be missed or over-detected. In addition, it cannot directly distinguish 5-methylcytosine from other forms of DNA modifications.

[0024] MeDIP-Seq uses a methylation DNA antibody to selectively enrich methylated DNA fragments, and then analyzes the enriched product by high-throughput sequencing technology. It can enrich the entire methylated genomic region and is suitable for genome-wide methylation analysis. However, it cannot provide high-resolution information on individual CpG sites.

[0025] MBD-Seq uses methylated DNA-binding proteins (such as MBD2 or MBD3) to enrich methylated DNA fragments, which are then analyzed by sequencing. It can provide a high enrichment efficiency and is suitable for genome-wide methylation analysis. However, similar to MeDIP-Seq, it cannot provide high-resolution information on individual CpG sites.

[0026] In the present invention, the methylation treatment is also referred to as methylation conversion. Common methylation treatment sequencing techniques include bisulfite sequencing (BS-seq). BS-seq uses bisulfite to treat DNA, converting unmethylated cytosine into uracil, while methylated cytosine remains unaffected, and then it is analyzed by sequencing. It can provide high-resolution information on individual CpG sites and can perform genome-wide methylation analysis. However, the experimental steps are relatively cumbersome.

[0027] In some embodiments of the present invention, the methylated DNA immunoprecipitation (MeDIP) technique is used to enrich methylated fragments. The methylated DNA antibody is selected from one of the 5-methylcytidine antibody, 5-methylcytosine (5-mC) antibody, 5-hydroxymethylcytosine (5-hmC) antibody, 5-formylcytosine (5-fC) antibody, and 5-carboxylcytosine (5-caC) antibody.

[0028] In some preferred embodiments of the present invention, the methylated region enrichment reagent includes a 5-methylcytosine antibody.

[0029] Furthermore, the qPCR detection reagent includes a primer pair and a probe targeting the specific region.

[0030] Furthermore, the specific region includes at least a part of chr2:181457330-181457528. Preferably, the methylated region is chr2:181457370-181457425. The primer pair is as shown in SEQ ID No.1 and SEQ ID No.2, and the probe is as shown in SEQ ID No.3.

[0031] In some other embodiments of the present invention, the methylation treatment reagent includes bisulfite.

[0032] Furthermore, the qPCR detection reagent includes a primer pair and a probe targeting the sequence of the specific region after the methylation treatment.

[0033] Furthermore, the specific region includes at least a part of chr2:181457330-181457528, preferably, the methylated region is chr2:181457370-181457425, the primer pair is shown as SEQ ID No.4 and SEQ IDNo.5, and the probe is shown as SEQ ID No.6.

[0034] It is possible to achieve early detection of cancer by performing relevant methylated DNA analysis on a certain type of cancer. The current mainstream methylation analysis method is bisulfite treatment, which includes denaturation, deamination and desulfonation. DNA is first denatured into a single strand, and then subjected to high temperature, high salt, acidic and alkaline environments, encountering two extremes of ice and fire. The morphology of the converted DNA obtained is: mainly single strand, double strand mixed, fragment nicks, gap damage, and uracil state nucleotides. This process generally causes 90% of the DNA template to be lost, and a large amount of methylation information cannot be detected by subsequent processes. At the same time, during the base conversion treatment, there is an incomplete sequence conversion or over-conversion, which produces artificial bias, and the subsequent PCR amplification will further amplify it, resulting in inaccurate signals. Therefore, the methylation markers obtained based on bisulfite treatment generally have the problem of low sensitivity, especially in blood samples. After the limited number of free DNA fragments are treated with bisulfite, the difficulty of detecting methylation levels is greatly increased.

[0035] Although the sensitivity for colorectal cancer can be improved by increasing the number of methylated genes based on bisulfite treatment, such as the combined detection of 3 colorectal cancer gene methylation by Beijing Aikelun Medical Technology Co., Ltd., which was approved by the National Medical Products Administration NMPA in 2022. The sensitivity of clinical trials using blood sample cfDNA treated with bisulfite was 84.75% (328 / 387), but the sensitivity still cannot meet the actual clinical needs. Therefore, trying to discover colorectal cancer-related gene methylation markers in human blood samples based on non-bisulfite treatment methods and effectively detect changes in their methylation levels has also become the most urgent need for early cancer screening.

[0036] The second aspect of the present invention provides the use of a detection reagent for the methylation level of a specific region of the ITGA4 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method:

[0037] S1, obtain cfDNA samples from biological samples of subjects;

[0038] S2, enriching methylated regions of cfDNA samples or performing methylation treatment;

[0039] S3. Using the product enriched or processed in step S2 as a template, perform qPCR amplification with a primer pair and a probe targeting the untreated or treated specific region.

[0040] If there is a typical amplification curve and the Ct value is not greater than the preset threshold, then it is diagnosed that the subject has colorectal cancer or is at risk of having colorectal cancer.

[0041] The specific region includes at least a part of chr2:181457330 - 181457528. Preferably, the methylation region includes at least a part of chr2:181457370 - 181457425.

[0042] An amplification curve is a graph that detects the accumulation of products during the polymerase chain reaction (PCR). It is generated by monitoring the increase in fluorescence signal in the reaction solution. The following are the characteristics of a typical PCR amplification curve:

[0043] Initial stage:

[0044] Threshold Cycle (Ct) value: In the early stage of the PCR reaction, the fluorescence signal may be low, but as the PCR products accumulate, the fluorescence signal gradually increases. The Ct value refers to the number of cycles required in the PCR reaction for the fluorescence signal to rise above a pre - set threshold. The lower the Ct value, the higher the starting amount of the target DNA in the sample.

[0045] Exponential growth stage:

[0046] Exponential phase: In the middle stage of the PCR reaction, the PCR products grow exponentially. At this time, the increase rate of the Ct value will accelerate, reflecting the exponential growth of the target DNA in the PCR reaction.

[0047] Plateau stage:

[0048] Plateau stage: In the later stage of the PCR reaction, the accumulation of PCR products reaches saturation and no longer grows exponentially. The PCR amplification curve in this stage forms a plateau, and the increase of the Ct value becomes slow.

[0049] In some embodiments of the present invention, the preset threshold is determined based on the representative value of the Ct values obtained by the same method from population non - colorectal cancer samples and / or population colorectal cancer samples. The representative value is selected from the mean, mode, median, first quartile or third quartile.

[0050] The third aspect of the present invention provides a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer by detecting the methylation level of a specific region of the ITGA4 gene based on DNA immunoprecipitation. The methylation region is chr2:181457370-181457425. The kit includes a 5-methylcytosine antibody, a primer pair targeting the methylation region, and a probe. The primer pair is shown as SEQ ID No.1 and SEQ ID No.2, and the probe is shown as SEQ ID No.3.

[0051] In some embodiments of the present invention, the diagnosis is early diagnosis. Specifically, the early stage is CRC I-II.

[0052] Advantages of the present invention

[0053] Compared with the prior art, the present invention has the following technical effects:

[0054] The present invention provides the methylation level of a specific region of the ITGA4 gene as a marker for early diagnosis or prediction of colorectal cancer, enriching the choices of those skilled in the art.

[0055] The present invention uses the methylation DNA immunoprecipitation technique to detect the methylation level of a specific region of the ITGA4 gene, with high sensitivity and strong specificity, and has very important clinical application value.

[0056] Based on the methylation level of a specific region of the ITGA4 gene, the present invention can experimentally detect early colorectal cancer and provide more marker choices for early colorectal screening. Description of the drawings

[0057] Figure 1 Shows the significantly different regions of the ITGA4 gene methylation level (visualized by IGV) in 8 randomly selected colorectal cancer positive samples and 8 normal healthy individuals.

[0058] Figure 2 Shows the significant difference in the ITGA4 methylation level (RPM index) between 548 colorectal cancer positive samples and 389 normal healthy individual samples.

[0059] Figure 3 Shows the ITGA4 methylation gene detection flow chart in Example 2 of the present invention.

[0060] Figure 4 Shows an ITGA4 amplification curve (A) based on qPCR detection by the methylation DNA immunoprecipitation enrichment method and an ITGA4 amplification curve (B) based on qPCR detection after bisulfite conversion treatment.

[0061] Figure 5The ROC curves (A) of qPCR detection based on the methylation DNA immunoprecipitation enrichment method and (B) of qPCR detection based on bisulfite conversion treatment are shown for 120 samples. Detailed implementation manners

[0062] Unless otherwise specified, implied from the context, or in accordance with the convention of the prior art, all parts and percentages in this application are based on weight, and the testing and characterization methods used are synchronized with the filing date of this application. Where applicable, any patents, patent applications, or published content referred to in this application are incorporated herein by reference in their entirety, and their equivalent family patents are also incorporated by reference, particularly the definitions of relevant terms in this field disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0063] The numerical ranges in this application are approximate values, so unless otherwise specified, they may include values outside the range. The numerical range includes all values from the lower limit value to the upper limit value increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or fractions greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (such as 1 to 5), 1 unit is usually regarded as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of the values between the lowest and highest values listed are considered to be clearly recorded in this application.

[0064] The terms "comprising", "including", "having" and their derivatives do not exclude the existence of any other components, steps or processes, and are independent of whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless expressly stated, all compositions using the terms "comprising", "including", or "having" in this application may contain any additional additives, excipients or compounds. In contrast, the term "consisting essentially of" excludes any other components, steps or processes from the scope described below any such term, except those necessary for the operating performance. The term "consisting of" does not include any components, steps or processes not specifically described or listed. Unless expressly stated, the term "or" refers to the individual members listed or any combination thereof.

[0065] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention will be further described in detail below with reference to the embodiments.

[0066] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention, and thus can be regarded as preferred embodiments for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, and still obtain the same or similar results, without departing from the spirit or scope of the present invention.

[0067] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All references cited herein and the materials to which they refer are hereby incorporated by reference.

[0068] Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein through routine experimentation. Such equivalents are intended to be encompassed by the claims.

[0069] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instrumentation and equipment used in the following examples are all conventional laboratory instrumentation and equipment unless otherwise specified; the test materials used in the following examples are all obtained from a conventional biochemical reagent store unless otherwise specified.

[0070] Example 1 Discovery of Colorectal Cancer-Specific Methylated Genes

[0071] To screen for biomarkers specifically methylated in colorectal cancer, the present invention collected blood samples, corresponding paired samples of homologous cancer tissues and adjacent tissues (if any) from a total of 548 clinical colorectal cancer patients from the First Affiliated Hospital of Zhejiang University School of Medicine and several other central hospitals, as well as blood samples and corresponding tissue samples (if any) from 389 clinical colorectal cancer-negative patients. It is required that the differentially methylated genes or regions screened in the blood samples be verified in the tissue samples at the same time to be methylation markers with more reliable clinical significance.

[0072] 1. Preparation of Methylated DNA Sample Library

[0073] (1) DNA Extraction

[0074] The extraction of cfDNA was performed using a commercial company's extraction kit according to the instructions in the manual.

[0075] Quality control of the concentration and fragment distribution of nucleic acids was performed using Qubit 4.0 and Qsep 100 respectively. The yield of cfDNA extracted from 2 mL of human plasma should be greater than 5 ng, and there should be an enrichment peak at or near 167 bp for cfDNA. When the yield is greater than 50 ng, fragment quality control was performed using Qsep 100 capillary electrophoresis. When there is large fragment contamination, magnetic beads were used for fragment screening to remove large fragments.

[0076] Genomic DNA of samples such as cancer tissues, adjacent tissues, and normal tissues was extracted using a conventional commercial kit or a self-prepared reagent according to the method described in the instruction manual. Genomic DNA can be fragmented by sonication or enzymatic digestion to obtain DNA with a length of about 200 bp for convenient library construction.

[0077] (2) Library construction

[0078] Commercial company library construction kits can be used according to the method described in the instruction manual. For example, Rapid Plus DNA LibPrep Kit for illumina (Cat.No.RK20208, ABclonal) or VAHTS Universal ProDNA Library Prep Kit for illuminaVazyme (Cat.No.ND608 - 02, Novoprotein) and other similar kits were used for end repair, adding an "A" tail, and connecting with Adapters. In this example, VAHTS Universal Pro DNA Library Prep Kit for illuminaVazyme (Cat.No.ND608 - 02, Novoprotein) was used for library construction.

[0079] (3) Methylated DNA immunoprecipitation

[0080] Methylation enrichment of cfDNA and tissue genomic DNA was carried out in different reactions. Each methylation enrichment reaction can simultaneously complete the mixing of 12 - 100 cfDNA libraries (the input amount of each cfDNA sample library is about 10 ng), and can simultaneously complete the mixing of 10 - 24 genomic DNA libraries (the input amount of each genomic DNA library is about 100 ng). Methylation enrichment based on the principle of 5mC antibody was performed using a commercial company methylation enrichment kit or a self-prepared reagent according to the method described in the instruction manual. In this example, zymoMeDIP kit (product number D5101 - A) was used. Then, after the methylation enrichment reaction, purification was carried out according to the instruction manual, and 10 - 12 rounds of conventional PCR amplification were performed using a universal sequencing primer pair to obtain a methylated DNA fragment library with a yield of more than 500 ng for each reaction.

[0081] 2. Prepare the DNA probe library

[0082] (1) Probe design

[0083] The inventors designed probes for the sequences including the ITGA4 gene, its promoter region, 5'-UTR, the first exon (exon1), and the sequence within 1 kb upstream of the start codon in accordance with the principle of full coverage without crossing.

[0084] The inventors designed a set of hybridization capture DNA probe libraries consisting of 21 DNA probes in total, covering all possible CpG sites, including coding genes and microRNA genes.

[0085] (2) Probe synthesis

[0086] The probe coverage information is shown in Table 1.

[0087] Table 1 Probe coverage information

[0088]

[0089] Customize the probe panel at Nuoanda (Nanjing) Biotechnology Co., Ltd.

[0090] 3. DNA capture probe hybridization

[0091] Use NadPrep hybridization capture reagent (Cat.No.REF1005101, Nuoanda) for liquid-phase hybridization capture. The hybridization capture reaction can be single hybridization or multiple hybridizations. The total input amount of the MeDIP amplification library for each hybridization capture reaction should be in the range of 300 ng to 8 μg. 500 ng of the purified library (if less than 500 ng, then all is input), and Human Cot DNA and Nano Blockers are added, and then placed in a vacuum concentrator preheated to 42°C and dried at a rotation speed of 1000 rpm; after drying, the prepared hybridization reaction solution (containing the above probe panel) is added, shaken and centrifuged instantaneously, and hybridized and captured for 4 - 16 hours under the hybridization program: 95°C / 30 sec; 65°C / Hold (100°C hot lid); then the washed streptavidin magnetic beads are added to the hybridization system and incubated for 40 minutes, during which vortex mixing is performed every 10 minutes to ensure that the magnetic beads are completely resuspended; it should be noted that the reaction temperature of the hybridization capture is the conventional 65°C, rather than 63°C of the methylation probe designed based on bisulfite conversion.

[0092] After the hybridization capture reaction is completed, the combined magnetic beads are washed with the four washing solutions provided in the kit, and the residual liquid needs to be discarded in each step; finally, 20 μL of nuclease-free water is added and gently vortexed and mixed evenly.

[0093] 4. PCR Amplification and Purification after Hybridization Capture

[0094] Perform PCR amplification on the products after hybridization capture. The amplification reagents used are those in the VAHTS Universal Pro DNA Library Prep Kit for illumina (Cat.No.ND608 - 02, Vazyme Novizan), and the number of cycles is 12 - 13. After the amplification is completed, use an equal volume of VAHTS DNA Clean Beads (Cat.No.N411 - 03, Vazyme Novizan) to purify the products to obtain a relatively pure hybridization capture library; use Qubit 4.0 to quantitatively measure the library concentration, and use the Qsep100 fully automatic nucleic acid and protein analyzer to detect the fragment size of the library.

[0095] 5. Library Loading for Sequencing and Bioinformatics Analysis

[0096] Dilute the concentration of the library to be loaded onto the machine to 4 nM, mix it according to the proportion of the required data volume, and the total data volume should not exceed 120 G. After mixing, take out 5 μL of the library, add 5 μL of 0.2 N NaOH, pipette and mix well, denature for 5 minutes, and immediately add 990 μL of HT1 Buffer (REF: 15058251, illumina) after completion. After vortexing and mixing, take out 105 μL and add another 1295 μL of HT1 Buffer, and vortex and mix well to obtain the library for loading onto the machine, with a concentration of 1.5 pM.

[0097] The sequencer is the NextSeq 550Dx produced by illumina. The reagents used are High Output Reagent Cartridge v2 (REF: 15057929, illumina) (300 cycles), High Output Flow Cell Cartridge v2.5 (REF: 20022408, illumina), and Buffer Cartridge v2 (REF: 15057941, illumina). Add 1300 μL of the library for loading onto the machine to the sample position of the High Output Reagent Cartridge v2, and then add each reagent in turn to start the sequencing; in this example, paired - end sequencing is used, and the total duration is about 30 hours.

[0098] 6. Quality Control of Sequencing Data

[0099] The off-machine data was quality controlled using Fastp (version 0.22.0) to remove low-quality bases. The overall Q20 of the clean data was above 90%, and the Q30 was above 85%. The average sequencing depth was about 300×. The average on-target rate of the probes in the above probe combination was above 80%, indicating that the detection of cancer-related methylation regions based on the combination of methylation immunoprecipitation and liquid hybridization capture probes in this example is feasible and effective.

[0100] 7. Analysis of methylation differential regions of ITGA4 gene related to colorectal cancer

[0101] The DiffBind tool (version 3.8.4) was used to screen for differential peaks between tumors and non-tumors. Two algorithms, DESeq and EdgeR, were used, and the regions within the intersection and within the panel were preferentially screened.

[0102] In this example, from the regions of the ITGA4 gene covered by 34 probes, two characteristic methylation regions targeted by the two probes with the most significant differences between the colorectal cancer group and the non-colorectal cancer group were screened out: chr2:181457330-181457528 (promoter-TSS, NM_000885). The visualization of the IGV map of this characteristic methylation region (i.e., the specific region) is shown in Figure 1 .

[0103] The methylation CpG sites of this characteristic methylation region are shown in Table 2:

[0104] Table 2 CpG sites of characteristic methylation region

[0105]

[0106] The RPM index (Reads per million mapped reads) of this characteristic methylation differential region was analyzed in 548 colorectal cancer positive samples and 389 normal healthy population samples, and the P value was 0.0074, indicating a significant difference, as shown in Figure 2 .

[0107] Example 2 Detection of ITGA4 methylated gene in clinical samples and comparison of different treatment regimens

[0108] To further verify the clinical performance of the ITGA4 gene methylation differential region related to colorectal cancer in CRC plasma samples, the inventors used qPCR to detect 48 plasma samples clinically diagnosed with CRC and 72 plasma control samples with negative colonoscopy results. Among the 48 plasma samples clinically diagnosed with CRC, there were 6 samples in CRC stage I, 10 samples in CRC stage II, 15 samples in CRC stage III, and 17 samples in CRC stage IV.

[0109] The flowchart of ITGA4 methylation gene detection is as Figure 3 shown, specifically:

[0110] (1) DNA extraction

[0111] The extraction of cfDNA was performed using a commercial company's extraction kit according to the instructions in the manual.

[0112] Qubit4.0 and Qsep100 were used to perform quality control on the concentration and fragment distribution of nucleic acids respectively. The yield of cfDNA extracted from 4 mL of human plasma should be greater than 10 ng, and there should be an enrichment peak at 167 bp or nearby for cfDNA. When the yield is greater than 50 ng, Qsep100 capillary electrophoresis is required for fragmentation quality control. When there is contamination with large fragments, magnetic beads are used for fragment screening to remove large fragments.

[0113] (2) Methylated DNA processing

[0114] ① Methylated DNA immunoprecipitation

[0115] Take 1 / 2 of the total amount of the extracted nucleic acids above for cfDNA methylation enrichment, which is carried out in different reactions. For methylation enrichment based on the principle of 5mC antibody, the zymoMeDIP kit (product number D5101-A) is used, and after the methylation enrichment reaction, purification is carried out according to the manual, with an elution volume of 50 μL.

[0116] ② Bisulfite conversion of methylated DNA

[0117] Take 1 / 2 of the total amount of the extracted nucleic acids above for bisulfite treatment of cfDNA methylation, which is carried out in different reactions. For the treatment of methylated DNA based on the principle of bisulfite conversion, the DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH biological company is used for bisulfite treatment of DNA. The elution volume is 50 μL.

[0118] (3) qPCR detection

[0119] The primers and probes were synthesized by Shanghai Bioer Technology Co., Ltd., and the specific sequence information is as follows:

[0120] The sequences of Taqman MGB probe primers enriched by methylated DNA immunoprecipitation are shown in Table 3.

[0121] Table 3 Taqman MGB probe primers enriched by methylated DNA immunoprecipitation

[0122]

[0123] The 3'-end of the probe is labeled with MGB.

[0124] Using the enriched product by methylated DNA immunoprecipitation as the template, PCR amplification was carried out. The final concentration of each primer was 10 μM. The PCR reaction system was 5 μL of the enriched template DNA, 1.25 μL of the premixed solution containing the above primers; 12.5 μL of the PCR reaction solution reagent (2×Rapid Taq Master Mix), and the total volume was made up to 25 μL with water. The PCR reaction conditions were as follows: 95°C for 30 seconds, 95°C for 10 seconds, 60°C for 30 seconds, and amplified for 45 cycles.

[0125] The sequences of Taqman MGB probe primers after bisulfite conversion are shown in Table 4.

[0126] Table 4 Taqman MGB probe primers after bisulfite conversion

[0127]

[0128] The 3'-end of the probe is labeled with MGB.

[0129] Using the DNA after bisulfite conversion as the template, PCR amplification was carried out. The final concentration of each primer was 10 μM. The PCR reaction system was 5 μL of the enriched template DNA, 1.25 μL of the premixed solution containing the above primers; 12.5 μL of the PCR reaction solution reagent (2×Rapid Taq Master Mix), and the total volume was made up to 25 μL with water. The PCR reaction conditions were as follows: 95°C for 30 seconds, 95°C for 10 seconds, 60°C for 30 seconds, and amplified for 45 cycles.

[0130] (4) Analysis of clinical sample test results

[0131] The off-machine data was analyzed. The detection results of 120 samples by using the method of enriching methylated DNA immunoprecipitation and the method of treating DNA with bisulfite followed by qPCR are shown in Table 5.

[0132] Table 5 Summary table of qPCR detection results of 120 samples

[0133]

[0134]

[0135]

[0136]

[0137] ITGA4 Figure 4 Examples of qPCR amplification curves of samples from the same CRC patient (sample number: 101712) using the methylated DNA immunoprecipitation enrichment method and the bisulfite-treated DNA method are shown. Among them, the Ct value of qPCR amplification after methylated DNA immunoprecipitation enrichment in Figure A is 31.59, and the Ct value of qPCR amplification of bisulfite-treated DNA in Figure B is 36.18. There is an obvious advantage in qPCR detection after methylated DNA immunoprecipitation enrichment.

[0138] Set the Ct value of samples with a detection result of Undetermined to 45, and draw ROC curves respectively, as Figure 5 shown. The areas under the ROC curves (AUC) of the ROC curves obtained by the two different methods are 0.797 and 0.699 respectively. According to the ROC curve, set the cut-off value for different methods: for qPCR detection based on the methylated DNA immunoprecipitation enrichment method, the cut-off value is set to Ct = 34.85; for qPCR detection based on bisulfite conversion treatment, the cut-off value is set to Ct = 36.55. If the Ct value of the ITGA4 gene amplification of the sample to be monitored is equal to or lower than the set cut-off value, the sample is determined to be a positive sample, otherwise it is determined to be a negative sample. Thus, the detection results of 120 samples are statistically analyzed.

[0139] Table 6 shows the comparison between qPCR detection based on the methylated DNA immunoprecipitation enrichment method and the colonoscopy examination results (gold standard). Table 7 shows the comparison between the qPCR detection results based on bisulfite conversion treatment and the colonoscopy examination results. Table 8 shows the comparison between the qPCR detection results based on the methylated DNA immunoprecipitation enrichment method and the qPCR detection results based on bisulfite conversion treatment.

[0140] Table 6 Comparison of qPCR detection results based on the methylated DNA immunoprecipitation enrichment method with colonoscopy examination results

[0141]

[0142] Table 7 Comparison of qPCR detection results based on bisulfite conversion treatment with colonoscopy examination results

[0143]

[0144]

[0145] Table 8 Comparison of enrichment methods based on methylated DNA immunoprecipitation and bisulfite conversion

[0146]

[0147] As can be seen from Tables 6 - 8, when the methylation differential region of the ITGA4 gene was verified on the qPCR detection platform based on the enrichment method of methylated DNA immunoprecipitation, it had higher sensitivity (75.00%) for CRC, and also maintained high specificity (86.11%) for non-colorectal cancer samples, with an accuracy of 81.67%. The overall performance was superior to that of the qPCR detection based on bisulfite conversion.

[0148] (5) Analysis of the detection results of clinical colorectal cancer stage samples

[0149] Among the above 48 plasma samples clinically diagnosed as CRC, there were 6 samples in CRC stage I, 10 samples in CRC stage II, 15 samples in CRC stage III, and 17 samples in CRC stage IV. The detection and statistical analysis of samples with different CRC pathological stages by qPCR detection based on the enrichment method of methylated DNA immunoprecipitation and qPCR detection based on bisulfite conversion are shown in Tables 9 - 10:

[0150] Table 9 Detection of samples with different CRC pathological stages

[0151]

[0152] Table 10 Statistical analysis of sensitivity for different CRC pathological stages

[0153]

[0154]

[0155] As can be seen from Tables 9 - 10, when the methylation differential region of the ITGA4 gene was verified on the qPCR detection platform based on the enrichment method of methylated DNA immunoprecipitation, it also maintained high sensitivity (83.33%) for CRC stage I samples, and its performance for early CRC detection was superior to that of the qPCR detection based on bisulfite conversion, providing a new potential biomarker for early detection of colorectal cancer.

[0156] All the documents mentioned in the present invention are cited herein for reference as if each document was individually cited for reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. Use of a reagent for detecting the methylation level of a specific region of the ITGA4 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer, characterized in that, The specific region includes at least a part of chr2:181457330-181457528.

2. The application according to claim 1, wherein The detection reagent includes a methylation region enrichment or methylation treatment reagent, and also includes a qPCR detection reagent.

3. The application according to claim 2, characterized in that, The methylation region enrichment reagent includes a methylated DNA antibody.

4. The application according to claim 3, characterized in that The qPCR detection reagent includes a primer pair and a probe targeting the specific region.

5. The application according to claim 4, characterized in that, The specific region is chr2:181457370-181457425, the primer pair is as shown in SEQ ID No.1 and SEQ ID No.2, and the probe is as shown in SEQ ID No.

3.

6. The application according to claim 2, characterized in that, The methylation treatment reagent includes bisulfite.

7. The application according to claim 6, wherein The qPCR detection reagent includes a primer pair and a probe targeting the treated specific region.

8. The application according to claim 7, characterized in that, The specific region is chr2:181457370-181457425, the primer pair is as shown in SEQ ID No.4 and SEQ ID No.5, and the probe is as shown in SEQ ID No.

6.

9. Application of a detection reagent for the methylation level of a specific region of the ITGA4 gene in preparing a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method: S1, obtaining a cfDNA sample from a biological sample of a subject; S2, enriching the methylation region of the cfDNA sample or performing methylation treatment; S3, using the product enriched or treated in step S2 as a template, and performing qPCR amplification with a primer pair and a probe targeting the untreated or treated specific region; If there is a typical amplification curve and the Ct value is not greater than a preset threshold, it is diagnosed that the subject has colorectal cancer or has a risk of developing colorectal cancer. The specific region includes at least a part of chr2:181457330-181457528.

10. A kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer by detecting specific regions of the ITGA4 gene based on DNA immunoprecipitation, characterized in that, The specific region is chr2:181457370-181457425, the kit includes a 5-methylcytosine antibody, a primer pair and a probe targeting the specific region, the primer pair is as shown in SEQ ID No.1 and SEQ ID No.2, and the probe is as shown in SEQ ID No.3.

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