A tumor marker located on chromosome 10 and its use

Abstract

The application is a divisional application of application No. CN202011608940.0. The present application provides a tumor marker located on chromosome 10 and an application thereof, wherein the tumor marker is located on human genome chromosome 10 and comprises one or more CpG sites which can be subjected to methylation modification, and the tumor marker comprises a region located at chr10: 124902392-124902455 with human genome hg19 as a reference version. The present application provides a tumor marker related to DNA epigenetic modification, and the marker shows a significant hypermethylation state in tumor patients. The tumor marker can be used for clinical auxiliary screening, diagnosis and prognosis of tumors, or can be used for designing diagnostic reagents and kits.

Description

[0001] This application is a divisional application of application No. CN202011608940.0, titled "Tumor marker and its application", filed on December 30, 2020. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to a tumor marker located on chromosome 10 and its application. BACKGROUND

[0003] The occurrence and development of tumors is a complex, multi-level and multi-factor dynamic process, involving the interaction of various factors such as external environment, genetic variation and epigenetic changes. External environmental factors include physical, chemical, biological and other carcinogenic factors, as well as unhealthy living habits, etc.; genetic variations include gene mutations, copy number variations, chromosomal translocations, etc.; epigenetic changes mainly include DNA methylation, histone modification, non-coding RNA and other changes. In the process of tumor occurrence and development, the above factors complement each other and work together to cause a series of inactivation of tumor suppressor genes and activation of proto-oncogenes, leading to tumors. Timely detection and diagnosis is of great significance in tumor treatment.

[0004] With the deepening of people's understanding of tumors and the progress of science and technology, many new tumor markers have been discovered and used for clinical diagnosis. Before 1980, tumor markers were mainly some hormones, enzymes, proteins and other cell secretions, such as carcinoembryonic antigen (CEA) and alpha-fetal protein (AFP), which can be used as markers for various tumors such as gastric cancer and liver cancer. Although this type of tumor marker is still used in clinical practice, its sensitivity and accuracy have been difficult to meet the current clinical needs. Now there are more and more mutation-based markers, such as mutations in tumor suppressor p53, BRCA gene mutations, and microsatellite instability in colorectal cancer, but these markers are not ideal in early tumor detection and diagnosis. Using DNA methylation-based markers can diagnose tumors in the early stages of tumor development.

[0005] Epigenomics is a discipline that studies the heritable changes in gene function without changes in DNA sequence, and ultimately leads to changes in phenotype. Epigenomics mainly includes biochemical processes such as DNA methylation, histone modification, microRNA level changes, etc. DNA methylation is a more in-depth epigenetic mechanism, which has application prospects in tumor clinical practice including diagnosis and treatment. DNA methylation refers to the process of transferring a methyl group to a specific base under the catalysis of DNA methyltransferase (DMT) in the body with S-adenosyl methionine (SAM) as the methyl donor. In mammals, DNA methylation mainly occurs on C of 5'-CpG-3', generating 5-methylcytosine (5mC).

[0006] In normal cells, some CpGs are in a highly methylated / transcriptionally silenced state, while in tumor cells these CpGs undergo extensive demethylation, leading to transcription of repetitive sequences, activation of transposons, making the genome highly unstable and enhancing the transcription of proto-oncogenes. In addition, some CpG islands that are in a low methylation state in normal cells will be hypermethylated in tumor cells, leading to transcriptional inactivation of genes, including DNA repair genes, cell cycle control genes and anti-apoptotic genes. After genomic DNA is treated with bisulfite, the methylation status of specific sites can be effectively determined by PCR or sequencing. Using the above techniques, these abnormal DNA methylation states can be detected in the early stages of tumor development.

[0007] Although the complete sequence of the human genome has been mastered, the complex genomic sequence, which genes or segments are closely related to diseases, is still not very clear. In addition, due to the complexity of tumors themselves, it is more difficult to find both sensitive and specific tumor markers. In the present application, the inventors find more accurate tumor markers by using a large amount of data and combining more optimized methods, which provides more ways for tumor diagnosis. SUMMARY

[0008] The present application overcomes the defects of the prior art and provides a class of DNA epigenetic modification-related tumor markers, which exhibit significant hypermethylation in tumor patients.

[0009] In a first aspect, the present application provides a tumor marker, wherein the tumor marker is located on chromosome 1, chromosome 2, chromosome 3, chromosome 5, chromosome 7, chromosome 8, chromosome 10, chromosome 11 and chromosome 21 of human genome, and comprises one or more CpG sites which can be subjected to methylation modification.

[0010] Preferably, the tumor marker is located on chromosome 10, and the tumor marker comprises a region of chr10:124902392-124902455 on human genome Hg19 (coordinates start from 0).

[0011] Preferably, the region of chr10:124902392-124902455 is at least one selected from the group consisting of: a) the base sequence shown in SEQ ID NO. 1; b) the complement sequence of the base sequence shown in SEQ ID NO. 1; and c) a nucleotide sequence homologous to SEQ ID NO. 1 at least 70%, or the complement sequence of the nucleotide sequence.

[0012] Preferably, the tumor marker comprises a marker for one or more of breast cancer, cervical cancer, esophageal cancer, head and neck cancer, lung cancer and pancreatic cancer.

[0013] Preferably, the tumor marker further comprises a marker for one or more of bladder cancer, cholangiocarcinoma, colon cancer, glioblastoma, renal clear cell carcinoma, papillary renal cell carcinoma, liver cancer, pheochromocytoma and paraganglioma, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer, endometrial cancer.

[0014] Preferably, the methylation modification comprises 5-formaldehyde methylation modification, 5-hydroxymethylation modification, 5-methylation modification or 5-carboxymethylation modification.

[0015] Preferably, the tumor marker further comprises one or more of the regions located at chr2: 105459135-105459190, chr3: 157812331-157812498, chr21: 38378275-38378539, chr8: 97170353-97170404, chr5: 134880362-134880455, chr10: 94835119-94835252, chr11: 31826557-31826963, chr3: 147114032-147114108, chr10: 50819227-50819589, chr2: 66809255-66809281, chr7: 97361393-97361461, chr8: 70984200-70984294, chr1: 6515341-6515409 on the human genome Hg19 (coordinates start from 0).

[0016] Preferably, each of the above regions is selected from at least one of the following groups: a) the base sequence shown in Table 1; b) the complement of the shown base sequence; c) a nucleotide sequence that is at least 70% homologous to the shown base sequence or the complement of the nucleotide sequence.

[0017] The target sequences of the above regions and their reverse complements are shown in Table 1 below. Among them, the CpG sites are represented in bold italic font, and the numbers under the emphasis marks represent the numbers of the detection sites.

[0018] Table 1

[0019]

[0020]

[0021]

[0022] In a second aspect of the present application, the use of the tumor marker described in the present application is provided, which is selected from one of the following uses: the use of the tumor marker in the preparation of a tumor screening reagent, a prognosis reagent, a detection reagent or a diagnostic reagent; the use of the tumor marker as a drug target for tumors; the use of the tumor marker in the preparation of a drug for inhibiting tumor proliferation; the use of a reagent for detecting the methylation level of the CpG site of the tumor marker in the preparation of a reagent for detecting tumors. Among them, the CpG site is shown in Table 1 above.

[0023] Preferably, the tumor includes bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer (esophageal carcinoma), glioblastoma, head and neck cancer, kidney clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung cancer (lung adenocarcinoma), pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer, endometrial cancer, breast cancer; further preferably, the tumor includes breast cancer, cervical cancer, esophageal cancer, head and neck cancer, lung cancer, pancreatic cancer. Specifically, for example, the tumor of SEQ ID NO. 1 or its antisense complementary sequence as a marker includes breast cancer, cervical cancer, esophageal cancer, head and neck cancer, lung cancer, pancreatic cancer.

[0024] Preferably, in the above-mentioned applications, the detection reagent is a tumor marker detection kit, which includes primers or probes for specifically detecting the tumor marker; and / or, the drug for inhibiting tumor proliferation is an inhibitor of the tumor marker. Further preferably, the primers or probes are primers or probes for specifically detecting the methylation of the CpG site of the tumor marker; and the inhibitor is a methylation inhibitor.

[0025] In a third aspect of the present application, a drug for inhibiting tumor proliferation is provided, which comprises an inhibitor of the tumor marker of the present application. Preferably, the inhibitor is a methylation inhibitor; further preferably, the inhibitor is an inhibitor for inhibiting the methylation of one or more CpG sites in Table 1. The above-mentioned drug can further comprise a pharmaceutically acceptable carrier.

[0026] In a fourth aspect of the present application, a method for detecting a tumor marker is provided, which comprises the following steps: S1, obtaining a tissue sample to be detected; S2, extracting DNA of the tissue sample to be detected to obtain a methylation value of the sample; S3, calculating the methylation of each CpG site in the sequence region of the tumor marker or the average methylation of the whole region.

[0027] Preferably, the method for obtaining the methylation value of the sample in step S2 includes sequencing, probes, antibodies, mass spectrometry, etc.

[0028] In a fifth aspect of the present application, a tumor marker detection kit is provided, which comprises primers or probes for specifically detecting the tumor marker of the present application. Preferably, the specific detection of the tumor marker includes the methylation of the CpG site of the tumor marker; wherein the methylation of the CpG site of the tumor marker is 5-methylation, including 5-formyl methylation, 5-hydroxymethylation or 5-carboxymethylation.

[0029] The present application has the following advantages over the prior art: the present application provides a DNA epigenetic modification related tumor marker, which shows a significant hypermethylation state in tumor patients. The tumor marker can be used for clinical auxiliary screening, diagnosis and prognosis of tumors, or can be used for designing diagnostic reagents and kits, etc. BRIEF DESCRIPTION OF DRAWINGS

[0030] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the principles of the application. In the drawings:

[0031] Figure 1 Figure 6 is a graph of the average methylation value of the target sequence of the chr2:105459135-105459190 region in Example 2 of the present application in the TCGA database;

[0032] Figure 2 Figure 7 is a graph of the average methylation value of the target sequence of the chr3:157812331-157812498 region in Example 2 of the present application in the TCGA database;

[0033] Figure 3 Figure 8 is a graph of the average methylation value of the target sequence of the chr21:38378275-38378539 region in Example 2 of the present application in the TCGA database;

[0034] Figure 4 Figure 9 is a graph of the average methylation value of the target sequence of the chr8:97170353-97170404 region in Example 2 of the present application in the TCGA database;

[0035] Figure 5 Figure 10 is a graph of the average methylation value of the target sequence of the chr5:134880362-134880455 region in Example 2 of the present application in the TCGA database;

[0036] Figure 6 Figure 11 is a graph of the average methylation value of the target sequence of the chr11:31826557-31826963 region in Example 2 of the present application in the TCGA database;

[0037] Figure 7 Figure 12 is a graph of the average methylation value of the target sequence of the chr10:50819227-50819589 region in Example 2 of the present application in the TCGA database;

[0038] Figure 8 Figure 13 is a graph of the average methylation value of the target sequence of the chr7:97361393-97361461 region in Example 2 of the present application in the TCGA database;

[0039] Figure 9 The average methylation value plot of the target sequence of the region chr8:70984200-70984294 in Example 2 of the present application in the TCGA database;

[0040] Figure 10 The average methylation value heat map of the target sequence of 14 regions in Example 3 of the present application on different tumor cell lines;

[0041] Figure 11 The average methylation value distribution and ROC plot of the target sequence of the region chr2:105459135-105459190 in Example 4 of the present application in six tumors;

[0042] Figure 12 The average methylation value distribution and ROC plot of the target sequence (SEQ ID NO. 1) of the region chr10:124902392-124902455 in Example 5 of the present application in six tumors;

[0043] Figure 13 The average methylation value distribution and ROC plot of the target sequence of the region chr3:157812331-157812498 in Example 6 of the present application in six tumors;

[0044] Figure 14 The average methylation value distribution and ROC plot of the target sequence of the region chr21:38378275-38378539 in Example 7 of the present application in six tumors;

[0045] Figure 15 The average methylation value distribution and ROC plot of the target sequence of the region chr8:97170353-97170404 in Example 8 of the present application in six tumors;

[0046] Figure 16 The average methylation value distribution and ROC plot of the target sequence of the region chr5:134880362-134880455 in Example 9 of the present application in five tumors;

[0047] Figure 17 The average methylation value distribution and ROC plot of the target sequence of the region chr10:94835119-94835252 in Example 10 of the present application in five tumors;

[0048] Figure 18 The average methylation value distribution and ROC plot of the target sequence of the region chr11:31826557-31826963 in Example 11 of the present application in six tumors;

[0049] Figure 19 The average methylation value distribution and ROC plot of the target sequence of the region chr3: 147114032-147114108 in Example 12 of the present application in six tumors;

[0050] Figure 20 The average methylation value distribution and ROC plot of the target sequence of the region chr10: 50819227-50819589 in Example 13 of the present application in six tumors;

[0051] Figure 21 The average methylation value distribution and ROC plot of the target sequence of the region chr2: 66809255-66809281 in Example 14 of the present application in six tumors;

[0052] Figure 22 The average methylation value distribution and ROC plot of the target sequence of the region chr7: 97361393-97361461 in Example 15 of the present application in six tumors;

[0053] Figure 23 The average methylation value distribution and ROC plot of the target sequence of the region chr8: 70984200-70984294 in Example 16 of the present application in six tumors;

[0054] Figure 24 The average methylation value distribution and ROC plot of the target sequence of the region chr1: 6515341-6515409 in Example 17 of the present application in six tumors. DETAILED DESCRIPTION

[0055] The specific embodiments of the present application will be further described below in conjunction with the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present application, and cannot be used to limit the protection scope of the present application. The experimental methods not specified in the following examples are selected according to the conventional methods and conditions in the art, or according to the product instructions.

[0056] The specific sequence information of the target sequence verified in the following examples is shown in Table 1 above.

[0057] Example 1 - Methylation level detection

[0058] The methylation detection methods such as RRBS (Reduced Representation Bisulfite Sequencing), WGBS (Whole Genome Bisulfite Sequencing) are used, and the main steps are as follows: obtaining samples: selecting tumor cell lines, or obtaining cancer tissues and adjacent normal tissues from clinics; using DNA extraction kit to extract DNA of samples; performing whole genome sequencing on the extracted DNA; after sequencing, aligning the sequence to the target sequence, and calculating the methylation of each CpG site in the target sequence region. The average methylation value of the sequenced CpG sites in the target sequence is taken as the average methylation value of the target sequence in the sample. The target sequence information is shown in Table 1 above.

[0059] Example 2 - Verification of 14 target sequences in TCGA database methylation chip data

[0060] 2.1, Target sequence and database

[0061] The target sequences of the 14 regions verified are shown in Table 1. The TCGA database is used, and DNA methylation chip data of cancer tissues and adjacent normal tissues of more than 20 tumor types are collected in the TCGA database, including: bladder cancer (BLCA), cervical cancer (CESC), cholangiocarcinoma (CHOL), colon cancer (COADREAD), esophageal cancer (ESCA), glioblastoma (GBM), head and neck squamous cell carcinoma (HNSC), kidney clear cell carcinoma (KIRC), papillary renal cell carcinoma (KIRP), liver cancer (LIHC), lung adenocarcinoma (LUNG), pancreatic cancer (PAAD), pheochromocytoma and paraganglioma (PCPG), prostate cancer (PRAD), sarcoma (SARC), melanoma (SKCM), gastric adenocarcinoma (STAD), thyroid cancer (THCA), thymus cancer (THYM), endometrial cancer (UCEC).

[0062] 2.2, The number of probes contained in each target sequence is obtained, and the results are shown in Table 3;

[0063] Table 3 Number of probes contained in each target sequence

[0064] Region Target Sequence Number of Probes Region Target Sequence Number of Probes chr2:105459135-105459190 1 chrll:31826557-31826963 1 chr10:124902392-124902455 0 chr3:147114032-147114108 0 chr3:157812331-157812498 1 chr10:50819227-50819589 1 chr21:38378275-38378539 2 chr2:66809255-66809281 0 chr8:97170353-97170404 1 chr7:97361393-97361461 1 chr5:134880362-134880455 1 chr8:70984200-70984294 2 chr10:94835119-94835252 0 chr1:6515341-6515409 0

[0065] 2.3, The average methylation values of all probes in all cancer samples and normal tissue samples in this target sequence are calculated according to the method of Example 1. The results are shown in Table 4. Figure 1 - Figure 9 The sample naming method is: cancer English name_C or N (number), where C represents tumor tissue, N represents normal tissue, and the number represents the sample amount.

[0066] Figure 1 For the average methylation value of the target sequence in the region of chr2: 105459135-105459190 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 17 cancers of bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, kidney clear cell carcinoma, papillary renal cell carcinoma, lung adenocarcinoma, pheochromocytoma and paraganglioma, prostate cancer, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer, endometrial cancer is significantly higher than the average methylation value of the normal sample.

[0067] Figure 2 For the average methylation value of the target sequence in the region of chr3: 157812331-157812498 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 20 cancers of bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, kidney clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer, endometrial cancer is higher than the average methylation value of the normal sample.

[0068] Figure 3 For the average methylation value of the target sequence in the region of chr21: 38378275-38378539 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 19 cancers of bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, kidney clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer, endometrial cancer is significantly higher than the average methylation value of the normal sample. The average methylation value of the above-mentioned target sequence in pheochromocytoma and paraganglioma is almost the same as the average methylation value of the normal sample.

[0069] Figure 4 For the average methylation value of the target sequence in the region of chr8: 97170353-97170404 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 19 cancers of bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, kidney clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer, endometrial cancer is significantly higher than the average methylation value of the normal sample. The average methylation value of the above-mentioned target sequence in pheochromocytoma and paraganglioma is lower than the average methylation value of the normal sample.

[0070] Figure 5For the average methylation value of the target sequence in the region of chr5: 134880362-134880455 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 18 kinds of cancers including bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, head and neck squamous cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer and endometrial cancer is significantly higher than the average methylation value of the normal sample. The average methylation value of the above-mentioned target sequence in glioblastoma, pheochromocytoma and paraganglioma is lower than the average methylation value of the normal sample.

[0071] Figure 6 For the average methylation value of the target sequence in the region of chr11: 31826557-31826963 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 19 kinds of cancers including bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer and endometrial cancer is significantly higher than the average methylation value of the normal sample. The average methylation value of the above-mentioned target sequence in pheochromocytoma and paraganglioma is lower than the average methylation value of the normal sample.

[0072] Figure 7 For the average methylation value of the target sequence in the region of chr10: 50819227-50819589 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 20 kinds of cancers including bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer and endometrial cancer is higher than the average methylation value of the normal sample.

[0073] Figure 8 For the average methylation value of the target sequence in the region of chr7: 97361393-97361461 in the TCGA database, it can be seen that the average methylation value of the above-mentioned target sequence in 20 kinds of cancers including bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, renal clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer and endometrial cancer is higher than the average methylation value of the normal sample.

[0074] Figure 9The average methylation values of the target sequence of the region of chr8:70984200-70984294 in the TCGA database can be seen from the above, the average methylation values of the target sequence in 20 kinds of cancers of bladder cancer, cervical cancer, cholangiocarcinoma, colon cancer, esophageal cancer, glioblastoma, head and neck squamous cell carcinoma, kidney clear cell carcinoma, papillary renal cell carcinoma, liver cancer, lung adenocarcinoma, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, sarcoma, melanoma, gastric adenocarcinoma, thyroid cancer, thymus cancer, endometrial cancer are higher than the average methylation value of the normal sample.

[0075] It can be seen that in the above-mentioned large number of tumor types, the average methylation value of the above-verified target sequence in the tumor sample is higher than the average methylation value in the normal tissue.

[0076] Example 3-Methylation level of 14 region target sequences in tumor cell lines

[0077] Eleven tumor cell line samples were collected, including cholangiocarcinoma, breast cancer, colorectal cancer, gallbladder cancer, kidney cancer, leukemia, liver cancer, lung cancer, pancreatic cancer, prostate cancer and gastric cancer. The average methylation values of the 14 target sequences in each sample were obtained by using the RRBS methylation sequencing technology according to the method of Example 1. The results are shown in Figure 10 The darker the color, the higher the average methylation value. It can be seen that the methylation of the target sequences of the regions of chr2:105459135-105459190, chr10:124902392-124902455 (SEQ ID No. 1), chr3:157812331-157812498, chr21:38378275-38378539, chr8:97170353-97170404, chr11:31826557-31826963, chr3:147114032-147114108, chr7:97361393-97361461, chr8:70984200-70984294 can be detected in 11 tumor cell lines, and the average methylation values in 11 tumor cell lines are very high. The methylation of the target sequences of the regions of chr5:134880362-134880455, chr10:94835119-94835252, chr10:50819227-50819589, chr2:66809255-66809281 and chr1:6515341-6515409 can be detected only in part of the tumor cell lines.

[0078] Example 4-Application of the target sequence of the region of chr2:105459135-105459190 in clinical detection

[0079] Samples: 6 kinds of tumor tissues and their adjacent normal tissue samples, respectively, breast cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases), cervical cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), esophageal cancer (cancer tissue 3 cases, adjacent normal tissue 1 case), head and neck cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), lung cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases) and pancreatic cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases).

[0080] The average methylation values of the target sequence of the region chr2:105459135-105459190 were obtained respectively in the 26 tumor tissues and 26 adjacent normal tissues by using the methylation sequencing technology of RRBS according to the method of Example 1. The results are shown in Figure 11 The results are shown in Figure 1. The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, where the left picture represents the box plot of the average methylation value distribution of the target sequence in the tumor sample and the normal sample (control), and through the picture, the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; The right picture represents the ROC curve diagram of the detection, and through the picture, the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that in the 6 kinds of tumors, the average methylation value of the target sequence of the region chr2:105459135-105459190 in the tumor sample is higher than that in the adjacent normal tissue. According to the average methylation value of the sample in the target sequence of the region chr2:105459135-105459190, the tumor sample and the normal tissue of the cancer can be accurately distinguished.

[0081] Example 5-Application of the target sequence (SEQ ID No. 1) of the region chr10:124902392-124902455 in clinical detection

[0082] Samples: 6 kinds of tumor tissues and their adjacent normal tissue samples, respectively, breast cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), cervical cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), esophageal cancer (cancer tissue 3 cases, adjacent normal tissue 1 case), head and neck cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), lung cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases) and pancreatic cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases).

[0083] The average methylation values of the target sequence of the region of chr10: 124902392-124902455 in the 25 tumor tissues and 26 normal tissues adjacent to the cancer were obtained respectively by using the RRBS methylation sequencing technology according to the method of Example 1. The results are shown in Figure 12 The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, wherein the left picture represents the box plot of the average methylation values of the target sequence in the tumor sample and the normal sample (control), and the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen from the picture; the right picture represents the ROC curve diagram of the detection, and the specificity of the difference result can be seen from the picture. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that in the 6 tumors, the average methylation value of the target sequence of the region of chr10: 124902392-124902455 in the tumor sample is higher than that in the normal tissue adjacent to the cancer. According to the average methylation value of the sample in the target sequence of the region of chr10: 124902392-124902455, the tumor sample and the normal tissue adjacent to the cancer can be accurately distinguished.

[0084] Example 6-Application of the target sequence of the region of chr3: 157812331-157812498 in clinical detection

[0085] Samples: 6 kinds of tumor tissues and their normal tissues adjacent to the cancer, which are breast cancer (5 cases of cancer tissues and 5 cases of normal tissues adjacent to the cancer), cervical cancer (5 cases of cancer tissues and 5 cases of normal tissues adjacent to the cancer), esophageal cancer (5 cases of cancer tissues and 4 cases of normal tissues adjacent to the cancer), head and neck cancer (5 cases of cancer tissues and 5 cases of normal tissues adjacent to the cancer), lung cancer (4 cases of cancer tissues and 2 cases of normal tissues adjacent to the cancer) and pancreatic cancer (2 cases of cancer tissues and 2 cases of normal tissues adjacent to the cancer).

[0086] The average methylation values of the target sequence of the region of chr3: 157812331-157812498 in the 26 tumor tissues and 23 normal tissues adjacent to the cancer were obtained respectively by using the RRBS methylation sequencing technology according to the method of Example 1. The results are shown in Figure 13The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, from which the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, from which the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that in the 5 tumors, the average methylation value of the target sequence in the chr3: 157812331-157812498 region of the tumor sample is higher than that of the normal tissue adjacent to the cancer; in esophageal cancer, the average methylation value of most tumor samples is higher than that of the normal tissue adjacent to the cancer, and according to the average methylation value of the sample on the target sequence in the chr3: 157812331-157812498 region, most of the tumor samples and the normal tissue adjacent to the cancer can be accurately distinguished.

[0087] Example 7-Application of target sequence in the region of chr21:38378275-38378539 in clinical detection

[0088] Samples: 6 kinds of tumor tissues and their normal tissue samples adjacent to the cancer, respectively breast cancer (4 cases of cancer tissue, 4 cases of normal tissue adjacent to the cancer), cervical cancer (5 cases of cancer tissue, 2 cases of normal tissue adjacent to the cancer), esophageal cancer (5 cases of cancer tissue, 4 cases of normal tissue adjacent to the cancer), head and neck cancer (2 cases of cancer tissue, 4 cases of normal tissue adjacent to the cancer), lung cancer (5 cases of cancer tissue, 5 cases of normal tissue adjacent to the cancer) and pancreatic cancer (5 cases of cancer tissue, 5 cases of normal tissue adjacent to the cancer).

[0089] According to the method of Example 1, the average methylation value of the target sequence in the region of chr21:38378275-38378539 of the 26 tumor tissues and 24 normal tissues adjacent to the cancer was obtained by using RRBS methylation sequencing technology. The results are as follows Figure 14The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, from which the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, from which the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that the average methylation value of the target sequence in the chr21:38378275-38378539 region of the breast cancer sample, the head and neck cancer sample and the esophageal cancer sample is higher than that of the normal tissue adjacent to the cancer. In the esophageal cancer sample, the cervical cancer sample and the lung cancer sample, the average methylation value of the target sequence in the chr21:38378275-38378539 region is mostly higher than that of the normal tissue adjacent to the cancer, and the average of the average methylation value is higher than that of the normal tissue adjacent to the cancer. According to the average methylation value of the sample in the chr21:38378275-38378539 region, most of the tumor samples and the cancer normal tissue can be distinguished.

[0090] Example 8-Application of the target sequence in the region of chr8:97170353-97170404 in clinical detection

[0091] Samples: 6 kinds of tumor tissues and their normal tissues adjacent to cancer, respectively, breast cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 5 cases), cervical cancer (cancer tissue 3 cases, normal tissue adjacent to cancer 4 cases), esophageal cancer (cancer tissue 4 cases, normal tissue adjacent to cancer 3 cases), head and neck cancer (cancer tissue 4 cases, normal tissue adjacent to cancer 5 cases), lung cancer (cancer tissue 2 cases, normal tissue adjacent to cancer 2 cases) and pancreatic cancer (cancer tissue 3 cases, normal tissue adjacent to cancer 2 cases).

[0092] According to the method of Example 1, the average methylation value of the target sequence in the region of chr8:97170353-97170404 of the 21 tumor tissues and the 21 normal tissues adjacent to cancer was obtained by RRBS methylation sequencing technology, respectively. The results are as follows Figure 15The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, from which the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, from which the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that the average methylation value of the target sequence in the chr8:97170353-97170404 region in the breast cancer sample, cervical cancer sample and lung cancer sample is higher than that of the normal tissue adjacent to the cancer. In esophageal cancer, head and neck cancer and lung cancer samples, the average methylation value of the target sequence in the chr8:97170353-97170404 region is mostly higher than that of the normal tissue adjacent to the cancer, and the average value of the average methylation value is higher than that of the normal tissue adjacent to the cancer. According to the average methylation value of the sample in the chr8:97170353-97170404 region 5 target sequences, most of the tumor samples and cancer normal tissues can be distinguished.

[0093] Example 9-Application of target sequences in the region of chr5:134880362- 134880455 in clinical detection

[0094] Samples: 5 kinds of tumor tissues and their normal tissues adjacent to cancer, respectively breast cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 5 cases), esophageal cancer (cancer tissue 2 cases, normal tissue adjacent to cancer 1 case), head and neck cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 5 cases), lung cancer (cancer tissue 3 cases, normal tissue adjacent to cancer 1 case) and pancreatic cancer (cancer tissue 2 cases, normal tissue adjacent to cancer 1 case).

[0095] According to the method of Example 1, the average methylation values of the target sequences in the region of chr5:134880362-134880455 in the 17 tumor tissues and 13 normal tissues adjacent to cancer were obtained by RRBS methylation sequencing technology, respectively. The results are as follows: Figure 16The figure contains A, B, C, D, E, 5 groups of pictures. Each group of pictures contains 2 pictures, where the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, and the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen from the picture; the right picture represents the ROC curve diagram of the detection, and the specificity of the difference result can be seen from the picture. Among them, A: breast cancer sample and its control, B: esophageal cancer sample and its control, C: head and neck cancer sample and its control, D: lung cancer sample and its control, E: pancreatic cancer sample and its control. It can be seen that in the 5 tumors, the average methylation value of the target sequence of the chr5: 134880362-134880455 region in the tumor sample is higher than that in the normal tissue adjacent to the cancer. According to the average methylation value of the sample on the target sequence of the chr5: 134880362-134880455 region, the tumor sample and the cancer normal tissue can be accurately distinguished.

[0096] Example 10-Application of the target sequence of the region of chr10: 94835119-94835252 in clinical detection

[0097] Samples: 5 kinds of tumor tissues and their normal tissues adjacent to cancer, respectively breast cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 4 cases), cervical cancer (cancer tissue 2 cases, normal tissue adjacent to cancer 1 case), head and neck cancer (cancer tissue 2 cases, normal tissue adjacent to cancer 2 cases), lung cancer (cancer tissue 3 cases, normal tissue adjacent to cancer 3 cases) and pancreatic cancer (cancer tissue 4 cases, normal tissue adjacent to cancer 5 cases).

[0098] According to the method of Example 1, the average methylation values of the target sequence of the region of chr10: 94835119-94835252 in the 16 tumor tissues and 15 normal tissues adjacent to cancer were obtained by using RRBS methylation sequencing technology. The results are shown in Figure 17 The figure contains A, B, C, D, E, 5 groups of pictures. Each group of pictures contains 2 pictures, where the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, and the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen from the picture; the right picture represents the ROC curve diagram of the detection, and the specificity of the difference result can be seen from the picture. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: head and neck cancer sample and its control, D: lung cancer sample and its control, E: pancreatic cancer sample and its control. It can be seen that in the 5 tumors, the average methylation value of the target sequence of the chr10: 94835119-94835252 region in the tumor sample is higher than that in the normal tissue adjacent to the cancer. According to the average methylation value of the sample on the target sequence of the chr10: 94835119-94835252 region, the tumor sample and the cancer normal tissue can be accurately distinguished.

[0099] Example 11 - Application of the target sequence in the region of chr11:31826557-31826963 in clinical detection

[0100] Samples: 6 kinds of tumor tissues and their paracancer normal tissue samples, respectively, breast cancer (cancer tissue 5 cases, paracancer normal tissue 5 cases), cervical cancer (cancer tissue 4 cases, paracancer normal tissue 5 cases), esophageal cancer (cancer tissue 4 cases, paracancer normal tissue 5 cases), head and neck cancer (cancer tissue 4 cases, paracancer normal tissue 5 cases), lung cancer (cancer tissue 5 cases, paracancer normal tissue 5 cases) and pancreatic cancer (cancer tissue 4 cases, paracancer normal tissue 5 cases).

[0101] The average methylation values of the target sequence in the region of chr11:31826557-31826963 in the 26 tumor tissues and 30 paracancer normal tissues were obtained respectively by using the RRBS methylation sequencing technology according to the method of Example 1. The results are shown in Figure 18 The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, where the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, and through the picture, the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, and through the picture, the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that the average methylation value of the target sequence in the region of chr11:31826557-31826963 in the breast cancer sample, cervical cancer sample, lung cancer, head and neck cancer and pancreatic cancer sample is higher than that of the paracancer normal tissue. In the esophageal cancer sample, the average methylation value of the target sequence in the region of chr11:31826557-31826963 is mostly higher than that of the paracancer normal tissue, and the average value of its average methylation value is higher than that of the paracancer normal tissue. According to the average methylation value of the sample on the target sequence in the region of chr11:31826557-31826963, most of the tumor samples and cancer normal tissues can be distinguished.

[0102] Example 12 - Application of the target sequence in the region of chr3:147114032-147114108 in clinical detection

[0103] Samples: 6 kinds of tumor tissues and their adjacent normal tissue samples, respectively, breast cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases), cervical cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), esophageal cancer (cancer tissue 5 cases, adjacent normal tissue 4 cases), head and neck cancer (cancer tissue 4 cases, adjacent normal tissue 4 cases), lung cancer (cancer tissue 5 cases, adjacent normal tissue 4 cases) and pancreatic cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases).

[0104] The average methylation values of the target sequence of the region chr3: 147114032-147114108 in the 28 tumor tissues and 27 adjacent normal tissues were obtained respectively by using the methylation sequencing technology of RRBS according to the method of Example 1. The results are shown in Figure 19 The results are shown in FIG. 1. FIG. 1 includes A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, where the left picture represents the box plot of the average methylation value distribution of the target sequence in the tumor sample and the normal sample (control), and through the picture, the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, and through the picture, the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that the average methylation value of the target sequence of the region chr3: 147114032-147114108 in the breast cancer sample, cervical cancer sample, lung cancer, head and neck cancer and pancreatic cancer sample is higher than that of the adjacent normal tissue. In the esophageal cancer sample, the average methylation value of the target sequence of the region chr3: 147114032-147114108 is mostly higher than that of the adjacent normal tissue, and the average value of the average methylation value is higher than that of the adjacent normal tissue. According to the average methylation value of the sample in the target sequence of the region chr3: 147114032-147114108, most of the tumor samples and the normal tissue of the cancer can be distinguished.

[0105] Example 13-Application of the target sequence of the region chr10: 50819227-50819589 in clinical detection

[0106] Samples: 6 kinds of tumor tissues and their adjacent normal tissue samples, respectively, breast cancer (cancer tissue 5 cases, adjacent normal tissue 5 cases), cervical cancer (cancer tissue 4 cases, adjacent normal tissue 3 cases), esophageal cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), head and neck cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases), lung cancer (cancer tissue 4 cases, adjacent normal tissue 3 cases) and pancreatic cancer (cancer tissue 4 cases, adjacent normal tissue 5 cases).

[0107] The average methylation values of the target sequence of the region of chr10:50819227-50819589 in the 25 tumor tissues and 26 normal tissues adjacent to cancer were obtained respectively by using the RRBS methylation sequencing technology according to the method of Example 1. The results are shown in Figure 20 Fig. 1. The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, wherein the left picture represents the box plot of the distribution of the average methylation values of the target sequence in the tumor samples and the normal samples (control), and through the picture, the difference in methylation of the target sequence in the tumor samples and the normal samples can be seen; the right picture represents the ROC curve diagram of the detection, and through the picture, the specificity of the difference result can be seen. Among them, A: breast cancer samples and their controls, B: cervical cancer samples and their controls, C: esophageal cancer samples and their controls, D: head and neck cancer samples and their controls, E: lung cancer samples and their controls, F: pancreatic cancer samples and their controls. It can be seen that the average methylation values of the target sequence of the region of chr10:50819227-50819589 in the breast cancer samples, cervical cancer samples, lung cancer, head and neck cancer and pancreatic cancer samples are higher than the average methylation values of the normal tissues adjacent to cancer. In the esophageal cancer samples, the average methylation values of the target sequence of the region of chr10:50819227-50819589 are mostly higher than the average methylation values of the normal tissues adjacent to cancer, and the average of the average methylation values is higher than the average of the average methylation values of the normal tissues adjacent to cancer. According to the average methylation values of the samples in the target sequence of the region of chr10:50819227-50819589, most of the tumor samples and the normal tissues adjacent to cancer can be distinguished.

[0108] Example 14-Application of the target sequence of the region of chr2:66809255-66809281 in clinical detection

[0109] Samples: 6 kinds of tumor tissues and their normal tissues adjacent to cancer, which are breast cancer (5 cases of cancer tissues and 5 cases of normal tissues adjacent to cancer), cervical cancer (2 cases of cancer tissues and 5 cases of normal tissues adjacent to cancer), esophageal cancer (1 case of cancer tissue and 3 cases of normal tissues adjacent to cancer), head and neck cancer (5 cases of cancer tissues and 5 cases of normal tissues adjacent to cancer), lung cancer (4 cases of cancer tissues and 4 cases of normal tissues adjacent to cancer) and pancreatic cancer (3 cases of cancer tissues and 3 cases of normal tissues adjacent to cancer).

[0110] The average methylation values of the target sequence of the region of chr2:66809255-66809281 in the 20 tumor tissues and 25 normal tissues adjacent to cancer were obtained respectively by using the RRBS methylation sequencing technology according to the method of Example 1. The results are shown in Figure 21The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, from which the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, from which the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that the average methylation value of the target sequence in the chr2:66809255-66809281 region in the breast cancer sample, cervical cancer sample, lung cancer, esophageal cancer and pancreatic cancer sample is higher than that of the normal tissue adjacent to the cancer. In the head and neck cancer sample, the average methylation value of the target sequence in the chr2:66809255-66809281 region is mostly higher than that of the normal tissue adjacent to the cancer, and the average of the average methylation value is higher than that of the normal tissue adjacent to the cancer. According to the average methylation value of the sample in the chr2:66809255-66809281 region, most of the tumor samples and cancer normal tissues can be distinguished.

[0111] Example 15-Application of the target sequence in the region of chr7:97361393-97361461 in clinical detection

[0112] Samples: 6 kinds of tumor tissues and their normal tissues adjacent to the cancer, respectively, breast cancer (cancer tissue 5 cases, normal tissue adjacent to the cancer 5 cases), cervical cancer (cancer tissue 3 cases, normal tissue adjacent to the cancer 5 cases), esophageal cancer (cancer tissue 4 cases, normal tissue adjacent to the cancer 3 cases), head and neck cancer (cancer tissue 5 cases, normal tissue adjacent to the cancer 5 cases), lung cancer (cancer tissue 5 cases, normal tissue adjacent to the cancer 5 cases) and pancreatic cancer (cancer tissue 5 cases, normal tissue adjacent to the cancer 5 cases).

[0113] According to the method of Example 1, the average methylation value of the target sequence in the region of chr7:97361393-97361461 in the 27 tumor tissues and 28 normal tissues adjacent to the cancer was obtained by RRBS methylation sequencing technology, respectively. The results are as follows: Figure 22The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, from which the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, from which the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that the average methylation value of the target sequence of the chr7:97361393-97361461 region in the breast cancer sample, the cervical cancer sample, the head and neck cancer, the esophageal cancer and the pancreatic cancer sample is higher than that of the normal tissue adjacent to the cancer. In the lung cancer sample, the average methylation value of the target sequence of the chr7:97361393-97361461 region is mostly higher than that of the normal tissue adjacent to the cancer, and the average of the average methylation value is higher than that of the normal tissue adjacent to the cancer. According to the average methylation value of the sample on the target sequence of the chr7:97361393-97361461 region, most of the tumor samples and the cancer normal tissue can be distinguished.

[0114] Example 16-Application of target sequence of chr8:70984200-70984294 region in clinical detection

[0115] Samples: 6 kinds of tumor tissues and their normal tissues adjacent to cancer samples, respectively breast cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 5 cases), cervical cancer (cancer tissue 1 case, normal tissue adjacent to cancer 5 cases), esophageal cancer (cancer tissue 2 cases, normal tissue adjacent to cancer 1 case), head and neck cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 5 cases), lung cancer (cancer tissue 4 cases, normal tissue adjacent to cancer 5 cases) and pancreatic cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 5 cases).

[0116] According to the method of Example 1, the average methylation value of the target sequence of the chr8:70984200-70984294 region was obtained by using RRBS methylation sequencing technology on the 22 tumor tissues and 26 normal tissues adjacent to cancer, respectively. The results are as follows Figure 23The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, from which the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, from which the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that the average methylation value of the target sequence of the chr8:70984200-70984294 region in the breast cancer sample, the cervical cancer sample, the head and neck cancer, the esophageal cancer and the pancreatic cancer sample is higher than that of the normal tissue adjacent to the cancer. In the lung cancer sample, the average methylation value of the target sequence of the chr8:70984200-70984294 region is mostly higher than that of the normal tissue adjacent to the cancer, and the average of the average methylation value is higher than that of the normal tissue adjacent to the cancer. According to the average methylation value of the sample on the target sequence of the chr8:70984200-70984294 region, most of the tumor samples and the cancer normal tissue can be distinguished.

[0117] Example 17-Application of the target sequence of the region of chr1:6515341-6515409 in clinical detection

[0118] Samples: 6 kinds of tumor tissues and their normal tissues adjacent to cancer samples, respectively, breast cancer (cancer tissue 5 cases, normal tissue adjacent to cancer 5 cases), cervical cancer (cancer tissue 3 cases, normal tissue adjacent to cancer 5 cases), esophageal cancer (cancer tissue 3 cases, normal tissue adjacent to cancer 1 case), head and neck cancer (cancer tissue 4 cases, normal tissue adjacent to cancer 5 cases), lung cancer (cancer tissue 3 cases, normal tissue adjacent to cancer 3 cases) and pancreatic cancer (cancer tissue 2 cases, normal tissue adjacent to cancer 2 cases).

[0119] According to the method of Example 1, the average methylation value of the target sequence of the region of chr1:6515341-6515409 in the 20 tumor tissues and 21 normal tissues adjacent to cancer was obtained by RRBS methylation sequencing technology, respectively. The results are as follows: Figure 24The figure contains A, B, C, D, E, F, 6 groups of pictures. Each group of pictures contains 2 pictures, the left picture represents the box plot of the average methylation value distribution of the tumor sample and the cancer normal sample (control) on the target sequence, from which the difference in methylation of the target sequence in the tumor sample and the normal sample can be seen; the right picture represents the ROC curve diagram of the detection, from which the specificity of the difference result can be seen. Among them, A: breast cancer sample and its control, B: cervical cancer sample and its control, C: esophageal cancer sample and its control, D: head and neck cancer sample and its control, E: lung cancer sample and its control, F: pancreatic cancer sample and its control. It can be seen that in the breast cancer sample, the cervical cancer sample, the head and neck cancer, the esophageal cancer and the lung cancer sample, the average methylation value of the target sequence in the chr1:6515341-6515409 region is higher than that of the normal tissue around the cancer. In the pancreatic cancer sample, the average methylation value of the target sequence in the chr1:6515341-6515409 region is mostly higher than that of the normal tissue around the cancer, and the average of the average methylation value is higher than that of the normal tissue around the cancer. According to the average methylation value of the sample on the target sequence in the chr1:6515341-6515409 region, most of the tumor samples and the cancer normal tissue can be distinguished.

[0120] The above detailed description of the specific embodiments of the present application is only as an example, and the present application is not limited to the above described specific embodiments. Any equivalent modification and substitution of the present application made by those skilled in the art is also within the scope of the present application. Therefore, any equivalent transformation and modification made without departing from the spirit and scope of the present application should be covered within the scope of the present application.

Claims

1. The application of a reagent for detecting the methylation level of tumor marker CpG sites in the preparation of tumor detection or diagnostic reagents, characterized in that, The tumor marker is located on human chromosome 10 and includes one or more CpG sites that can undergo methylation modification; wherein the tumor marker is located in the chr10:124902392-124902455 region with human genome hg19 as a reference version, and its sequence is the base sequence shown in SEQ ID NO.1; the tumor marker is used as a marker for one or more of breast cancer, cervical cancer, esophageal cancer, head and neck cancer, lung cancer, and pancreatic cancer.

2. The application according to claim 1, characterized in that, The methylation modification includes 5-aldehyde methylation, 5-hydroxymethylation, 5-methylation, or 5-carboxymethylation.

3. The application according to claim 1, characterized in that, The reagent used to detect the methylation level of the tumor marker CpG site is a tumor marker detection kit, which includes primers or probes that specifically detect the methylation of the tumor marker CpG site.

4. The application according to claim 3, characterized in that, The method of using the reagent for detecting the methylation level of the tumor marker CpG site includes the following steps: S1. Obtain the tissue sample to be tested; S2. Extract DNA from the tissue sample to be tested and obtain the methylation value of the sample; S3. Calculate the methylation status of each CpG site within the tumor marker sequence region or the average methylation status of the entire region.

5. The method according to claim 4, characterized in that, The methods for obtaining the methylation value of a sample as described in step S2 include: sequencing, probes, antibodies, and mass spectrometry.

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