Novel DNA methylation marker tagme-3 capable of being used for tumor identification, and use thereof

Abstract

A novel DNA methylation marker TAGMe-3 capable of being used for tumor identification, and a use thereof. The TAGMe-3 gene sequence region has significant methylation differences between cancer tissue and paracancerous tissue, and it is only necessary for an abnormal high methylation state of the TAGMe-3 gene sequence region to be detected for a subject to be determined to be in a high-risk group for tumors. Moreover, the significant difference between tumor tissue and non-tumor tissue presented by TAGMe-3 is widely present in different types of tumors (pan-cancer).

Description

A novel DNA methylation marker TAGMe-3 for tumor identification and its use

[0001] This application claims priority to patent application No. CN 202311286423.X, filed on October 5, 2023; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of epigenetics and biomedicine, and more specifically, relates to a novel DNA methylation marker TAGMe-3 that can be used for tumor identification and its use. Background Art

[0003] A tumor is a neoplasm formed when, under the influence of various carcinogenic factors, a cell in a local tissue loses normal genetic regulation of its growth, leading to its clonal abnormal proliferation. With the increasing aging population, the number of cancer patients worldwide has begun to increase continuously. Cancer has become a global public health challenge. Its etiology is extremely complex, and most tumors are difficult to prevent. Tumor development is a gradual process, and most patients are already in the middle or late stages when they seek medical treatment, having lost the opportunity for fundamental treatment. Early detection, diagnosis, and treatment are key to improving cure rates and prognosis. Therefore, early cancer screening and timely intervention can effectively block cancer progression and reduce morbidity and mortality.

[0004] Epigenomics is a branch of genetics that studies the heritable changes in gene expression that ultimately lead to phenotypic changes without altering the nucleotide sequence of the gene. Epigenetics encompasses DNA methylation, histone modification, genomic imprinting, chromosome remodeling, and non-coding RNA regulation. It primarily influences gene function and characteristics by regulating gene transcription or translation, thereby impacting the development and progression of tumors.

[0005] DNA methylation is a key epigenetic modification of genes. It is a chemical modification process catalyzed by DNA methyltransferases (DNMTs) that transfers a reactive methyl group to specific bases within the DNA chain using S-adenosylmethionine as a methyl donor. In mammals, DNA methylation primarily occurs at the 5′-terminal cytosine of cytosine-phosphate-guanine (CpG) islands, generating 5-methylcytosine (m5C). As a key epigenetic phenomenon, DNA methylation participates in a variety of important biological processes, playing a crucial role in regulating gene expression, maintaining genome stability, modulating DNA spatial conformation, and influencing higher-order chromatin structure. Numerous studies have demonstrated that the early stages of tumorigenesis are accompanied by increased methylation levels of tumor suppressor genes or decreased methylation levels of proto-oncogenes. Alterations in methylation patterns are considered the first detectable tumor-associated marker and further alter with increasing tumor malignancy.

[0006] Aberrant DNA methylation is closely linked to the development, progression, and carcinogenesis of tumors. The main causes are as follows: 1. Cytosine in methylated CpG island dinucleotides is frequently deaminated to thymine, causing gene mutations; 2. Hypermethylation silences tumor suppressor genes and DNA repair genes; 3. Oncogenes are activated by reduced methylation levels; and 4. Reduced overall methylation levels in the genome activate transposons and repetitive sequences, leading to decreased chromosomal stability. Therefore, DNA methylation can serve as a biomarker and prognostic indicator for early diagnosis of tumors and other diseases, and is crucial for cancer screening and risk assessment, early diagnosis, staging, prognosis, and treatment monitoring. While early cancer screening based on DNA methylation molecular markers is gaining increasing attention, clinical applications are limited, relegating cancer screening to specialized testing programs. Furthermore, most existing tumor markers are specific to specific tumor types, and few can be used for multi-cancer screening.

[0007] Therefore, finding molecular targets that can be used to diagnose, prognose, and predict the occurrence and development of cancer is of great significance for early cancer screening, clinical intervention, and guiding patient treatment.

[0008] Summary of the Invention

[0009] The purpose of the present invention is to provide a tumor marker related to epigenetic modification, which utilizes the abnormal hypermethylation phenomenon of marker-specific sites in tumors to detect tumors.

[0010] In a first aspect of the present invention, there is provided a use of an isolated polynucleotide or a polynucleotide converted therefrom in the preparation of a reagent or kit for identifying a tumor; wherein the polynucleotide comprises: (1) a polynucleotide TAGMe-3 having a nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein, or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

[0011] In one embodiment, the identification includes diagnosis, detection, screening or prognostic assessment.

[0012] As an embodiment, the SEQ ID NO: 1 also includes sequence variants or homologous sequences thereof.

[0013] In one embodiment, the sequence variant or homologous sequence is a sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 92% or more, 95% or more, 96% or more, 98% or more, 99% or more, 99.5% or more, or 99.8% or more to the sequence shown in SEQ ID NO: 1. Accordingly, polynucleotides derived from the sequence variant or homologous sequence by conversion (conversion of the unmodified cytosine to T or U, while the cytosine C at the modified CpG site remains unchanged) are also included.

[0014] In one embodiment, the modification includes 5-methylation modification (5mC), 5-hydroxymethylation modification (5hmC), 5-aldehydemethylation modification (5-fC) or 5-carboxymethylation modification (5-caC).

[0015] As an embodiment, the polynucleotide converted from the isolated polynucleotide is a polynucleotide having a nucleotide sequence shown in SEQ ID NO: 2.

[0016] As one embodiment, the at least one modified CpG site is any one "CpG" or a combination thereof selected from No. 1 to No. 48 (such as 2 to 48, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45) in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, it is any one CpG site or a combination thereof selected from No. 9 to No. 19, CpG sites or a combination thereof, or CpG sites No. 1 to No. 8 or a combination thereof, or CpG sites No. 20 to No. 48 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1.

[0017] In one embodiment, the polynucleotide fragment is a polynucleotide having a nucleotide sequence shown at positions 302 to 428 or positions 272 to 458 in SEQ ID NO: 1.

[0018] As an embodiment, the tumor includes (but is not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, blood system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors or skeletal system tumors.

[0019] As an embodiment, the tumor includes: lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumor, gastric cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal cancer, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

[0020] As an embodiment, the tumor identification is directed to a tumor (including an early, middle or late stage tumor) or a precancerous lesion thereof.

[0021] As an embodiment, the samples targeted for tumor identification include (but are not limited to): tissue samples, body fluid samples, and blood samples.

[0022] As an embodiment, the samples include (but are not limited to): paraffin-embedded samples, pleural effusion samples and alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brush samples, tissue and cell biopsy samples, etc.

[0023] In another aspect of the present invention, a method for preparing a reagent for identifying a tumor is provided, comprising: (a) providing an isolated polynucleotide or a polynucleotide converted therefrom, comprising (1) the polynucleotide TAGMe-3 of the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the converted polynucleotide corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the at least one modified CpG site is any one CpG site selected from Nos. 1 to 48 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, it is any one CpG site selected from Nos. 9 to 19 or a combination thereof, Nos. 1 to 8 or a combination thereof, or Nos. 20 to 48 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is SEQ ID NO: 1. (b) using the polynucleotide of (a) as a target sequence, designing a detection reagent that specifically detects the CpG site modification status of the target sequence.

[0024] As an embodiment, the reagents for identifying tumors include but are not limited to: primers, probes, chips or test strips.

[0025] As an embodiment, one or more sets of reagents may be prepared for the target sequence.

[0026] As an embodiment, the detection reagent is integrated on a chip.

[0027] In another aspect of the present invention, a reagent or reagent combination for specifically detecting the modification of CpG sites of a target sequence is provided, wherein the target sequence is: (1) a polynucleotide TAGMe-3 having a nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the converted polynucleotide corresponds to a polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the reagent or reagent combination is directed against a gene sequence comprising the target sequence, preferably, the gene sequence comprises a gene panel or a gene group; preferably, the reagent or reagent combination comprises: a primer for amplifying positions 302 to 428 or positions 272 to 458 in the nucleotide sequence shown in SEQ ID NO: 1.

[0028] As an embodiment, the reagent or reagent combination is: a primer containing the sequence at positions 29 to 58 in SEQ ID NO: 3 and the sequence at positions 28 to 57 in SEQ ID NO: 4.

[0029] As an embodiment, the reagent or reagent combination is: primers of SEQ ID NO: 3 and SEQ ID NO: 4 sequences.

[0030] As an embodiment, the reagent or reagent combination is: a primer formed by connecting any sequence shown in SEQ ID NO: 7-16 to the sequence at positions 29-58 in SEQ ID NO: 3, and a primer formed by connecting any sequence shown in SEQ ID NO: 17-26 to the sequence at positions 28-57 in SEQ ID NO: 4.

[0031] As an embodiment, the reagent or reagent combination is: primers with sequences shown in SEQ ID NO: 27 and SEQ ID NO: 28.

[0032] As an embodiment, the reagent or reagent combination is: primers with sequences shown in SEQ ID NO: 29 and SEQ ID NO: 30.

[0033] In another embodiment, the reagent or reagent combination further comprises primers of SEQ ID NO: 5 and SEQ ID NO: 6.

[0034] In another aspect of the present invention, there is provided a use of the reagent or reagent combination for preparing a kit for identifying tumors.

[0035] In another aspect of the present invention, a kit for identifying tumors is provided, comprising the reagent or reagent combination.

[0036] As an embodiment, the kit may further include, but not limited to: DNA purification reagents, DNA extraction reagents, Bisulfite, and PCR amplification reagents.

[0037] As an embodiment, the kit further includes: instructions for indicating the detection operation steps and result judgment criteria.

[0038] In another aspect of the present invention, a method for analyzing the methylation level of a sample to be tested is provided, comprising: (i) obtaining a polynucleotide from the sample to be tested; and (ii) analyzing the CpG site modification of a target sequence or a fragment thereof in the extracted polynucleotide, wherein the target sequence is: (1) a polynucleotide TAGMe-3 having a nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the converted polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the unmodified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; and the converted polynucleotide having a nucleotide sequence shown in SEQ ID NO: 1 corresponds to a polynucleotide (1) or (2), wherein the unmodified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

[0039] As an embodiment, the method for detecting the CpG site modification status of the target sequence in the extracted polynucleotide includes: pyrophosphate sequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion method, methylation chip method, qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole-genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, or a combination thereof.

[0040] As an embodiment, the method for analyzing the modification status of the CpG sites of the target sequence in the extracted polynucleotide comprises: (i) treating the extracted polynucleotide to convert the unmodified cytosine therein into uracil; preferably, the modification comprises 5-methylation modification, 5-hydroxymethylation modification, 5-aldehydemethylation modification or 5-carboxymethylation modification; preferably, treating the nucleic acid described in step (i) with Bisulfite; and (ii) analyzing the modification status of the target sequence described in the nucleic acid treated in (i).

[0041] In one embodiment, the polynucleotide of step (i) is treated with Bisulfite; and (ii) the modification of the target sequence in the polynucleotide treated in step (i) is analyzed.

[0042] As an embodiment, abnormal methylation level refers to high methylation of C in the CpG of the polynucleotide.

[0043] As an embodiment, other methylation detection methods and methylation detection methods newly developed in the future can also be applied to the present invention.

[0044] As an embodiment, the method for analyzing methylation levels is not a diagnostic method, that is, it is not intended to directly obtain a diagnosis result of a disease.

[0045] As an embodiment, the method for detecting the methylation level of a sample is an in vitro method.

[0046] As an embodiment, the methylation-sensitive restriction endonuclease is a restriction endonuclease whose recognition site is sensitive to methylated bases; including but not limited to: HhaI, BmgBI, HaeII, RruI, TaiI, Bsu15I, Hin6I, HpyCH4IV, NarI, etc., one or more combinations thereof.

[0047] In another aspect of the present invention, an isolated polynucleotide or a polynucleotide converted therefrom is provided, wherein the polynucleotide comprises: (1) the polynucleotide TAGMe-3 having the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; preferably, the at least one modified CpG site is selected from any one of CpG sites 9 to 19 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 20 to 48 or a combination thereof in the polynucleotide having the nucleotide sequence shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is a polynucleotide having the nucleotide sequence shown in positions 302 to 428 or positions 272 to 458 in SEQ ID NO: 1; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, and the cytosine C of the modified CpG site remains unchanged.

[0048] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in clinical lung cancer samples (left panel) and sensitivity and specificity analysis (right panel).

[0050] Figure 2. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in clinical liver cancer samples (left panel) and sensitivity and specificity analysis (right panel).

[0051] Figure 3. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in prostate cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0052] Figure 4. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in cervical cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0053] Figure 5. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in endometrial cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0054] Figure 6. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in clinical samples of urothelial carcinoma (left panel) and sensitivity and specificity analysis (right panel).

[0055] Figure 7. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in clinical samples of biliary tract tumors (left panel) and sensitivity and specificity analysis (right panel).

[0056] Figure 8. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in clinical gastric cancer samples (left panel) and sensitivity and specificity analysis (right panel).

[0057] Figure 9. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in breast cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0058] Figure 10. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in esophageal cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0059] Figure 11. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in clinical glioma samples (left panel) and sensitivity and specificity analysis (right panel).

[0060] Figure 12. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in colorectal cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0061] Figure 13. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in leukemia clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0062] Figure 14. Comparison of TAGMe-3 methylation values ​​in pancreatic cancer tissues and controls (left panel) and sensitivity and specificity analysis (right panel) in clinical pancreatic cancer samples.

[0063] Figure 15. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in thyroid cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0064] Figure 16. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in melanoma clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0065] Figure 17. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in nasopharyngeal carcinoma clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0066] Figure 1 8. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in oral cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0067] FIG19 . Comparison of TAGMe-3 methylation values ​​in laryngeal cancer tissues and controls (left panel) and sensitivity and specificity analysis (right panel) in clinical laryngeal cancer samples.

[0068] Figure 20. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in osteosarcoma clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0069] Figure 21. Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in lymphoma clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0070] FIG22 . Comparison of TAGMe-3 methylation values ​​in renal cell carcinoma tissues and controls (left panel) and sensitivity and specificity analysis (right panel) in clinical samples of renal cell carcinoma.

[0071] FIG23 . Comparison of TAGMe-3 methylation values ​​in cancer tissues and controls in ovarian cancer clinical samples (left panel) and sensitivity and specificity analysis (right panel).

[0072] FIG24 is a BSP validation of the methylation levels of methylation sites 1-8 (CpG) in SEQ ID NO: 1 in cancer cells and normal control cells.

[0073] FIG25 . BSP verification of the methylation levels of methylation sites 20-48 in SEQ ID NO: 1 in cancer cells and normal control cells. DETAILED DESCRIPTION

[0074] In order to find a DNA methylation tumor marker molecular target that can be used for multi-cancer screening, the inventors have determined the target of TAGMe-3 after extensive and in-depth research, analysis and clinical trials. There is a significant methylation difference between cancer tissue and adjacent tissue in the TAGMe-3 gene sequence region. As long as the abnormal high methylation state is detected in the TAGMe-3 gene sequence region, it can be determined that the subject belongs to a high-risk group for tumors. Moreover, the significant difference between tumor tissue and non-tumor tissue presented by TAGMe-3 is widely present in different types of tumors (Pan-cancer), including solid tumors and non-solid tumors.

[0075] As used herein, "sample" or "specimen" includes any material obtained from any individual (preferably a human) or isolated tissue, cell, or body fluid (such as plasma), suitable for DNA extraction and useful for methylation detection. For example, the sample may include, but is not limited to, tissue samples, paraffin-embedded samples, blood samples, pleural effusion samples and alveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, stool samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brush samples, and tissue and cell biopsy samples.

[0076] As used herein, the term "hypermethylation (level)" refers to the presence of high methylation, hydroxymethylation, aldehyde methylation, or carboxymethylation modifications at CpG sites in a gene sequence. For example, in the context of methylation-specific PCR (MSP) analysis, a positive PCR result obtained in a PCR reaction using methylation-specific primers indicates that the DNA (gene) region under test is in a hypermethylated state. For example, in the context of real-time quantitative methylation-specific PCR, the determination of a hypermethylated state can be based on the statistical difference in the relative value of the methylation state of a control sample.

[0077] In the present invention, the term "tumor" refers to a relatively broad range of tumors (Pan-cancer), in which the SEQ ID NO: 1 segment in the genome presents a high methylation state as described in the present invention, which can be a solid tumor or a non-solid tumor, and can include (but is not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, blood system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors or skeletal system tumors.

[0078] In the present invention, the methylation status of the nucleotide sequence represented by SEQ ID NO: 1 or a partial region (fragment) thereof significantly differs between tumor tissue and non-tumor tissue. When abnormally high methylation is detected in the gene sequence region, the subject can be considered to have a tumor or be at high risk for cancer. This significant difference in methylation status of the gene sequence represented by SEQ ID NO: 1 or a partial region thereof is very prominent in a variety of tumors (including those at early stages).

[0079] The present invention also includes "conservative variant sequences" of SEQ ID NO: 1 (or its reverse complementary sequence) that are conservative or have a higher sequence identity with the nucleotide sequence shown in SEQ ID NO: 1 (or its reverse complementary sequence). The "high sequence identity" is, for example, higher than 90%, higher than 92%, higher than 95%, higher than 98%, higher than 99%, etc. It should be understood that different biological individuals may have differences at individual sequence sites (for example, there may be some meaningless SNPs), but this does not affect the detection based on the overall scheme of the present invention.

[0080] Based on the above, the present invention provides a nucleic acid derived from a specific region of the human genome, comprising the gene sequence set forth in SEQ ID NO: 1 or a portion thereof, and also including its antisense strand. In tumor cells, 5-methylcytosine (5mC) or other similar epigenetic modifications are generated at multiple 5'-CpG-3' C positions within the nucleic acid sequence.

[0081] Detection of one or more CpG sites provided by the present invention is possible, and thus the present invention also encompasses nucleic acid fragments of the nucleotide sequence, including at least one methylated CpG site. The at least one CpG site may include 1 to 48, more specifically 2, 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, or 45, of SEQ ID NO: 1 or its reverse complement. It will be appreciated by those skilled in the art that, after the present invention provides CpG numbering based on a DNA strand, the numbering of each CpG site in the complementary DNA strand corresponding to the sense strand can be readily obtained based on the information provided by the present invention.

[0082] Based on the information provided herein regarding the specific fragments in the human genome, those skilled in the art can readily obtain and utilize the CpG sites. The embodiments of the present invention provide a series of sequence fragments containing CpG sites, which serve as examples of preferred embodiments. However, it should be understood that variations can be made based on the information provided herein, such as selecting longer sequences containing the sequences of the present invention, or selecting sequences that overlap with the sequences of the present invention in certain regions.

[0083] The present invention also includes a gene panel or gene group having the nucleotide sequence or sequence fragment shown in SEQ ID NO: 1 or its complementary sequence. For the gene panel or gene group, the characteristics of normal cells and tumor cells can also be obtained by detecting the DNA methylation status.

[0084] It should be understood that a variety of techniques for analyzing methylation status can be applied to the present invention, and the present invention is not particularly limited to such detection techniques. The nucleic acids provided by the present invention can serve as key regions for analyzing methylation status in the genome, and their methylation status can be analyzed using various techniques known in the art, thereby analyzing the occurrence or progression of tumors.

[0085] The nucleic acid described in SEQ ID NO: 1 of the present invention, or a fragment thereof, or a sequence complementary thereto, can be treated with bisulfite, wherein unmethylated cytosine is converted to uracil, while methylated cytosine remains unchanged. Therefore, the present invention also provides nucleic acids obtained by bisulfite treatment of the aforementioned nucleic acid (including its complementary strand (antisense strand)), including nucleic acids or nucleic acid fragments having the nucleotide sequence of SEQ ID NO: 2. These nucleic acids can serve as more direct targets for designing detection reagents or kits.

[0086] The nucleic acid of the nucleotide sequence set forth in SEQ ID NO: 1 and / or its complementary nucleic acid and / or one or more fragments thereof of the present invention can be incorporated into one or more entities, such as one or more nucleic acid collections, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid collection to design targeted analytical reagents. The designed targeted analytical reagents can also be incorporated into one or more entities, such as one or more kits.

[0087] The nucleic acids of the present invention, derived from the nucleic acid of SEQ ID NO: 1 and / or its complementary nucleic acid and / or one or more fragments thereof (e.g., by bisulfite conversion), can also be incorporated into one or more entities, such as one or more nucleic acid collections, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid collection to design targeted analytical reagents. The designed targeted analytical reagents can also be incorporated into one or more entities, such as one or more kits, or one or more chips.

[0088] Based on the target gene and its epigenetic characteristics provided in the present invention, these techniques known in the art and some technologies to be developed can all be applied to the present invention to implement the detection of methylation levels. The methylation profile of nucleic acids can be determined using existing technologies (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or other technologies that are still under development or to be developed. For example, quantitative methylation-specific PCR (QMSP) can be used to detect methylation levels. It is based on the continuous optical monitoring of a fluorescent PCR and is more sensitive than the MSP method. It has a high throughput and avoids the use of electrophoresis to analyze its results. In addition, other available technologies include: qPCR (Me-qPCR), second-generation sequencing, pyrosequencing, Sanger sequencing, bisulfite conversion sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing technology or HPLC, and combined gene group detection methods, etc. Conventional methods in this field. Although some preferred methods are provided in the embodiments of the present invention, the overall scheme of the present invention is not limited thereto.

[0089] As a preferred embodiment of the present invention, a method for detecting the methylation pattern of nucleic acids in a sample in vitro is also provided. This method is based on the principle that bisulfite can convert unmethylated cytosine into uracil, which is then converted to thymine during the subsequent PCR amplification process, while methylated cytosine remains unchanged. Therefore, after bisulfite treatment of nucleic acids, the methylated sites produce a single nucleotide polymorphism (SNP) similar to a C / T. Identifying the methylation pattern of nucleic acids in a test sample based on this principle can effectively distinguish between methylated and unmethylated cytosines.

[0090] The method of the present invention comprises: first providing a sample and extracting genomic DNA; secondly, treating the genomic DNA described in step (a) with bisulfite, thereby converting unmethylated cytosine in the genomic DNA into uracil; and thirdly, analyzing whether there is any abnormal methylation spectrum in the genomic DNA treated in step (b).

[0091] The methods of the present invention can be used to: test samples from subjects to assess whether they have a tumor; or to identify high-risk populations for tumors. These methods can be used in situations where the goal is not to obtain a direct disease diagnosis, such as situations where the goal is not to determine the final outcome of a disease, regional population analysis studies, scientific research, or censuses.

[0092] In a preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing. However, the present invention is not limited to this method in practical applications, and other DNA methylation detection methods known in the art or being improved may also be used. In performing PCR amplification, the primers used are not limited to those provided in the embodiments. Primers that differ in sequence from the primers provided in the embodiments of the present invention but are still specific to the nucleic acids or corresponding CpG sites indicated by the present invention may also be obtained.

[0093] As a preferred embodiment of the present invention, a method for detecting the methylation of nucleic acid in an in vitro detection sample is also provided, which is a methylation-sensitive restriction endonuclease (MSRE) method. When a methylated base is contained in its cleavage site, the methylation-sensitive restriction endonuclease cannot cut DNA. The MSRE method is based on the fundamental principle that methylation-sensitive type II restriction endonucleases cannot cut sequences containing one or more methylated cleavage sites. Fragments containing one or more methylated CpG sequences are cut using methylation-sensitive type II endonucleases and their isozymes (insensitive to methylation), and then analyzed using Southern blotting. The advantages of this method include: no need to know the detailed information of the target DNA primary structure, and direct evaluation of the methylation status of the CpG island can be provided, including obtaining some quantitative analysis information on the methylation of the gene being tested.

[0094] Regarding the marker nucleic acids provided by the present invention, other detection methods and reagents for determining the sequence of a genome, its variation and methylation status known to those skilled in the art can be included in the present invention.

[0095] The present invention provides a method for preparing a tumor detection reagent, comprising: providing a nucleic acid, using the full-length or fragmented nucleic acid as a target sequence, and designing a detection reagent that specifically detects the target sequence; wherein the target sequence includes at least one methylated CpG site. The detection reagent may include, but is not limited to, chips, primers, probes, etc. After obtaining the marker, the selection of the detection reagent is within the skill of those skilled in the art.

[0096] After knowing the sequence of the nucleic acid, it is known to those skilled in the art to design primers, with two primers flanking the specific sequence of the target gene to be amplified (including CpG sequences, complementary to CpG sequences for previously methylated gene regions, and complementary to TpG sequences for previously demethylated gene regions). In preferred embodiments of the present invention, the reagents are primers, preferably those listed in the Examples. In addition to primers, other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.

[0097] The reagent may also be a reagent combination, such as a primer combination. For example, the combination includes more than one set of primers, thereby being able to amplify the above-mentioned multiple nucleic acids separately.

[0098] The present invention also provides a kit for detecting the methylation profile of nucleic acid in a sample in vitro. The kit comprises: a container and the above primer pair located in the container.

[0099] The kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification, and other reagents, such as sample processing reagents. In addition, the kit may also include instructions for use, which indicate the detection steps and result judgment criteria for use by those skilled in the art.

[0100] The methods and reagents of the present invention demonstrate high accuracy in clinical tumor diagnosis, as demonstrated in the examples of the present invention, using a variety of clinical tumor samples. The present invention can be applied to areas such as early-stage tumor screening, therapeutic efficacy assessment, auxiliary diagnosis, and prognosis monitoring, as well as in situations where obtaining a direct disease diagnosis is not the primary objective, as previously mentioned.

[0101] The present invention provides multi-cancer markers suitable for detecting cervical exfoliated cells, among other things. Obtaining test samples is easy and non-invasive. Compared to surgical tissue sampling in clinical settings, this significantly reduces patient pain, improves compliance, and makes the procedure easier for clinicians. Clearly, this represents a significant advancement.

[0102] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0103] Example 1: Determination of methylation detection targets

[0104] 1.1 Obtain human DNA sequence SEQ ID NO: 1, which is the TAGMe-3 gene sequence;

[0105] In the above sequence (sense strand), each "CG" underlined by a solid line represents a methylated CpG site, numbered from 5' to 3' as CpG sites 1 to 48 (methylated CpG sites 1 to 48). The dotted underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold region corresponds to the detection target region in some embodiments.

[0106] 1.2 Obtain the DNA sequence SEQ ID NO: 2 after bisulfite conversion, wherein Y represents C or U (T):

[0107] In the above sequence (sense strand), each "YG" underlined in the solid line represents a converted methylated CpG site, numbered from 5' to 3' as "YG" 1 to 48 (converted methylated CpG sites 1 to 48). The dotted underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold region corresponds to the detection target region in some embodiments.

[0108] 1.3 Determine the detection region and design primers upstream and downstream of the detection region.

[0109] Example 2: Design and synthesis of detection reagents

[0110] 2.1 Design first-round PCR primers with a length of 25-35 bp and a moderate CG content, and amplify the length of 100-300 bp;

[0111] 2.2 Add a barcode (Sample-ID) to the end of the first round primers (Table 1);

[0112] 2.3 Add a tag sequence to the end of the first round of primers for library construction;

[0113] 2.4 Synthesize primers for the first-round PCR reaction and primers for the second-round PCR reaction with Illumina adapters and indexes (Table 1).

[0114] Table 1

[0115] Among them, F1 and R1 amplify the sequence segment corresponding to positions 272 to 458 in SEQ ID NO: 1 or SEQ ID NO: 2, wherein positions 302 to 428 contain CpG sites 9 to 19.

[0116] Example 3: Validation of Detection Reagents

[0117] Synthesize a pair of primers to perform two rounds of PCR reaction on the positive and negative reference products:

[0118] The first-round PCR reaction system is shown in Table 2.

[0119] Table 2

[0120] The second-round PCR reaction system is shown in Table 3.

[0121] Table 3

[0122] Example 4: Construction of a primer pool

[0123] After verifying the efficiency of primer PCR, a primer pool with different barcodes was synthesized, and the primers were diluted and combined (Table 4). For the first round of PCR, there are M×N (10×10) combinations of F and R primers, enabling simultaneous detection and localization of multiple samples. In Table 4, the lowercase bases in the F1 primer correspond to positions 1-21 (Illumina adapter) in the upstream primer F1 in Table 1, and the uppercase bases are the sequencing tag. The tag is followed by bases complementary to the target detection sequence segment (in subsequent examples, it is connected to positions 29-58 in SEQ ID NO: 3); the lowercase bases in the R1 primer correspond to positions 1-20 in the upstream primer R1 in Table 1, and the uppercase bases are the sequencing tag. The tag is followed by bases complementary to the target detection sequence segment (in subsequent examples, it is connected to positions 28-57 in SEQ ID NO: 4). The second round primers are F2 and R2 in Table 1.

[0124] Table 4

[0125] Here, R represents A or G.

[0126] Example 5: Methylation differences of TAGMe-3 CpG sites between tumor tissues and non-tumor cells - NGS sequencing

[0127] 5.1 Obtaining Clinical Samples: Obtain paracancerous / non-cancerous-cancer tissue samples from the clinic. Paracancerous / non-cancerous samples serve as the control group, and cancerous tissue samples serve as the tumor detection experimental group.

[0128] 5.2 DNA extraction: DNA was extracted from the experimental and control groups respectively; this experiment used the adsorption column method (but this method is not limited to this method);

[0129] 5.3 Bisulfite Treatment: Treat the extracted DNA samples with bisulfite, strictly following the instructions; this experiment used the ZYMO Research EZ DNA Methylation-Gold Kit, Catalog No. D5006 (this kit is not limited to this one).

[0130] 5.4 Use primers in the primer pool (first-round PCR primers) and Illumina system universal sequencing primers (second-round PCR primers) to perform two rounds of PCR amplification using conventional methods to construct the NGS library.

[0131] 5.5 After PCR amplification, 2% agarose gel electrophoresis was used to test the specificity of the PCR fragments. 5 μl of PCR product was collected from each sample, mixed, and purified to recover the target fragment library for NGS sequencing.

[0132] 5.6 Analyze sequencing results: Extract sample sequencing information based on primer barcode sequences;

[0133] 5.7 Calculation of TAGMe-3 methylation values: NGS sequencing can independently detect the methylation status of individual CpG sites within the target region and calculate the median methylation value of all CpG sites as the methylation value of TAGMe-3 in the sample;

[0134] 5.8 Result Analysis: Compare the TAGMe-3 methylation values ​​in non-tumor tissues and tumor tissues and determine the cutoff value using the ROC curve.

[0135] Example 6. TAGMe-3: Lung Cancer Clinical Sample Validation - NGS Sequencing

[0136] Forty clinical samples were obtained, including 20 normal lung cancer samples as a control group and 20 lung cancer samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical lung cancer samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0137] The results are shown in Figure 1, which shows that in lung cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001). The sensitivity and specificity were both 100%.

[0138] Example 7, TAGMe-3: Validation of Liver Cancer Clinical Samples - NGS Sequencing

[0139] Thirty clinical samples were obtained, including 15 liver cancer adjacent to the tumor samples as a control group and 15 liver cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of the liver cancer clinical samples were constructed and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0140] The results are shown in Figure 2, showing that in clinical liver cancer samples, the methylation value of TAGMe-3 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001). The sensitivity was 86.67% and the specificity was 100%.

[0141] Example 8, TAGMe-3: Prostate cancer clinical sample validation - NGS sequencing method

[0142] Thirty-four clinical samples were obtained, including 17 prostate cancer adjacent to the cancer sample as a control group and 17 prostate cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of prostate cancer clinical samples were constructed and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0143] The results are shown in Figure 3, showing that in prostate cancer clinical samples, the methylation value of TAGMe-3 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001), with a sensitivity of 76.47% and a specificity of 94.12%.

[0144] Example 9, TAGMe-3: Validation of cervical cancer clinical samples - NGS sequencing

[0145] 40 clinical samples were obtained, including 20 adjacent cervical adenocarcinoma samples as a control group and 20 cervical cancer samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of cervical cancer clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0146] The results are shown in Figure 4, which shows that in cervical cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001). The sensitivity was 90% and the specificity was 95%.

[0147] Example 10, TAGMe-3: Endometrial cancer clinical sample validation - NGS sequencing method

[0148] Twenty-four clinical samples were obtained, including 12 endometrial cancer para-cancer samples as a control group and 12 endometrial cancer samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of endometrial cancer clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0149] The results are shown in Figure 5, showing that in endometrial cancer clinical samples, the methylation value of TAGMe-3 in cancer tissue was significantly higher than that in adjacent tissue (****P<0.0001), with a sensitivity of 91.67% and a specificity of 91.67%.

[0150] Example 11. TAGMe-3: Validation of urothelial carcinoma clinical samples - NGS sequencing

[0151] Thirty-eight clinical samples were obtained, including 19 urothelial carcinoma paracancerous tissue samples as a control group and 19 urothelial carcinoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of urothelial carcinoma clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0152] The results are shown in Figure 6, which shows that in clinical samples of urothelial carcinoma, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001). The sensitivity was 100% and the specificity was 100%.

[0153] Example 12, TAGMe-3: Validation of Biliary Tract Tumor Clinical Samples - NGS Sequencing

[0154] Thirty-six clinical samples were obtained, including 18 adjacent biliary tract tumor samples as a control group and 18 biliary tract tumor samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of clinical biliary tract tumor samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0155] The results are shown in Figure 7, which shows that in clinical samples of biliary tract tumors, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (****P<0.0001), with a sensitivity of 83.33% and a specificity of 83.33%.

[0156] Example 13, TAGMe-3: Validation of Gastric Cancer Clinical Samples - NGS Sequencing

[0157] Fourteen clinical samples were obtained, including seven gastric cancer para-cancer samples as a control group and seven gastric cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of gastric cancer clinical samples were constructed and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0158] The results are shown in Figure 8, which show that in gastric cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 71.43% and a specificity of 85.71%.

[0159] Example 14, TAGMe-3: Breast cancer clinical sample validation - NGS sequencing method

[0160] Sixteen clinical samples were obtained, including eight adjacent breast cancer samples as a control group and eight breast cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical breast cancer samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0161] The results are shown in Figure 9, which show that in breast cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (***P<0.001), with a sensitivity of 100% and a specificity of 100%.

[0162] Example 15. TAGMe-3: Validation of Esophageal Cancer Clinical Samples - NGS Sequencing

[0163] Twelve clinical samples were obtained, including six esophageal cancer adjacent to the cancer sample as a control group and six esophageal cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of clinical esophageal cancer samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0164] The results are shown in Figure 10, which show that in esophageal cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 100%.

[0165] Example 16, TAGMe-3: Validation of Clinical Samples of Brain Glioma - NGS Sequencing

[0166] Twelve clinical samples were obtained, including 6 adjacent glioma samples as a control group and 6 glioma samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 and the second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical glioma samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0167] The results are shown in Figure 11, which show that in clinical samples of glioma, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 66.67% and a specificity of 100%.

[0168] Example 17, TAGMe-3: Validation of colorectal cancer clinical samples - NGS sequencing

[0169] Twelve clinical samples were obtained, including six colorectal cancer adjacent to the cancer sample as a control group and six colorectal cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of colorectal cancer clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0170] The results are shown in Figure 12, which show that in colorectal cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 83.33%.

[0171] Example 18, TAGMe-3: Leukemia Clinical Sample Validation - NGS Sequencing

[0172] Twelve clinical samples were obtained, including 6 non-leukemia bone marrow smear samples as a control group and 6 leukemia bone marrow smear samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 and the second-round primers F2 and R2 in Table 1) to construct an NGS library of leukemia clinical samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0173] The results are shown in Figure 13, which show that in leukemia clinical samples, the methylation value of TAGMe-3 in leukemia bone marrow smears was significantly higher than that in non-leukemia bone marrow smears (**P<0.01), with a sensitivity of 100% and a specificity of 83.33%.

[0174] Example 19, TAGMe-3: Validation of pancreatic cancer clinical samples - NGS sequencing

[0175] Twelve clinical samples were obtained, including six pancreatic cancer adjacent tissue samples as a control group and six pancreatic cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of liposarcoma clinical samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0176] The results are shown in Figure 14, which show that in pancreatic cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0177] Example 20, TAGMe-3: Validation of thyroid cancer clinical samples - NGS sequencing

[0178] Twelve clinical samples were obtained, including six thyroid cancer adjacent tissue samples as a control group and six thyroid cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of thyroid tumor clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0179] The results are shown in Figure 15, which show that in thyroid cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 83.33% and a specificity of 83.33%.

[0180] Example 21, TAGMe-3: Melanoma clinical sample validation - NGS sequencing

[0181] Twelve clinical samples were obtained, including six normal skin tissue samples as a control group and six skin melanoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of melanoma clinical samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0182] The results are shown in Figure 16, which show that in melanoma clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0183] Example 22, TAGMe-3: Validation of nasopharyngeal carcinoma clinical samples - NGS sequencing

[0184] Twelve clinical samples were obtained, including six nasopharyngeal carcinoma paracancerous samples as a control group and six nasopharyngeal carcinoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of nasopharyngeal carcinoma clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0185] The results are shown in Figure 17, which show that in NPC clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 83.33%.

[0186] Example 23, TAGMe-3: Validation of oral cancer clinical samples - NGS sequencing

[0187] Twelve clinical samples were obtained, including six oral cancer paracancerous samples as a control group and six oral cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of oral cancer clinical samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0188] The results are shown in Figure 18, which show that in oral cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05). The sensitivity was 100% and the specificity was 83.33%.

[0189] Example 24, TAGMe-3: Laryngeal cancer clinical sample validation - NGS sequencing method

[0190] Twelve clinical samples were obtained, including six paracancerous laryngeal cancer samples as a control group and six laryngeal cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 was selected, and the second-round primers were F2 and R2 in Table 1). NGS libraries of laryngeal cancer clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0191] The results are shown in Figure 19, which show that in clinical samples of laryngeal cancer, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0192] Example 25, TAGMe-3: Validation of Osteosarcoma Clinical Samples - NGS Sequencing

[0193] Twelve clinical samples were obtained, including 6 adjacent osteosarcoma samples as a control group and 6 osteosarcoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and the second-round primers F2 and R2 in Table 1) to construct an NGS library of osteosarcoma clinical samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0194] The results are shown in Figure 20, which shows that in osteosarcoma clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 66.67% and a specificity of 83.33%.

[0195] Example 26, TAGMe-3: Lymphoma Clinical Sample Validation - NGS Sequencing

[0196] Twelve clinical samples were obtained, including 6 normal lymphoid samples as a control group and 6 lymphoma samples as an experimental group. Two rounds of PCR were performed according to the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of the lymphoma clinical samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0197] The results are shown in Figure 21, which show that in lymphoma clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 83.33%.

[0198] Example 27, TAGMe-3: Validation of Renal Cell Carcinoma Clinical Samples - NGS Sequencing

[0199] Twelve clinical samples were obtained, including six adjacent renal cell carcinoma samples as a control group and six renal cell carcinoma samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and second-round primers F2 and R2 in Table 1) to construct an NGS library of renal cell carcinoma clinical samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0200] The results are shown in Figure 22, which show that in clinical samples of renal cell carcinoma, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (**P<0.01), with a sensitivity of 100% and a specificity of 100%.

[0201] Example 28, TAGMe-3: Validation of ovarian cancer clinical samples - NGS sequencing

[0202] Twelve clinical samples were obtained, including six adjacent ovarian cancer samples as a control group and six ovarian cancer samples as an experimental group. Two rounds of PCR were performed using the primer combination described in Example 4 (one pair of first-round primers in Table 4 and the second-round primers F2 and R2 in Table 1) to construct an NGS library of clinical ovarian cancer samples. TAGMe-3 methylation levels were analyzed according to the NGS sequencing protocol.

[0203] The results are shown in Figure 23, which shows that in ovarian cancer clinical samples, the methylation value of TAGMe-3 in cancer tissues was significantly higher than that in adjacent tissues (*P<0.05), with a sensitivity of 83.33% and a specificity of 66.67%.

[0204] Example 29: Detection Performance Analysis of Single CpG Sites

[0205] Using the clinical samples obtained in the previous examples, the feasibility of analyzing CpG sites 9 to 19 (CpG sites within positions 302 to 428 in SEQ ID NO: 1) as single CpG sites for cancer detection was assessed. Methylation at the single CpG sites was analyzed using NGS sequencing.

[0206] The results are shown in Tables 5 to 9. It can be seen that a single CpG site has high sensitivity and specificity, and can also be used as a target for methylation modification analysis, which is meaningful for the diagnosis of cancer.

[0207] Table 5

[0208] Table 6

[0209] Table 7

[0210] Table 8

[0211] Table 9

[0212] Example 30: Methylation Differences of TAGMe-3 CpG Sites between Tumor Cells and Non-Tumor Cells—Bisulfite Sequencing PCR (BSP)

[0213] The steps of the bisulfite treatment and sequencing method are as follows:

[0214] 1. Extract genomic DNA from hematological tumor cell lines (myeloid leukemia cell line K562), colorectal cancer cell line (HCT116), pancreatic cancer cell line (SW1990), human renal clear cell adenocarcinoma cell line (786-O), gastric cancer cell line (BGC-823), breast cancer cell line (BT-549), and cervical cancer cell line (HeLa) and their corresponding normal cell genomic DNA;

[0215] 2. Treat the extracted genomic DNA of cancer cell lines and normal cell lines with bisulfite and use them as templates for subsequent PCR amplification;

[0216] 3. Design amplification primers according to the sequence of SEQ ID NO: 2, as shown in Table 10, and perform amplification.

[0217] 4. After PCR amplification, the specificity of the PCR fragments was tested by 2% agarose gel electrophoresis. The target fragments were recovered by gel excision, inserted into the T vector, transformed into competent E. coli, and plated. The next day, clones were selected for sequencing. More than 10 clones were selected for each fragment for Sanger sequencing.

[0218] Table 10. BSP primers

[0219] BSP verification of the methylation levels of cancer cells and normal control cells at methylation sites 1-8 in SEQ ID NO: 1 is shown in Figure 24. The results show that the methylation level of TAGMe-3 in cancer cells is significantly higher than that in normal cells.

[0220] BSP verification of the methylation levels of cancer cells and normal control cells at the 20-48 methylation sites in SEQ ID NO: 1 is shown in Figure 25. The results show that the methylation level of TAGMe-3 in cancer cells is significantly higher than that in normal cells.

[0221] The above-described embodiments merely represent several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art may make several modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be based on the appended claims. At the same time, all documents mentioned in this application are cited as references in this application, just as if each document was cited as a reference individually.

Claims

1. Use of an isolated polynucleotide or a polynucleotide converted therefrom in the preparation of a reagent or kit for identifying a tumor; wherein: The polynucleotide comprises: (1) a polynucleotide TAGMe-3 having a nucleotide sequence as shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); The polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

2. The use according to claim 1, characterized in that The polynucleotide converted from the isolated polynucleotide is a polynucleotide with a nucleotide sequence shown in SEQ ID NO: 2; or The at least one modified CpG site is any one "CpG" selected from No. 1 to No. 48 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; preferably any one CpG site selected from No. 9 to No. 19 or a combination thereof, No. 1 to No. 8 or a combination thereof, or No. 20 to No. 48 or a combination thereof in the polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1; or The polynucleotide fragment is a polynucleotide having a nucleotide sequence shown at positions 302 to 428 or positions 272 to 458 in SEQ ID NO:

1.

3. The use according to claim 1, characterized in that The tumors include: respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, blood system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors or bone system tumors; Preferably, the tumor includes: lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, gastric cancer, breast cancer, esophageal cancer, glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

4. The use according to claim 1, characterized in that The samples targeted for tumor identification include: tissue samples, body fluid samples, and blood samples.

5. A method for preparing a reagent for identifying a tumor, comprising: (a) providing an isolated polynucleotide or a polynucleotide converted therefrom, comprising (1) a polynucleotide TAGMe-3 having a nucleotide sequence as shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the converted polynucleotide corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the at least one modified CpG site is any one of CpG sites 1 to 48 or a combination thereof selected from the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; preferably, any one of CpG sites 9 to 19 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 20 to 48 or a combination thereof selected from the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is any one of CpG sites 9 to 19 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 20 to 48 or a combination thereof selected from the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; A polynucleotide having the nucleotide sequence shown at positions 302 to 428 or 272 to 458 in NO: 1; (b) Using the polynucleotide of (a) as the target sequence, a detection reagent is designed to specifically detect the CpG site modification status of the target sequence.

6. A reagent or reagent combination for specifically detecting the modification of a CpG site of a target sequence, wherein the target sequence is: (1) a polynucleotide TAGMe-3 having a nucleotide sequence as shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the converted polynucleotide corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; preferably, the reagent or reagent combination is directed to a gene sequence comprising the target sequence, preferably, the gene sequence comprises a gene panel or a gene group; preferably, the reagent or reagent combination comprises: Primers for amplifying the nucleotide sequence at positions 302 to 428 or 272 to 458 in SEQ ID NO:

1.

7. Use of the reagent or reagent combination according to claim 6 for preparing a kit for identifying a tumor; preferably, a respiratory system tumor, a digestive system tumor, a urinary system tumor, a gynecological and reproductive system tumor, a blood system tumor, a nervous system tumor, a head and neck tumor, a skin system tumor, an endocrine system tumor or a skeletal system tumor; more preferably, the tumor comprises: Lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial carcinoma, biliary tract tumors, gastric cancer, breast cancer, esophageal cancer, brain glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal cancer, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

8. A kit for identifying a tumor, comprising the reagent or reagent combination according to claim 6.

9. A method for analyzing the methylation level of a sample to be tested, comprising: (i) obtaining polynucleotides from a sample to be tested; and (ii) analyzing the CpG site modification of the target sequence or its fragment in the extracted polynucleotide, wherein the target sequence is: (1) the polynucleotide TAGMe-3 of the nucleotide sequence shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); wherein the converted polynucleotide corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged; the converted polynucleotide of the nucleotide sequence shown in SEQ ID NO: 1 corresponds to the polynucleotide of (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

10. The method according to claim 9, characterized in that Methods for detecting the modification of CpG sites in the target sequence of the extracted polynucleotide include: pyrophosphate sequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, methylation chip method, qPCR method, digital PCR method, second-generation sequencing method, third-generation sequencing method, whole-genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, MassArray, or a combination thereof; or The method for analyzing the modification of the CpG site of the target sequence in the extracted polynucleotide comprises: (i) treating the extracted polynucleotide to convert the unmodified cytosine therein into uracil; preferably, the modification comprises 5-methylation modification, 5-hydroxymethylation modification, 5-aldehyde methylation modification or 5-carboxymethylation modification; preferably, treating the nucleic acid described in step (i) with Bisulfite; and (ii) analyzing the modification of the target sequence described in the nucleic acid treated by (i).

11. An isolated polynucleotide or a polynucleotide converted therefrom, the polynucleotide comprising: (1) a polynucleotide TAGMe-3 having a nucleotide sequence as shown in SEQ ID NO: 1, or a polynucleotide fragment containing at least one modified CpG site therein; preferably, the at least one modified CpG site is selected from any one of CpG sites 9 to 19 or a combination thereof, CpG sites 1 to 8 or a combination thereof, or CpG sites 20 to 48 or a combination thereof in the polynucleotide having a nucleotide sequence as shown in SEQ ID NO: 1; preferably, the polynucleotide fragment is a polynucleotide having a nucleotide sequence as shown in positions 302 to 428 or positions 272 to 458 in SEQ ID NO: 1; or (2) a polynucleotide complementary in sequence to the polynucleotide or fragment of (1); The polynucleotide converted from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein the non-modified cytosine is converted to T or U, while the cytosine C of the modified CpG site remains unchanged.

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