Application of TAC1 gene methylation level in diagnosis and prediction of colorectal cancer

Abstract

The invention discloses application of a TAC1 gene methylation level detection reagent in preparation of a kit for diagnosing colorectal cancer or predicting colorectal cancer risk, and belongs to the technical field of cancer molecular diagnosis. The invention provides a TAC1 gene methylation level as a marker for early diagnosis or prediction of colorectal cancer, and enriches choices of technicians in the field. The methylation level of the TAC1 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 specifically relates to the application of the methylation level of the TAC1 gene in the diagnosis and prediction of colorectal cancer. Background Art

[0002] Colorectal cancer is one of the most common malignant tumors globally, and its incidence and mortality rates show an increasing trend year by year. In China, according to the data of the National Cancer Center in 2023, colorectal cancer has surpassed gastric cancer to become the second most common cancer in terms of incidence, and nearly 80% of patients are found to be in the middle and advanced stages, with nearly half of the patients having a survival period of less than 5 years. Therefore, reducing the incidence and mortality of colorectal cancer has become a major public health problem that urgently needs to be solved in China and even globally.

[0003] According to the multi-stage theory of the carcinogenesis process, the occurrence of colorectal cancer morphologically shows a staged evolution from normal mucosal hyperplasia, adenoma formation, adenoma carcinogenesis to invasion and metastasis. It takes 10 - 15 years for an adenoma to evolve into colorectal cancer. The cure rate for early detection of colorectal cancer can reach over 90%, while that for 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 strong invasiveness and cumbersome bowel preparation, the compliance of Chinese people with colonoscopy screening is relatively low. In addition, the demand for colonoscopy is large. With the aging of the population, the number of the elderly and people over 40 years old is increasing, and the number of colonoscopy examinations shows a blowout growth. Large-scale application of colonoscopy as a screening method will also cause a great waste of resources. The traditional screening program adopts a two-step screening mode combining questionnaire surveys with two fecal occult blood tests (FIT). For those with any positive result, they are determined to be positive in the primary screening, indicating a high-risk population and requiring colonoscopy examination. This screening has problems such as too high false positives and low detection rate of colorectal cancer, and this screening mode results in insufficient human input in hospitals, thus causing slow progress of the project.

[0005] For the colorectal cancer auxiliary diagnosis technology based on blood DNA detection, the currently marketed products generally have low sensitivity, all less than 85%, and cannot meet the clinical needs. Clinical practice shows that the main limitation of blood Septin9 methylation detection lies in its relatively low sensitivity in identifying both colorectal cancer and precancerous lesions (adenomas). 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 sensitivity for stage 0-II early cancers is still below 90%, and the detection rate of 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 habits. The public still needs a very long time 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 a new colorectal cancer marker with higher sensitivity based on blood samples with the highest user compliance in clinical practice. Summary of the Invention

[0008] To solve the above technical problems, the inventors of the present invention have made great efforts to develop a colorectal cancer methylation biomarker effective in blood based on non-bisulfite technology, which makes early diagnosis of cancer and cancer risk possible. Unexpectedly, it was found that the TAC1 gene is methylated in colorectal cancer cells. Using this gene as a biomarker, the methylation level of this gene is detected by a non-bisulfite-treated methylation detection system, which has high sensitivity and can diagnose colorectal cancer, thus completing the present invention.

[0009] The first aspect of the present invention provides the application of a detection reagent for the methylation level of the TAC1 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer.

[0010] The TAC1 (tachykinin precursor 1) gene encodes four products of the tachykinin hormone family, substance P and neurokinin A, as well as related peptides, neuropeptide K and neuropeptide γ. The expression of the TAC1 gene is particularly abundant in the muscular layer. Since TAC1 mRNA is prevalent in the intestine, SP and NKA are the main tachykinins in this organ system.

[0011] Almost all tumors are caused and promoted by genetic changes 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, and CpG dinucleotides are unevenly distributed in the human genome. Regions with 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 the occurrence and development of tumors 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 each 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 of the TAC1 gene is significantly different between colorectal cancer and non-colorectal cancer.

[0015] In some embodiments of the present invention, the methylation region of the TAC1 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] Serial number Chromosome position Probe start position Probe end position 1 chr7 97731047 97731166 2 chr7 97731167 97731286 3 chr7 97731287 97731406 4 chr7 97731407 97731526 5 chr7 97731527 97731646 6 chr7 97731647 97731766 7 chr7 97731767 97731886 8 chr7 97731887 97732006 9 chr7 97732007 97732126 10 chr7 97732127 97732246 11 chr7 97732247 97732366 12 chr7 97732367 97732486 13 chr7 97732487 97732606 14 chr7 97732607 97732726 15 chr7 97732967 97733086 16 chr7 97733087 97733206 17 chr7 97737527 97737646 18 chr7 97737647 97737766

[0017] In some specific embodiments of the present invention, the methylation region includes at least a part of chr7:97733087-97733206, and there are 10 methylation sites included in chr7:97733087-97733206, which are respectively: chr7:97733095-97733096; chr7:97733109-97733110; chr7:97733118-97733119; chr7:97733120-97733121; chr7:97733130-97733131; chr7:97733138-97733139; chr7:97733140-97733141; chr7:97733142-97733143; chr7:97733166-97733167; chr7:97733200-97733201.

[0018] In some preferred embodiments of the present invention, the methylation region may include one or more of the above methylation sites, which may be continuous or spaced. In some more preferred embodiments of the present invention, the methylation region includes 8 methylation sites, namely chr7:97733109-97733110; chr7:97733118-97733119; chr7:97733120-97733121; chr7:97733130-97733131; chr7:97733138-97733139; chr7:97733140-97733141; chr7:97733142-97733143; chr7:97733166-97733167. In some most preferred embodiments of the present invention, the methylation region includes chr7:97733109-97733167. Further, the methylation region can be extended by several bases on this basis. In some specific embodiments of the present invention, the methylation region includes chr7:97733110-97733172.

[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 used to study methylation modifications on DNA. DNA methylation is an important epigenetic modification, which involves the addition of a methyl group to the cytosine ring in the DNA molecule. This modification plays a key role in biological processes such as regulating gene expression, cell differentiation, and genome stability. Therefore, understanding the state 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 them:

[0022] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments through PCR. It is simple and fast, 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 methylated sites may be missed or over-detected. In addition, it cannot directly distinguish 5-methylcytosine from other forms of DNA modification.

[0024] MeDIP-Seq uses methylated DNA antibodies to selectively enrich methylated DNA fragments, and then analyzes the enriched products through 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, and then analyzes them through 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 analysis is performed through sequencing. It can provide high-resolution information on individual CpG sites and can perform methylation analysis on the whole genome. 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 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 5-methylcytosine antibody.

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

[0030] Furthermore, the methylated region includes chr7:97733087-97733206. Preferably, the methylated region is chr7:97733110-97733172. 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 after methylation treatment.

[0033] Even further, the methylated region includes chr7:97733087-97733206. Preferably, the methylated region is chr7:97733110-97733172. 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.

[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 the TAC1 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 treated in step S2 as a template, and using primer pairs and probes targeting the untreated or treated methylated regions to perform qPCR amplification;

[0040] If there is a typical amplification curve and the Ct value is not greater than the preset threshold, the test subjects are diagnosed with colorectal cancer or have a risk of colorectal cancer.

[0041] The methylation region includes at least a part of chr7:97733087-97733206. Preferably, the methylation region includes at least a part of chr7:97733110-97733172.

[0042] The 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. A lower Ct value indicates a higher 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 the methylation region of the TAC1 gene based on DNA immunoprecipitation. The methylation region is chr7:97733110-97733172. 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 an early diagnosis. Specifically, the early stage is stage 0-I or stage II of CRC.

[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 the methylation region of the TAC1 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 the methylation region of the TAC1 gene, which has high sensitivity and strong specificity and has very important clinical application value.

[0056] Based on the methylation level of the TAC1 gene, the present invention can perform early detection of colorectal cancer and provide more marker choices for early screening of colorectal cancer. Brief description of the drawings

[0057] Figure 1 Shows the significantly different regions of the methylation level of the TAC1 gene (visualized by IGV) in 5 randomly selected colorectal cancer positive samples and 5 normal healthy individuals.

[0058] Figure 2 Shows the significant difference in the TAC1 methylation level between 548 colorectal cancer positive samples and 389 normal healthy individual samples (RPM index).

[0059] Figure 3 Shows the flow chart of the TAC1 methylation gene detection in Example 2 of the present invention.

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

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

[0062] Unless otherwise specified, implied from the context, or conventional in the art, all parts and percentages in this application are by weight, and the testing and characterization methods used are contemporaneous with the filing date of this application. Where applicable, any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, as are their equivalent foreign counterparts, particularly the definitions of relevant terms in the art 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 provided in this application shall control.

[0063] The numerical ranges in this application are approximate values, and thus, unless otherwise specified, may include values outside the range. The numerical ranges include all values from the lower value to the upper value in increments of one unit, provided that there is a separation of at least two units between any lower value and any higher value. For ranges containing values less than 1 or containing fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered to be 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered to be 0.1. These are merely specific examples of what is intended, and all possible combinations of values between the lowest and highest values recited are considered to be clearly recited in this application.

[0064] The terms "comprising," "including," "having," and their derivatives do not exclude the presence of any other component, step, or process, whether or not such other component, step, or process is disclosed in this application. To the extent any doubt exists, unless expressly stated otherwise, all compositions in this application that use the terms "comprising," "including," or "having" may include any additional additives, excipients, or compounds. In contrast, the term "consisting essentially of" excludes from the scope of any definition hereinafter set forth any other component, step, or process, except for those that are necessary for operability. The term "consisting of" does not include any component, step, or process not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed individual members or any combination thereof.

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

[0066] The following examples are used herein to illustrate 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 publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

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

[0069] Unless otherwise specified, 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 conventional biochemical reagent stores.

[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 and 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 more reliable methylation markers with 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] The concentration and fragment distribution of nucleic acids were quality controlled 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, Qsep 100 capillary electrophoresis was used for fragmentation quality control. When there was 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 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 "A" tails, 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] The methylation enrichment of cfDNA and tissue genomic DNA was carried out in different reactions. Each methylation enrichment reaction can simultaneously mix 12 - 100 cfDNA libraries (the input amount of each cfDNA sample library is about 10 ng), and can simultaneously mix 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 carried out using a commercial company methylation enrichment kit or self-prepared reagent according to the method described in the instruction manual. In this example, zymoMeDIP kit (product number D5101 - A) was used. Then, according to the instruction manual, the methylation enrichment reaction was purified, and 10 - 12 rounds of conventional PCR amplification were carried out 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 TAC1 (tachykinin precursor 1) gene encodes four products of the tachykinin hormone family, substance P and neurokinin A, as well as related peptides, neuropeptide K and neuropeptide γ. The expression of the TAC1 gene is particularly abundant in the muscular layer. Since TAC1 mRNA prevails in the intestine, SP and NKA are the main tachykinins in this organ system.

[0084] The inventors selected the longest TAC1 gene transcript (NM_003182.3) in the database to confirm the gene position. The sequence information of its promoter region, the first exon (exon1), 1 kb upstream of the start codon, as well as the gene intron and the region between adjacent genes (a total of 6.8 Kb) was screened and obtained.

[0085] Probes were designed for the obtained target methylation region of the TAC1 gene.

[0086] Probe design principles:

[0087] (1) Full coverage of the target region, no gaps;

[0088] (2) No overlap;

[0089] (3) Each probe is 120 nt in length.

[0090] The specific genomic corresponding positions list of its targeting region and the detailed list of the specific genomic corresponding positions covered by the probes designed therefrom can be obtained by those skilled in the art through the description of this patent specification.

[0091] (2) Probe synthesis

[0092] Using the above probe design principles and removal criteria, a total of 18 probes were designed for the methylation region of the TAC1 gene, covering all possible CpG sites. The coverage region annotation includes coding genes and microRNA genes. The probe coverage information is shown in Table 1.

[0093] Table 1 Probe coverage information

[0094] Serial number Chromosome position Probe start position Probe end position 1 chr7 97731047 97731166 2 chr7 97731167 97731286 3 chr7 97731287 97731406 4 chr7 97731407 97731526 5 chr7 97731527 97731646 6 chr7 97731647 97731766 7 chr7 97731767 97731886 8 chr7 97731887 97732006 9 chr7 97732007 97732126 10 chr7 97732127 97732246 11 chr7 97732247 97732366 12 chr7 97732367 97732486 13 chr7 97732487 97732606 14 chr7 97732607 97732726 15 chr7 97732967 97733086 16 chr7 97733087 97733206 17 chr7 97737527 97737646 18 chr7 97737647 97737766

[0095] Customize the probe panel at Nanoand (Nanjing) Biotechnology Co., Ltd.

[0096] 3. DNA capture probe hybridization

[0097] Perform liquid hybridization capture using the NadPrep hybridization capture reagent (Cat.No.REF1005101, Nanoand). 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. For the purified library, 500 ng (if less than 500 ng, add all of it) is added to Human Cot DNA and Nad Nano Blockers, and then placed in a vacuum concentrator preheated to 42°C to dry, with the rotation speed set at 1000 rpm. After drying, add the prepared hybridization reaction solution (containing the above probe panel), shake and centrifuge instantaneously. Hybridization capture is carried out for 4 - 16 hours under the hybridization program: 95°C / 30 sec; 65°C / Hold (100°C hot lid). Then add the washed streptavidin magnetic beads to the hybridization system and incubate for 40 minutes, vortexing and mixing evenly every 10 minutes during this period to ensure that the magnetic beads are completely resuspended. It should be noted that the reaction temperature for hybridization capture is the conventional 65°C, rather than 63°C for the methylation probe designed based on bisulfite conversion.

[0098] After the hybridization capture reaction is completed, wash the bound magnetic beads with the four washing solutions provided in the kit, and discard the residual liquid in each step. Finally, add 20 μL of nuclease-free water and gently vortex and mix evenly.

[0099] 4. PCR Amplification and Purification after Hybridization Capture

[0100] Perform PCR amplification on the product after hybridization capture. Use the amplification reagents in the VAHTS Universal Pro DNA LibraryPrep Kit for illumina (Cat.No.ND608-02, Vazyme) for the experiment, and the number of cycles is 12 - 13. After the amplification is completed, purify the product using an equal volume of VAHTS DNA Clean Beads (Cat.No.N411-03, Vazyme) 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.

[0101] 5. Library Sequencing on the Machine and Bioinformatics Analysis

[0102] Dilute the concentration of the library to be sequenced 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. Immediately after that, add 990 μL of HT1 Buffer (REF: 15058251, illumina), vortex and mix well. Then take out 105 μL and add 1295 μL of HT1 Buffer, vortex and mix well to obtain the library for sequencing, with a concentration of 1.5 pM.

[0103] The sequencer is 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 sequencing to the sample position of High Output Reagent Cartridge v2, and then put each reagent in turn to start sequencing. In this example, paired-end sequencing is used, and the total duration is about 30 hours.

[0104] 6. Quality control of sequencing data

[0105] Use Fastp (version 0.22.0) to perform quality control on the data after sequencing, remove low-quality bases. The overall Q20 of the clean data is above 90%, and Q30 is above 85%. The average sequencing depth is about 300×. The average on-target rate of the probes in the above probe combination is above 80%, which reflects the feasibility and effectiveness of detecting cancer-related methylation regions in this example based on the combination of methylation immunoprecipitation and liquid hybridization capture probes.

[0106] 7. Analysis of methylation differential regions of TAC1 gene related to colorectal cancer

[0107] Use the DiffBind tool (version 3.8.4) to screen for differential peaks between tumors and non-tumors, use two algorithms, DESeq and EdgeR, and preferentially screen the regions within the intersection and panel.

[0108] In this example, from each region of the TAC1 gene covered by 18 probes, 2 probe-targeted characteristic methylation regions with the most significant differences between the colorectal cancer group and the non-colorectal cancer group were screened out: chr7:97733087-97733206. The visualization of the IGV map of this characteristic methylation region is shown in Figure 1 .

[0109] The methylated CpG sites of this characteristic methylation region are shown in Table 2:

[0110] Table 2 CpG sites in the characteristic methylation region

[0111] Chromosome position Start site End site chr7 97733095 97733096 chr7 97733109 97733110 chr7 97733118 97733119 chr7 97733120 97733121 chr7 97733130 97733131 chr7 97733138 97733139 chr7 97733140 97733141 chr7 97733142 97733143 chr7 97733166 97733167 chr7 97733200 97733201

[0112] Analyze the RPM index (Reads per million mapped reads, that is, reads per million mapped) of this characteristic methylation difference region in 548 colorectal cancer positive samples and 389 normal healthy population samples. The P value is 2×10 -11 , and the difference is significant, as shown in Figure 2 .

[0113] Example 2 Detection of TAC1 methylated gene in clinical samples and comparison of different treatment regimens

[0114] In order to further verify the clinical performance of the TAC1 gene methylation difference region related to colorectal cancer in CRC plasma samples, the inventors used qPCR method to detect 48 plasma samples clinically diagnosed as CRC and 72 plasma control samples with negative colonoscopy results. Among the 48 plasma samples clinically diagnosed as CRC, there are 6 samples in CRC stage 0-I, 10 samples in CRC stage II, 15 samples in CRC stage III, and 17 samples in CRC stage IV.

[0115] The flow chart of TAC1 methylated gene detection is shown in Figure 3 , specifically:

[0116] (1) DNA extraction

[0117] The extraction of cfDNA was performed using a commercial company's extraction kit according to the instructions in the manual.

[0118] The concentration and fragment distribution of nucleic acids were quality controlled using Qubit4.0 and Qsep100 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 large fragment contamination, magnetic beads are required for fragment screening to remove large fragments.

[0119] (2) Methylated DNA treatment

[0120] ① Methylated DNA immunoprecipitation

[0121] Take 1 / 2 of the total amount of the extracted nucleic acids above for cfDNA methylation enrichment. Conduct the reaction in different batches. For methylation enrichment based on the principle of 5mC antibody, use the zymoMeDIP kit (product number D5101-A). Purify the reaction after methylation enrichment according to the instruction manual, and the elution volume is 50 μL.

[0122] ② Bisulfite conversion of methylated DNA

[0123] Take 1 / 2 of the total amount of the extracted nucleic acids above for bisulfite treatment of cfDNA methylation. Conduct the reaction in different batches. For the treatment of methylated DNA based on the principle of bisulfite conversion, use the DNA conversion kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH biotechnology company to conduct bisulfite treatment of DNA. The elution volume is 50 μL.

[0124] (3) qPCR detection

[0125] The primers and probes were synthesized by Shanghai BioGlory Biotechnology Co., Ltd. The specific sequence information is as follows:

[0126] The sequence of the Taqman MGB probe primer pair for methylated DNA immunoprecipitation enrichment is shown in Table 3.

[0127] Table 3 Taqman MGB probe primer pair for methylated DNA immunoprecipitation enrichment

[0128]

[0129] The 3' end of the probe is labeled with MGB.

[0130] Using the methylated DNA immunoprecipitation enrichment as the template, perform PCR amplification. The final concentration of each primer is 10 μM. The PCR reaction system is 5 μL of the enriched template DNA, 2.5 μL of the premixed solution containing the above primers; 17.5 μL of the PCR reaction solution reagent (2×Rapid Taq Master Mix), and the total volume is made up to 35 μL with water. The PCR reaction conditions are as follows: 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and amplify for 45 cycles.

[0131] The sequence of the Taqman MGB probe primer pair after bisulfite conversion is shown in Table 4.

[0132] Table 4 Taqman MGB probe primer pair after bisulfite conversion

[0133]

[0134] The 3'-end of the probe is labeled with MGB.

[0135] Using the bisulfite-converted DNA as a template, PCR amplification was performed. The final concentration of each primer was 10 μM. The PCR reaction system was 5 μL of enriched template DNA, 2.5 μL of the premixed solution containing the above primers; 17.5 μL of the PCR reaction solution reagent (2×RapidTaq Master Mix), and the total volume was made up to 35 μL with water. The PCR reaction conditions were as follows: 95°C for 5 minutes, 95°C for 15 seconds, 60°C for 40 seconds, and amplified for 45 cycles.

[0136] (4) Analysis of clinical sample test results

[0137] The off-machine data was analyzed. 120 samples were respectively detected by methylation DNA immunoprecipitation enrichment method and bisulfite-treated DNA method and then by qPCR. The test results are shown in Table 5.

[0138] Table 5 Summary of qPCR test results for 120 samples

[0139]

[0140]

[0141]

[0142]

[0143] Figure 4 Examples of qPCR amplification curves of samples from the same CRC patient (sample number: 101668) detected by methylation DNA immunoprecipitation enrichment method and bisulfite-treated DNA method are shown. Among them, the Ct value of qPCR amplification after methylation DNA immunoprecipitation enrichment in Figure A is 34.61, and the Ct value of qPCR amplification of bisulfite-treated DNA in Figure B is 37.77. There is an obvious advantage in qPCR detection after methylation DNA immunoprecipitation enrichment.

[0144] Set the Ct value of the samples with the test result of Undetermined to 45, and draw the ROC curves respectively, as Figure 5As shown in the figure, the areas under the ROC curves (AUC) obtained based on two different methods are 0.909 and 0.807 respectively. According to the ROC curves, cut-off values are set for different methods: for qPCR detection based on the methylation DNA immunoprecipitation enrichment method, the cut-off value is set at Ct = 37.33; for qPCR detection based on bisulfite conversion treatment, the cut-off value is set at Ct = 37.86. If the Ct value of the TAC1 gene amplification in 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.

[0145] Table 6 shows the comparison between the qPCR detection results based on the methylation 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 methylation DNA immunoprecipitation enrichment method and the qPCR detection results based on bisulfite conversion treatment.

[0146] Table 6 Comparison of qPCR method detection results based on methylation DNA immunoprecipitation enrichment method with colonoscopy examination results

[0147]

[0148] Table 7 Comparison of qPCR method detection results based on bisulfite conversion treatment with colonoscopy examination results

[0149]

[0150] Table 8 Comparison between methylation DNA immunoprecipitation enrichment method and bisulfite conversion treatment method

[0151]

[0152] As can be seen from Tables 6 - 8, compared with the traditional bisulfite treatment method, when the methylation differential region of the TAC1 gene is verified on the qPCR detection platform, it has higher sensitivity (83.3%) for CRC, and also maintains high specificity (90.3%) for non-colorectal cancer samples, with an accuracy of 87.5%. The overall performance is superior to the bisulfite-treated DNA method.

[0153] (5) Analysis of detection results of clinical colorectal cancer staging samples

[0154] Among the above 48 plasma samples clinically diagnosed with CRC, there are 6 samples in the CRC stage 0-I, 10 samples in the CRC stage II, 15 samples in the CRC stage III, and 17 samples in the CRC stage IV. Based on the comparison between qPCR detection using the methylation DNA immunoprecipitation enrichment method and qPCR detection using bisulfite conversion treatment, the detection and statistical analysis of samples in different CRC pathological stages are shown in Tables 9 to 10 as follows:

[0155] Table 9 Detection of Samples in Different CRC Pathological Stages

[0156]

[0157]

[0158] Table 10 Statistical Analysis of Sensitivity in Different CRC Pathological Stages

[0159]

[0160] As can be seen from Tables 9 to 10, when the methylation difference region of the TAC1 gene is verified on the qPCR detection platform using the methylation DNA immunoprecipitation enrichment method, it also maintains a high sensitivity (66.7%) for CRC stage 0-I samples, and its performance in the early detection of CRC is superior to that of qPCR detection using bisulfite conversion treatment, providing a new potential biomarker for the early detection of colorectal cancer.

[0161] All the documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by 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 the present application.

Claims

1. Use of a reagent for detecting the methylation level of the TAC1 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer.

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 methylation region.

5. The application according to claim 4, characterized in that, The methylation region is chr7:97733110-97733172, 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 methylation region.

8. The application according to claim 7, characterized in that, The methylation region is chr7:97733110-97733172, 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. Use of a reagent for detecting the methylation level of the TAC1 gene in the preparation of 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 methylation region of the untreated or treated TAC1 gene; 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 is at risk of developing colorectal cancer. The methylation region includes at least a part of chr7:97733110-97733172.

10. A kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer by detecting the TAC1 gene based on DNA immunoprecipitation, characterized in that, The methylation region of the TAC1 gene is chr7:97733110-97733172, the kit includes a 5-methylcytosine antibody, a primer pair and a probe targeting the methylation 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.

Images