Application of SIX6 gene methylation level in diagnosis and prediction of colorectal cancer

Abstract

The invention discloses application of a SIX6 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 SIX6 gene methylation level provided by the invention is used as a marker for early diagnosis or prediction of colorectal cancer, and the choices of technicians in the field are enriched. The methylation level of the SIX6 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. Specifically, it relates to the application of the methylation level of the SIX6 gene in the diagnosis and prediction of colorectal cancer. Background Art

[0002] The incidence and mortality rates of colorectal cancer (CRC) are both on the rise. The 5-year survival rate for colorectal cancer with intervention at the precancerous lesion stage is approximately 100%, the 5-year survival rate for intervention at the early cancer stage is as high as 90%, while the 5-year survival rate drops sharply to 14% when colorectal cancer progresses to stage IV. Therefore, achieving early screening of colorectal cancer and then performing early intervention is of great significance for reducing the incidence and mortality of colorectal cancer.

[0003] The gold standard for colorectal cancer screening is colonoscopy. However, as an invasive examination method, it is more painful and invasive of privacy, resulting in a relatively low overall compliance rate. The fecal immunochemical test (FIT) is a commonly used non-invasive examination method in colorectal cancer screening. It has a relatively high sensitivity to colorectal cancer, but its sensitivity to precancerous lesions such as non-progressive adenomas is relatively low.

[0004] Colorectal mucosal surface cells are constantly shed into the intestinal lumen. After shedding, cancer cells have a survival advantage over normal colorectal mucosal epithelial cells. Normal colorectal mucosal epithelial cells undergo apoptosis in situ or degenerate rapidly when separated from the basement membrane and are then phagocytosed by macrophages; while colorectal cancer cells can usually resist and escape anoikis apoptosis and remain intact, and can continue to shed from the colorectal mucosa. The shed cancer cells can be temporarily protected in the mucus layer during excretion. However, it is difficult to collect complete colon cells from patients with right colon tumors. Therefore, the detection rate of dysplastic cancer cell components is higher than that of whole cells. At the same time, a large number of developmentally abnormal cells and their components in tumors shed into the intestinal lumen, which also provides an analysis specimen for fecal detection. However, fecal sampling is relatively private and inconvenient, which is different from the medical habits of the public. The public still needs a very long time for health education and publicity to accept fecal DNA testing. Therefore, there are still certain problems with the detection compliance during the promotion of screening. Therefore, developing a new colorectal cancer marker with higher sensitivity based on blood samples, which have the highest user compliance in clinical practice, has become an urgent problem to be solved.

[0005] Elucidating the alterations of key genes or products that control the carcinogenic process is an essential basis for developing early cancer screening and diagnosis technologies. However, there are certain limitations in using mutant DNA for early cancer screening. Studies on DNA mutations in 30 types of tumors and normal organs have shown that DNA mutations in tumor driver genes can be detected in more than 30% of normal tissue samples, indicating that if DNA mutations are used as cancer screening indicators, false positive results are bound to occur. In this context, DNA methylation is currently the most promising cancer early screening indicator. Since there are significant differences in the DNA methylation patterns between normal cells and cancer cells, DNA methylation can be used as a screening indicator. Moreover, DNA methylation modifications change in the early stage of cancer, and DNA methylation is relatively stable, allowing the detection of DNA methylation through cell-free DNA in the blood.

[0006] DNA methylation refers to the addition of a methyl group to the 5th carbon atom of cytosine through the action of DNA methyltransferase, which can change the chromatin spatial conformation and has an obvious inhibitory effect on DNA transcription and expression, facilitating its long-term silencing. When tumor suppressor genes show abnormal methylation, normal transcription and translation are significantly inhibited, and tumor suppressor proteins cannot be formed, easily leading to tumor diseases. Therefore, using DNA methylation for early cancer screening is of great significance.

[0007] The bisulfite conversion method has the advantages of high sensitivity, high coverage, and high resolution, making it the preferred method for detecting methylation sites. This method can effectively deaminate methylated cytosine and then convert it into uracil without affecting methylated cytosine. Although bisulfite sequencing is the gold standard for studying DNA methylation, this conversion method can damage DNA, resulting in DNA breakage, loss, and GC bias. At the same time, the high cost and large amount of data also limit its wide application. The method of immunoprecipitation of DNA methylation to enrich methylated DNA mainly relies on antibodies or methylated DNA-binding proteins that specifically bind to methylated cytosine, and then enriches methylated DNA. This method can quickly and effectively detect DNA methylation across the genome.

[0008] Therefore, attempting 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. Summary of the Invention

[0009] To solve the above technical problems, the inventors of the present invention have made great efforts to develop colorectal cancer methylation markers effective in blood based on non-bisulfite technology, and such markers enable early diagnosis of cancer and cancer risk. Unexpectedly, it was found that the SIX6 gene is methylated in colorectal cancer cells. Using this gene as a biomarker and detecting the methylation level of this gene through a methylation detection system without bisulfite treatment has high sensitivity and can diagnose colorectal cancer, thus completing the present invention.

[0010] The first aspect of the present invention provides the use of a detection reagent for the methylation level of the SIX6 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer.

[0011] Sine oculis homeobox homolog-6 (SIX6) is a transcriptional regulatory protein that can regulate the expression of multiple downstream genes and has certain effects on the proliferation, migration, and apoptosis of tumor cells.

[0012] 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.

[0013] Methylation of cytosine on DNA is a covalent "acquired" 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 occurs almost specifically at CpG dinucleotide positions, and CpG dinucleotides are unevenly distributed in the human genome. The regions of concentrated enrichment are generally called CpG islands. Such CpGs exist in repetitive sequences of the human genome and the regulatory regions at the 5' ends of many genes. DNA methylation abnormalities in tumors include 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.

[0014] 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 tumor suppressor genes hypermethylated and proto-oncogenes hypomethylated, 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.

[0015] 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 SIX6 gene is significantly different between colorectal cancer and non-colorectal cancer.

[0016] In some embodiments of the present invention, the methylation region of the SIX gene is obtained in colorectal cancer population samples and normal samples using a probe combination, and the coverage information of each probe is as follows:

[0017] Serial number Chromosome position Probe start position Probe end position 1 chr14 60509307 60509426 2 chr14 60509568 60509687

[0018] In some specific embodiments of the present invention, the methylation region includes at least a part of chr14:60509521-60509604, and chr14:60509521-60509604 contains 10 methylation sites, namely chr14:60509523-60509524, chr14:60509530-60509531, chr14:60509553-60509554, chr14:60509561-60509562, chr14:60509563-60509564, chr14:60509567-60509568, chr14:60509575-60509576, chr14:60509587-60509588, chr14:60509600-60509601, and chr14:60509602-60509603.

[0019] In some aspects of the present invention, the methylation region may include one or more of the above methylation sites, which may be continuous or spaced.

[0020] 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.

[0021] 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 biological processes such as regulating gene expression, cell differentiation, and genomic stability. Therefore, understanding the state of DNA methylation is very important for understanding biological processes and the occurrence and development of diseases.

[0022] 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), among which:

[0023] MSP uses methylation-specific primers to selectively amplify methylated DNA fragments by 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.

[0024] 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.

[0025] MeDIP-Seq uses methylated DNA antibodies to selectively enrich methylated DNA fragments, and then analyzes the enriched products 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.

[0026] MBD-Seq uses methylated DNA-binding proteins (such as MBD2 or MBD3) to enrich methylated DNA fragments, and then analyzes them by sequencing. It can provide a higher 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.

[0027] 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 genome-wide methylation analysis. However, the experimental steps are relatively cumbersome.

[0028] In some embodiments of the present invention, the methylated DNA immunoprecipitation (MeDIP) technique is used for enrichment of 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.

[0029] In some preferred embodiments of the present invention, the methylated region enrichment reagent includes 5-methylcytosine antibody.

[0030] Furthermore, the qPCR detection reagent includes a primer pair and a probe targeting the methylated region.

[0031] Furthermore, the methylated region is chr14:60509521-60509604, 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.

[0032] In some other embodiments of the present invention, the methylation treatment reagent includes bisulfite.

[0033] Furthermore, the qPCR detection reagent includes a primer pair and a probe targeting the sequence after methylation treatment.

[0034] Even further, the methylated region is chr14:60509521-60509604, 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.

[0035] Early detection of cancer may be achieved through the analysis of methylated DNA in a certain type of cancer. The current mainstream methylation analysis method is bisulfite treatment, and the process includes denaturation, deamination, and desulfonation. DNA is first denatured into single strands, and then undergoes high temperature, high salt, acidic, and alkaline environments, experiencing extreme conditions. The transformed DNA obtained has the following forms: mainly single strands, mixed with double strands, fragment nicks, gap damages, and uracil-state nucleotides. This process generally results in the loss of 90% of the DNA template, and a large amount of methylation information cannot be detected by subsequent processes. At the same time, during base conversion treatment, there are situations where sequence conversion is incomplete or excessive, resulting in artificial bias, which will be further amplified by subsequent PCR amplification, causing inaccurate signals. Therefore, the methylation markers obtained based on bisulfite treatment currently generally have the problem of low sensitivity. Especially in blood samples, the detection of methylation levels becomes much more difficult after the already limited free DNA fragments are treated with bisulfite.

[0036] Although it is possible to improve the sensitivity for colorectal cancer by increasing the number of methylated genes based on bisulfite treatment. For example, the combined detection of 3-gene methylation in colorectal cancer by Beijing Aikelun Medical Technology Co., Ltd. was approved by the National Medical Products Administration (NMPA) in 2022. The clinical trial sensitivity using blood sample cfDNA after bisulfite treatment was 84.75% (328 / 387), but the sensitivity still cannot meet the actual clinical needs. Therefore, trying to discover methylation markers of colorectal cancer-related genes in human blood samples based on non-bisulfite treatment methods and effectively detecting changes in their methylation levels has also become the most urgent need for early cancer screening.

[0037] The second aspect of the present invention provides the application of a detection reagent for the methylation level of the SIX6 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method:

[0038] S1, obtain the cfDNA sample in the biological sample of the subject;

[0039] S2, enrich the methylation region of the cfDNA sample or perform methylation treatment;

[0040] S3, using the product enriched or treated in step S2 as a template, perform qPCR amplification with primer pairs and probes targeting the untreated or treated methylation region;

[0041] If there is a typical amplification curve and the Ct value is not greater than the preset threshold, then diagnose that the subject has colorectal cancer or has the risk of developing colorectal cancer.

[0042] The methylation region includes at least a part of chr14:60509521 - 60509604.

[0043] 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:

[0044] Initial stage:

[0045] 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.

[0046] Exponential growth stage:

[0047] Exponential stage: 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.

[0048] Plateau stage:

[0049] 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.

[0050] 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 for 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.

[0051] The third aspect of the present invention provides a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on detecting the methylation level of the methylation region of the SIX6 gene by DNA immunoprecipitation. The methylation region is chr14:60509521 - 60509604. 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.

[0052] In some embodiments of the present invention, the diagnosis is early diagnosis. Specifically, the early stage is CRC stage 0 - I or II.

[0053] Advantages of the present invention

[0054] Compared with the prior art, the present invention has the following technical effects:

[0055] The present invention provides the methylation level of the methylation region of the SIX6 gene as a marker for the early diagnosis or prediction of colorectal cancer, enriching the choices of those skilled in the art.

[0056] The present invention uses the methylation DNA immunoprecipitation technique to detect the methylation level of the methylation region of the SIX6 gene, which has high sensitivity and strong specificity and has very important clinical application value.

[0057] Based on the methylation level of the SIX6 gene, the present invention can experimentally detect early colorectal cancer and provide more marker choices for early colorectal screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Shows the significantly different regions of the methylation level of the SIX6 gene (visualized by IGV) in 8 randomly selected colorectal cancer positive samples and 8 samples with negative colonoscopy.

[0059] Figure 2 Shows the significant difference in the methylation level of SIX6 between 548 colorectal cancer positive samples and 389 normal healthy population samples (RPM index).

[0060] Figure 3 Shows the flow chart of the SIX6 methylation gene detection in Example 2 of the present invention.

[0061] Figure 4 Shows an amplification curve of the SIX6 gene based on the methylation DNA immunoprecipitation enrichment method (A) and an amplification curve of the SIX6 gene based on the bisulfite conversion method (B).

[0062] Figure 5 Shows the ROC curve (A) of the qPCR detection based on the methylation DNA immunoprecipitation enrichment method and the ROC curve (B) of the qPCR detection based on the bisulfite conversion treatment for 120 samples. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] 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 test and characterization methods used are synchronized with the filing date of this application. Where applicable, any patents, patent applications or disclosures involved in this application are incorporated herein by reference in their entirety, and their equivalent family patents are also incorporated by reference, especially 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 of the term provided in this application shall prevail.

[0064] The numerical ranges in this application are approximate values, so unless otherwise stated, 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-digit numbers 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 numerical values between the lowest and highest values listed are considered to be clearly recited in this application.

[0065] The terms "comprising", "including", "having" and their derivatives do not exclude the existence of any other components, steps or processes, and are irrelevant to 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.

[0066] 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 in conjunction with embodiments.

[0067] 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 that the inventors have found 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 the specific embodiments disclosed herein can be modified in many ways and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs, and all materials cited herein and their cited materials will be incorporated by reference.

[0069] Those skilled in the art will recognize or be able to learn many equivalent techniques of many of the specific embodiments of the invention described herein through routine experimentation. These equivalents will be included in the claims.

[0070] Unless otherwise specified, the experimental methods in the following examples are all conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the test materials used in the following examples are all obtained from regular biochemical reagent stores.

[0071] Example 1 Discovery of Colorectal Cancer-Specific Methylated Genes

[0072] In order to screen for biomarkers specifically methylated in colorectal cancer, the present invention collected blood samples 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 corresponding paired samples of homologous cancer tissues and adjacent tissues (if any), and 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.

[0073] 1. Preparation of Methylated DNA Sample Library

[0074] (1) DNA Extraction

[0075] The extraction of cfDNA was carried out using a commercial company's extraction kit according to the instructions in the manual.

[0076] 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 cfDNA has an enrichment peak at 167 bp or nearby. When the yield is greater than 50 ng, Qsep 100 capillary electrophoresis was used for fragment quality control. When there was contamination with large fragments, magnetic beads were used for fragment screening to remove large fragments.

[0077] The 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 instructions in the manual. The genomic DNA can be fragmented by ultrasound or enzymatic digestion to obtain DNA with a length of about 200 bp for convenient library construction.

[0078] (2) Library Construction

[0079] Commercial company library construction kits can be used and operated according to the methods indicated in the instructions. For example, the Rapid Plus DNALibPrep Kit for illumina (Cat.No.RK20208, ABclonal) or the VAHTS Universal ProDNALibrary Prep Kit for illuminaVazyme (Cat.No.ND608-02, Novoprotein) and other similar kits are used for end repair, adding "A" tails, and connecting with Adapters. In this example, the VAHTS Universal ProDNALibrary Prep Kit for illuminaVazyme (Cat.No.ND608-02, Novoprotein) is used for library construction.

[0080] (3) Methylated DNA immunoprecipitation

[0081] The methylation enrichment of cfDNA and tissue genomic DNA is 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). The methylation enrichment based on the principle of 5mC antibody is operated according to the methods indicated in the commercial company methylation enrichment kits or self-prepared reagent instructions. In this example, the zymoMeDIP kit (product number D5101-A) is used. Then, after the methylation enrichment reaction, it is purified according to the instructions, and 10 - 12 rounds of conventional PCR amplification are carried out with universal sequencing primer pairs to obtain a methylated DNA fragment library with a yield of more than 500 ng for each reaction.

[0082] 2. Prepare the DNA probe library

[0083] (1) Probe design

[0084] The SIX6 gene is a gene encoding the SIX homeobox 6 protein. This gene belongs to the SIX family and plays an important regulatory role in embryonic development and organ formation. The protein encoded by the SIX6 gene is a transcription factor that contains the specific domains of the SIX family, namely the SIX domain and the homeobox domain.

[0085] The inventors designed probes for a total of 380 bp of the CpG site enrichment region (methylated region) containing the SIX6 gene. The probe design principles are as follows:

[0086] (1) Full coverage of the target region, without gaps;

[0087] (2) No overlap;

[0088] (3) Each probe is 120 nt in length.

[0089] (2) Probe synthesis

[0090] Using the above probe design principles, a total of 2 probes were designed for the methylation region of the SIX6 gene, and the probe coverage information is shown in Table 1.

[0091] Table 1 Probe coverage information

[0092] Serial number Chromosome position Probe start position Probe end position 1 chr14 60509307 60509426 2 chr14 60509568 60509687

[0093] Customize probe panels at Nanoda (Nanjing) Biotechnology Co., Ltd.

[0094] 3. DNA capture probe hybridization

[0095] NadPrep hybrid capture reagent (Cat. No. REF1005101, Naonda) was used for liquid phase hybrid capture. The hybrid capture reaction can be single hybrid or multi-hybrid. The total amount of MeDIP amplified library input for each hybrid capture reaction should be between 300ng and 8μg. 500ng of the purified library (if less than 500ng, all of it should be input), human Cot DNA and Nad Nano Blockers were placed in a vacuum concentrator preheated to 42°C for drying, and the speed was set to 1000 rpm; after drying, the prepared hybridization reaction solution (including the above-mentioned probe panel) was added, and the mixture was oscillated and centrifuged instantaneously. Hybridization capture was performed for 4-16 hours under the hybridization program: 95°C / 30sec; 65°C / Hold (100°C hot cover); then the washed streptavidin magnetic beads were added to the hybridization system and incubated for 40 minutes, during which time they were vortexed every 10 minutes to ensure that the magnetic beads were completely resuspended; it is worth noting that the reaction temperature of hybridization capture is the conventional 65°C, rather than the 63°C of the methylation probe designed based on bisulfite conversion.

[0096] After the hybridization capture reaction is completed, the bound magnetic beads are washed with the four washing solutions provided by the kit. The residual liquid needs to be discarded in each step; finally, 20 μL of nuclease-free water is added and gently vortexed to mix.

[0097] 4. PCR amplification and purification after hybridization capture

[0098] The products after hybridization capture were subjected to PCR amplification. The amplification reagents used were those in the VAHTS Universal Pro DNA Library Prep Kit for illumina (Cat.No.ND608-02, Vazyme) for the experiment, and the number of cycles was 12 - 13. After the amplification was completed, the products were purified using an equal volume of VAHTS DNA Clean Beads (Cat.No.N411-03, Vazyme) to obtain a relatively pure hybridization capture library; Qubit 4.0 was used for the quantitative determination of the library concentration, and the Qsep100 fully automatic nucleic acid and protein analyzer was used to detect the fragment size of the library.

[0099] 5. Library loading for sequencing and bioinformatics analysis

[0100] The concentration of the library to be loaded was diluted to 4 nM, and mixed according to the proportion of the required data volume. The total data volume should not exceed 120 G. After mixing, 5 μL of the library was taken out, 5 μL of 0.2 N NaOH was added, and pipetted and mixed well. After denaturation for 5 minutes, immediately 990 μL of HT1 Buffer (REF: 15058251, illumina) was added, vortexed and mixed well. Then, 105 μL was taken out and another 1295 μL of HT1 Buffer was added, vortexed and mixed well to obtain the library for loading, with a concentration of 1.5 pM.

[0101] The sequencer was NextSeq 550Dx produced by illumina. The reagents used were 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). 1300 μL of the library for loading was added to the sample position of High Output Reagent Cartridge v2, and each reagent was placed in turn to start sequencing; the paired-end sequencing method was adopted in this example, and the total duration was about 30 hours.

[0102] 6. Quality control of sequencing data

[0103] 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.

[0104] 7. Analysis of methylation difference regions of SIX6 gene related to colorectal cancer

[0105] 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.

[0106] In this example, from each region of the SIX6 gene covered by 2 probes, the most significantly differentially methylated region between the colorectal cancer group and the non-colorectal cancer group was screened out, namely chr14:60509521-60509604 (exon NM_007374). The visualization of the IGV map of this characteristic methylation region is shown in Figure 1 .

[0107] The methylated CpG sites in the methylation difference region of this characteristic methylation region are shown in Table 2:

[0108] Table 2 CpG sites in methylation difference regions

[0109] Chromosome position Start site End site chr14 60509523 60509524 chr14 60509530 60509531 chr14 60509553 60509554 chr14 60509561 60509562 chr14 60509563 60509564 chr14 60509567 60509568 chr14 60509575 60509576 chr14 60509587 60509588 chr14 60509600 60509601 chr14 60509602 60509603

[0110] The RPM index (Reads per million mapped reads) of this characteristic differential region was analyzed in 548 colorectal cancer positive samples and 389 normal healthy population samples. The P value was 0.005, indicating a significant difference, as shown in Figure 2 .

[0111] Example 2 Detection of SIX6 methylated gene in clinical samples and comparison of different treatment regimens

[0112] To further verify the clinical performance of the SIX6 gene methylation differential region related to colorectal cancer in CRC plasma samples, the inventors used the qPCR method to detect 35 plasma samples clinically diagnosed with CRC and 50 plasma control samples with negative colonoscopy results. Among the 35 plasma samples clinically diagnosed with CRC, there were 5 samples in the CRC stage 0-I, 7 samples in the CRC stage II, 12 samples in the CRC stage III, and 11 samples in the CRC stage IV.

[0113] The flowchart of SIX6 methylation gene detection is as Figure 3 shown, specifically:

[0114] (1) DNA extraction

[0115] The extraction of cfDNA was performed using a commercial company's extraction kit according to the instructions in the manual.

[0116] 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 was used for fragmentation quality control. When there was contamination with large fragments, magnetic beads were used for fragment screening to remove large fragments.

[0117] (2) Methylated DNA processing

[0118] ① Methylated DNA immunoprecipitation

[0119] Take 1 / 2 of the total amount of the extracted nucleic acids above for cfDNA methylation enrichment, which was carried out in different reactions. The methylation enrichment based on the 5mC antibody principle used the zymoMeDIP kit (product number D5101-A). After the methylation enrichment reaction, purification was carried out according to the manual, and the elution volume was 50 μL.

[0120] ② Bisulfite conversion of methylated DNA

[0121] Take 1 / 2 of the total amount of the extracted nucleic acids above for bisulfite treatment of cfDNA methylation, which was carried out in different reactions. The methylation DNA treatment based on the bisulfite conversion principle used the DNA conversion kit (EZ DNA Methylation Kit, D5002) from ZYMO RESEARCH Biotechnology Company for bisulfite treatment of DNA. The elution volume was 50 μL.

[0122] (3) qPCR detection

[0123] The primers and probes were synthesized by Shanghai BioGeno Technology Co., Ltd. The specific sequence information is as follows:

[0124] The sequences of Taqman MGB probe primers enriched by methylated DNA immunoprecipitation are shown in Table 3.

[0125] Table 3 Taqman MGB probe primers enriched by methylated DNA immunoprecipitation

[0126]

[0127] The 3'-end of the probe is labeled with MGB.

[0128] Using the enriched product by methylated DNA immunoprecipitation as the template, PCR amplification was carried out, and the final concentration of each primer was 10 μM. The PCR reaction system was 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 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.

[0129] The sequences of Taqman MGB probe primers after bisulfite conversion are shown in Table 4.

[0130] Table 4 Taqman MGB probe primers after bisulfite conversion

[0131]

[0132] The 3'-end of the probe is labeled with MGB.

[0133] Using the DNA after bisulfite conversion as the template, PCR amplification was carried out, and the final concentration of each primer was 10 μM. The PCR reaction system was 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 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.

[0134] (4) Analysis of clinical sample test results

[0135] The off-machine data was analyzed. The detection results of 85 samples by using the methods of enriching methylated DNA immunoprecipitation and bisulfite-treated DNA and then performing qPCR are shown in Table 5.

[0136] Table 5 Summary table of qPCR detection results of 85 samples

[0137]

[0138]

[0139]

[0140] Figure 4 Examples of qPCR amplification curves of samples from the same CRC patient (sample number: 101678) 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 33.87, and the Ct value of qPCR amplification of bisulfite-treated DNA in Figure B is 35.81. There is an obvious advantage in qPCR detection after methylated DNA immunoprecipitation enrichment.

[0141] Set the Ct value of the sample with the detection result of Undetermined to 45, and draw the ROC curves respectively, as Figure 5 shown. The areas under the curves (AUC) of the ROC curves obtained based on the two different methods are 0.862 and 0.796 respectively. According to the ROC curves, set the cut-off values for different methods: for qPCR detection based on the methylated DNA immunoprecipitation enrichment method, the cut-off value is set to Ct = 35.38; for qPCR detection based on the bisulfite conversion treatment, the cut-off value is set to Ct = 35.79. If the Ct value of the amplified SIX6 gene 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 85 samples are statistically analyzed.

[0142] Table 6 shows the comparison between the methylated DNA immunoprecipitation enrichment method and the colonoscopy results (gold standard). Table 6 shows the comparison between the detection results of the bisulfite conversion treatment qPCR method and the colonoscopy results. Table 8 shows the comparison between the detection results of the methylated DNA immunoprecipitation enrichment method and the bisulfite conversion treatment method.

[0143] Table 6 Comparison of the detection results of the methylated DNA immunoprecipitation enrichment method qPCR method with the colonoscopy results

[0144]

[0145] Table 7 Comparison of the detection results of the bisulfite conversion treatment qPCR method with the colonoscopy results

[0146]

[0147] Table 8 Comparison between the methylated DNA immunoprecipitation enrichment method and the bisulfite conversion treatment method

[0148]

[0149]

[0150] As can be seen from Tables 6 to 8, compared with the traditional bisulfite treatment method, when the methylation differential region of the SIX6 gene is verified on the qPCR detection platform, it has higher sensitivity (82.9%) for CRC, and also maintains high specificity (84.0%) for samples of non-colorectal cancer, with an accuracy of 83.5%. The overall performance is superior to the bisulfite-treated DNA method.

[0151] (5) Analysis of the detection results of clinical colorectal cancer staging samples

[0152] Among the above 35 plasma samples clinically diagnosed as CRC, there are 5 samples in the CRC 0-I stage, 7 samples in the CRC II stage, 12 samples in the CRC III stage, and 11 samples in the CRC IV stage. Based on the qPCR detection by methylation DNA immunoprecipitation enrichment method and the comparison with the qPCR detection by bisulfite conversion treatment, the detection and statistical analysis of samples in different CRC pathological stages are shown in Tables 9 to 10:

[0153] Table 9 Detection of samples in different CRC pathological stages

[0154]

[0155] Table 10 Statistical analysis of sensitivity in different CRC pathological stages

[0156]

[0157] As can be seen from Tables 9 to 10, when the methylation differential region of the SIX6 gene is verified on the qPCR detection platform based on the methylation DNA immunoprecipitation enrichment method, it also maintains high sensitivity (80%) for CRC 0-I stage samples, and its performance for early detection of CRC is superior to that of the qPCR detection by bisulfite conversion treatment, providing a new potential biomarker for the early detection of colorectal cancer.

[0158] 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 detection reagent for the methylation level of the SIX6 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, wherein The methylation region enrichment reagent includes a methylated DNA antibody.

4. The application according to claim 3, wherein The qPCR detection reagent includes a primer pair and a probe targeting the methylation region.

5. The application according to claim 4, wherein The methylation region is chr14:60509521 - 60509604, 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, wherein The methylation treatment reagent includes bisulfite.

7. The application according to claim 6, characterized in that, The qPCR detection reagent includes a primer pair and a probe targeting the treated methylation region.

8. The application according to claim 7, wherein The methylation region is chr14:60509521 - 60509604, 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 detection reagent for the methylation level of the SIX6 gene in the preparation of a kit for diagnosing colorectal cancer or predicting the risk of colorectal cancer based on the following method: S1, Obtain a cfDNA sample from a biological sample of a subject; S2, Enrich the methylation region of the cfDNA sample or perform methylation treatment; S3, Using the product enriched or treated in step S2 as a template, perform qPCR amplification with a primer pair and a probe targeting the methylation region of the untreated or treated SIX6 gene; If there is a typical amplification curve and the Ct value is not greater than a preset threshold, then diagnose that the subject has colorectal cancer or is at risk of developing colorectal cancer. The methylation region includes at least a part of chr14:60509521 - 60509604.

10. A kit for detecting SIX6 gene by DNA immunoprecipitation to diagnose colorectal cancer or predict the risk of colorectal cancer, characterized in that, The methylation region of the SIX6 gene is chr14:60509521 - 60509604, 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.

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