TAGMe-3, a novel DNA methylation marker for tumor identification, and its applications.

Abstract

The application provides a novel DNA methylation marker TAGMe-3 for tumor identification and use thereof. The TAGMe-3 gene sequence region has significant methylation difference between cancer tissues and paracancer tissues. As long as abnormal high methylation state of the TAGMe-3 gene sequence region is detected, the subject is determined to belong to a tumor high-risk population. Moreover, the significant difference of the TAGMe-3 presented between tumor tissues and non-tumor tissues exists in different types of tumors (Pan-cancer) in a wide range.

Description

Technical Field

[0001] This invention belongs to the fields of epigenetics and biomedicine, and more specifically, this invention relates to TAGMe-3, a novel DNA methylation marker that can be used for tumor identification, and its uses. Background Technology

[0002] A tumor is a new growth formed when a cell in a local tissue loses its normal regulation of growth at the gene level, leading to abnormal clonal proliferation under the influence of various carcinogenic factors. With the increasing aging population, the number of cancer patients worldwide is constantly rising, making cancer a global public health challenge. Its etiology is extremely complex, and prevention of most cancers from their causes is difficult. Furthermore, the development of cancer is a gradual process, and most patients are already in the middle or late stages when they seek medical attention, missing the opportunity for fundamental treatment. Early detection, diagnosis, and treatment are key to improving cure rates and treatment prognosis. Therefore, early cancer screening and timely intervention can effectively block cancer progression and reduce morbidity and mortality.

[0003] Epigenetics is a branch of genetics that studies heritable changes in gene expression without altering the nucleotide sequence of the gene, ultimately leading to phenotypic changes. Epigenetics includes DNA methylation, histone modification, genomic imprinting, chromosomal remodeling, and non-coding RNA regulation. It primarily influences gene function and characteristics by regulating gene transcription or translation, thereby affecting tumorigenesis and development.

[0004] DNA methylation is an important epigenetic modification, referring to the chemical modification process in which an active methyl group is transferred to a specific base in the DNA strand under the catalysis of DNA methyltransferase (DNMT), using S-adenosylmethionine as a methyl donor. In mammals, DNA methylation mainly occurs at the 5′ end of the cytosine island of cytosine-phosphate-guanine (CpG), generating 5′-methylcytosine (m5C). As an important epigenetic phenomenon, DNA methylation participates in various important biological processes, playing a crucial role in regulating gene expression, maintaining genome stability, regulating DNA spatial conformation, and influencing the higher-order structure of chromatin. Numerous studies have shown that early tumorigenesis is 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-related indicator, and these patterns further change with increasing tumor malignancy.

[0005] Aberrant DNA methylation is closely related to the occurrence, development, and carcinogenesis of tumors, mainly due to the following reasons: 1. Cytosine in methylated CpG island dinucleotides undergoes deamination to thymine at a higher frequency, causing gene mutations; 2. Tumor suppressor genes and DNA repair genes are silenced due to hypermethylation; 3. Oncogenes are activated due to decreased methylation levels; 4. Decreased overall genomic methylation levels lead to the activation of transposons and repetitive sequences, resulting in decreased chromosome stability. Therefore, DNA methylation can serve as a biomarker and prognostic indicator for early diagnosis of tumors, and is of great significance for tumor screening and risk assessment, early diagnosis, staging and typing, prognosis, and treatment monitoring. Although early tumor screening based on DNA methylation molecular markers has gradually gained attention, very few programs are actually applied clinically, so tumor screening is considered a specialized test. Furthermore, most existing tumor markers are only for specific tumor types, and there are almost no markers that can be used for multi-cancer screening.

[0006] Therefore, finding molecular targets that can be used for the diagnosis, prognosis, and prediction of cancer development is of great significance for early cancer screening, clinical intervention, and guiding patient treatment. Summary of the Invention

[0007] The purpose of this invention is to provide an epigenetic modification-related tumor marker that utilizes the abnormal hypermethylation at specific sites of the marker in the tumor to detect the tumor.

[0008] In a first aspect of the invention, the use of isolated polynucleotides or polynucleotides derived therefrom in the preparation of reagents or kits for identifying tumors is provided; wherein the polynucleotides comprise: (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 that is sequence-complementary to the polynucleotide or fragment of (1); wherein the polynucleotide derived from the isolated polynucleotide is a polynucleotide corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C at its modified CpG site remains unchanged.

[0009] In one implementation, the identification includes diagnosis, testing, screening, or prognostic assessment.

[0010] As one implementation, SEQ ID NO:1 also includes its sequence variants, or homologous sequences.

[0011] In one embodiment, the sequence variant or homologous sequence is a sequence having more than 80%, 85%, 90%, 92%, 95%, 96%, 98%, 99%, 99.5%, or 99.8% sequence identity with the sequence shown in SEQ ID NO:1. Accordingly, it also includes polynucleotides derived from the transformation of the sequence variant or homologous sequence (conversion of unmodified cytosine to T or U, while the cytosine C at the modified CpG site remains unchanged).

[0012] In one embodiment, the modification includes 5-methylation (5mC), 5-hydroxymethylation (5hmC), 5-aldehyde methylation (5-fC), or 5-carboxymethylation (5-caC).

[0013] In one embodiment, the polynucleotide derived from the isolated polynucleotide is a polynucleotide with the nucleotide sequence shown in SEQ ID NO:2.

[0014] In one embodiment, the at least one modified CpG site is any CpG or combination thereof selected from the polynucleotide sequence of SEQ ID NO:1, specifically from CpGs 1 to 48 (e.g., 2 to 48, more specifically 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45); preferably, it is any CpG site or combination thereof selected from the polynucleotide sequence of SEQ ID NO:1, specifically from CpG sites 9 to 19, CpG sites 1 to 8, or CpG sites 20 to 48.

[0015] In one embodiment, the polynucleotide fragment is a polynucleotide of the nucleotide sequence shown at positions 302-428 or 272-458 in SEQ ID NO:1.

[0016] In one implementation, the tumors include (but are not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, hematological system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors, or skeletal system tumors.

[0017] In one implementation, 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 carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma, or ovarian cancer.

[0018] In one implementation, the tumor identification refers to tumors (including early, intermediate, or late-stage tumors) or their precancerous lesions.

[0019] As one implementation method, the samples used for tumor identification include (but are not limited to): tissue samples, body fluid samples, and blood samples.

[0020] In one implementation, the samples include (but are not limited to): paraffin-embedded samples, pleural effusion samples and bronchoalveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, sputum samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brushing samples, tissue and cell biopsy samples, etc.

[0021] In another aspect of the present invention, a method for preparing a reagent for identifying tumors is provided, comprising: (a) providing an isolated polynucleotide or a polynucleotide derived therefrom, including (1) a 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 sequence complementary to the polynucleotide or fragment of (1); wherein the derived polynucleotide is a polynucleotide corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged; preferably, the at least one modified CpG site is any CpG site selected from SEQ ID NO:1 from CpG sites 1 to 48 or a combination thereof; preferably, it is any CpG site selected from SEQ ID NO:1 from 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; preferably, the polynucleotide fragment is SEQ ID NO:1 from 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; preferably, the polynucleotide fragment is SEQ ID NO:1 from CpG sites 1 to 48 or a combination thereof. (a) A polynucleotide of the nucleotide sequence shown at positions 302–428 or 272–458 in NO:1; (b) Using the polynucleotide of (a) as the target sequence, design a detection reagent to specifically detect the CpG site modification of the target sequence.

[0022] As one implementation method, the reagents for identifying tumors include, but are not limited to, primers, probes, chips, or test strips.

[0023] As one implementation method, one or more sets of reagents can be prepared for the target sequence.

[0024] In one implementation, the detection reagent is integrated onto a chip.

[0025] In another aspect of the present invention, a reagent or reagent combination is provided for specifically detecting the CpG site modification of a target sequence, 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; or (2) a polynucleotide that is sequence-complementary to the polynucleotide or fragment of (1); wherein the converted polynucleotide is a polynucleotide corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged; preferably, the reagent or reagent combination is directed to a gene sequence containing the target sequence, preferably, the gene sequence includes a gene panel or gene group; preferably, the reagent or reagent combination includes: a primer for amplifying positions 302-428 or 272-458 of the nucleotide sequence shown in SEQ ID NO:1.

[0026] As one embodiment, the reagent or reagent combination is a primer containing the sequence of positions 29-58 in SEQ ID NO:3 and the sequence of positions 28-57 in SEQ ID NO:4.

[0027] As one embodiment, the reagent or reagent combination is: primers of the sequences SEQ ID NO:3 and SEQ ID NO:4.

[0028] As one 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 of SEQ ID NO:3, or a primer formed by connecting any sequence shown in SEQ ID NO:17-26 to the sequence at positions 28-57 of SEQ ID NO:4.

[0029] As one embodiment, the reagent or reagent combination is: primers of the sequences shown in SEQ ID NO:27 and SEQ ID NO:28.

[0030] As one embodiment, the reagent or reagent combination is: primers with the sequences shown in SEQ ID NO:29 and SEQ ID NO:30.

[0031] In another embodiment, the reagent or reagent combination further includes primers of the sequences SEQ ID NO:5 and SEQ ID NO:6.

[0032] In another aspect of the invention, the use of the aforementioned reagent or combination of reagents for preparing a kit for identifying tumors is provided.

[0033] In another aspect of the invention, a kit for identifying tumors is provided, comprising the aforementioned reagents or combinations thereof.

[0034] As one implementation, the kit may also include, but is not limited to: DNA purification reagents, DNA extraction reagents, Bisulfite, and PCR amplification reagents.

[0035] As one implementation method, the kit also includes an instruction manual that specifies the detection procedure and result determination criteria.

[0036] 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 fragment thereof 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 that is sequence complementary to the polynucleotide or fragment of (1); wherein the transformed polynucleotide is a polynucleotide corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged; the polynucleotide transformed from the nucleotide sequence shown in SEQ ID NO:1, corresponding to the polynucleotide of (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged.

[0037] As one implementation method, the methods for detecting CpG site modifications of target sequences in extracted polynucleotides include: pyrosequencing, bisulfite conversion sequencing, methylation-specific PCR, methylation-sensitive restriction endonuclease digestion, methylation microarray, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, MassArray, or combinations thereof.

[0038] As one implementation, the method for analyzing the CpG site modification status of the target sequence in the extracted polynucleotide includes: (i) treating the extracted polynucleotide to convert unmodified cytosine into uracil; preferably, the modification includes 5-methylation, 5-hydroxymethylation, 5-aldehyde methylation, or 5-carboxymethylation; preferably, treating the nucleic acid in step (i) with Bisulfite; and (ii) analyzing the modification status of the target sequence in the nucleic acid treated in (i).

[0039] In one implementation, the polynucleotide described in step (i) is treated with Bisulfite; and (ii) the modification of the target sequence described in the polynucleotide treated in (i) is analyzed.

[0040] As one implementation method, abnormal methylation level refers to the high methylation of the C in the polynucleotide CpG.

[0041] As one implementation method, other methylation detection methods and future newly developed methylation detection methods can also be applied to this invention.

[0042] As one implementation method, the method for analyzing methylation levels is not a diagnostic method, that is, it is not intended to directly obtain a diagnosis of a disease.

[0043] As one implementation method, the method for detecting the methylation level of the sample is an in vitro method.

[0044] As one embodiment, the methylation-sensitive restriction endonuclease is a restriction endonuclease that is sensitive to methylated bases at its recognition site; including but not limited to one or more of the following: HhaI, BmgBI, HaeII, RruI, TaiI, Bsu15I, Hin6I, HpyCH4IV, NarI, etc.

[0045] In another aspect of the invention, a polynucleotide or a polynucleotide derived therefrom is provided, the polynucleotide 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; preferably, the at least one modified CpG site is any CpG site selected from 9 to 19 or a combination thereof, 1 to 8 or a combination thereof, or 20 to 48 or a combination thereof from the polynucleotide sequence shown in SEQ ID NO:1; preferably, the polynucleotide fragment is a polynucleotide of the nucleotide sequence shown at positions 302 to 428 or 272 to 458 in SEQ ID NO:1; or (2) a polynucleotide that is sequence-complementary to the polynucleotide or fragment of (1); wherein the polynucleotide derived from the polynucleotide is a polynucleotide corresponding to (1) or (2), wherein its unmodified cytosine is converted to T or U, while the cytosine C of its modified CpG site remains unchanged.

[0046] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0047] Figure 1In clinical lung cancer samples, the methylation values ​​of TAGMe-3 in cancer tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0048] Figure 2 In clinical samples of liver cancer, the methylation values ​​of TAGMe-3 in cancer tissue and control were compared (left figure), and the sensitivity and specificity were analyzed (right figure).

[0049] Figure 3 In clinical samples of prostate cancer, the methylation values ​​of TAGMe-3 in cancer tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0050] Figure 4 In cervical cancer clinical samples, the methylation values ​​of TAGMe-3 in cancer tissue and control were compared (left figure), and the sensitivity and specificity were analyzed (right figure).

[0051] Figure 5 In clinical samples of endometrial cancer, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0052] Figure 6 In clinical samples of urothelial carcinoma, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0053] Figure 7 In clinical samples of biliary tract tumors, the methylation values ​​of TAGMe-3 in cancerous tissues and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0054] Figure 8 In clinical samples of gastric cancer, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0055] Figure 9 In clinical samples of breast cancer, the comparison of methylation values ​​of TAGMe-3 in cancer tissue and controls (left figure) and sensitivity and specificity analysis (right figure).

[0056] Figure 10 In esophageal cancer clinical samples, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and the sensitivity and specificity were analyzed (right figure).

[0057] Figure 11 In clinical samples of glioma, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0058] Figure 12 In clinical samples of colorectal cancer, the methylation values ​​of TAGMe-3 in cancer tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0059] Figure 13 In clinical leukemia samples, the methylation values ​​of TAGMe-3 in cancerous tissues and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0060] Figure 14 In clinical pancreatic cancer samples, the methylation values ​​of TAGMe-3 in cancer tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0061] Figure 15 In clinical samples of thyroid cancer, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0062] Figure 16 In clinical melanoma samples, TAGMe-3 methylation values ​​were compared between cancerous tissue and controls (left figure), along with sensitivity and specificity analysis (right figure).

[0063] Figure 17 In nasopharyngeal carcinoma clinical samples, the methylation values ​​of TAGMe-3 in cancer tissue and controls were compared (left figure), and the sensitivity and specificity were analyzed (right figure).

[0064] Figure 18 In clinical samples of oral cancer, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0065] Figure 19 In clinical samples of laryngeal cancer, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0066] Figure 20 In osteosarcoma clinical samples, TAGMe-3 methylation values ​​were compared between cancerous tissue and controls (left figure), and sensitivity and specificity were analyzed (right figure).

[0067] Figure 21 In clinical lymphoma samples, TAGMe-3 methylation values ​​were compared between cancerous tissue and controls (left figure), along with sensitivity and specificity analysis (right figure).

[0068] Figure 22 In clinical samples of renal cell carcinoma, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0069] Figure 23 In clinical samples of ovarian cancer, the methylation values ​​of TAGMe-3 in cancerous tissue and controls were compared (left figure), and sensitivity and specificity were analyzed (right figure).

[0070] Figure 24 BSP verification of the methylation levels of methylation sites 1-8 (CpG) in SEQ ID NO:1 in cancer cells and normal control cells.

[0071] Figure 25 BSP verification of methylation levels at methylation sites 20-48 in SEQ ID NO:1 in cancer cells and normal control cells. Detailed Implementation

[0072] To identify DNA methylation tumor biomarker targets for multi-cancer screening, the inventors conducted extensive and in-depth research, analysis, and clinical trials, ultimately identifying TAGMe-3 as a target. The TAGMe-3 gene sequence region exhibits significant methylation differences between cancerous and adjacent normal tissues. Detection of abnormally high methylation in the TAGMe-3 gene sequence region indicates that the individual is at high risk for cancer. Furthermore, this significant difference in TAGMe-3 between tumor and non-tumor tissues is broadly present in various types of pan-cancer, including both solid and non-solid tumors.

[0073] As used herein, "sample" or "sample" includes any substance obtained from any individual (preferably a human) or isolated tissue, cells, or bodily fluid (such as plasma) that is suitable for DNA extraction and can be used 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 bronchoalveolar lavage fluid samples, ascites and lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, cervical scraping or brushing samples, and tissue and cell biopsy samples.

[0074] As used herein, the term "high methylation" refers to the presence of high methylation, hydroxymethylation, aldehyde methylation, or carboxymethylation of CpG in a gene sequence. For example, in methylation-specific PCR (MSP) analysis, a positive PCR result obtained using methylation-specific primers indicates that the tested DNA (gene) region is in a high methylation state. For example, in real-time quantitative methylation-specific PCR, the determination of high methylation can be based on statistically significant differences in the relative methylation levels of control samples.

[0075] In this invention, the term "tumor" refers to a broad range of tumors (pan-cancers) whose genome exhibits a hypermethylated state as described in this invention in the SEQ ID NO:1 region. These tumors can be solid or non-solid tumors and may include (but are not limited to): respiratory system tumors, digestive system tumors, urinary system tumors, gynecological and reproductive system tumors, hematological system tumors, nervous system tumors, head and neck tumors, skin system tumors, endocrine system tumors, or skeletal system tumors.

[0076] In this invention, the methylation status of the nucleotide sequence or a portion thereof shown in SEQ ID NO:1 differs significantly between tumor and non-tumor tissues. When an abnormally high methylation status is detected in the gene sequence region, the subject can be considered to have a tumor or belong to a high-risk group for tumors. This significant difference in methylation status presented by the gene sequence or a portion thereof shown in SEQ ID NO:1 is very significant in a variety of tumors (including early stages).

[0077] This invention also includes "conservative variant sequences" of the sequence SEQ ID NO:1 (or its reverse complementary sequence) that have conservation or high sequence identity with the nucleotide sequence shown in SEQ ID NO:1 (or its reverse complementary sequence). "High sequence identity" is defined as, 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 (e.g., some meaningless SNPs may exist), but this does not affect the detection based on the overall scheme of this invention.

[0078] Based on the above, the present invention provides a nucleic acid derived from a specific region of the human genome, having the gene sequence shown in SEQ ID NO:1 or a portion thereof, and also including its antisense strand. Within tumor cells, 5-methylcytosine (5mC) or other similar epigenetic modifications are generated at multiple 5'-CpG-3' base C positions in this nucleic acid sequence.

[0079] Detection of one or more CpGs provided by this invention is possible; therefore, this invention also includes nucleic acid fragments of the nucleotide sequence, including at least one methylated CpG site. The at least one may include 1 to 48 of SEQ ID NO:1 or its reverse complementary sequence, more specifically 2, 3, 5, 10, 11, 12, 15, 20, 25, 30, 35, 40, and 45. Those skilled in the art will understand that after this invention provides CpG numbering based on a single DNA strand, the corresponding numbering of each CpG site on the positive strand in the complementary DNA strand is readily obtainable according to the content provided by this invention.

[0080] With the information on the specific segments in the human genome provided by the present invention, those skilled in the art can easily obtain and apply the CpG sites. The embodiments of the present invention provide a series of sequence fragments containing CpG sites, which may serve as examples of preferred embodiments. However, it should be understood that variations can be made based on the information provided by the present invention, such as selecting longer sequences that contain the sequences of the present invention, or selecting sequences that overlap regionally with the sequences of the present invention.

[0081] This invention also includes gene panels or gene groups containing the nucleotide sequence or sequence fragment shown in SEQ ID NO:1 or its complementary sequence. For the aforementioned gene panels or gene groups, characteristics of normal cells and tumor cells can also be obtained through DNA methylation status detection.

[0082] It should be understood that a wide variety of techniques for analyzing methylation status can be applied in this invention, and this invention does not impose any particular limitation on such detection techniques. The nucleic acids provided by this invention can serve as key regions in the genome for analyzing methylation status, and their methylation status can be analyzed using various techniques known in the art, thereby analyzing the occurrence or development of tumors.

[0083] The nucleic acid or its fragment, or its complementary sequence, described in SEQ ID NO:1 of the present invention can be converted to uracil by bisulfite treatment, while the methylated cytosine remains unchanged. Therefore, the present invention also provides nucleic acids obtained by treating the above-mentioned nucleic acid (including its complementary strand (antisense strand)) with bisulfite, comprising: nucleic acids or nucleic acid fragments of the nucleotide sequence shown in SEQ ID NO:2. These nucleic acids can serve as more direct targets for designing detection reagents or detection kits.

[0084] The nucleic acids and / or their complementary nucleic acids and / or one or more fragments thereof of the nucleotide sequence shown in SEQ ID NO:1 of the present invention can be integrated into one or more wholes, such as one or more nucleic acid sets, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from the nucleic acid set to design targeted analytical reagents. The designed targeted analytical reagents can also be integrated into one or more wholes, such as one or more kits.

[0085] The nucleic acids of this invention, derived from the nucleotide sequence shown in SEQ ID NO:1 and / or its complementary nucleic acids and / or one or more fragments thereof (e.g., via bisulfite conversion), can also be integrated into one or more whole units, such as one or more nucleic acid sets, for use by those skilled in the art, such as selecting one or more nucleic acids or nucleic acid fragments from such nucleic acid sets to design targeted analytical reagents. The designed targeted analytical reagents can also be integrated into one or more whole units, such as one or more kits, or one or more chips.

[0086] Based on the target genes and their epigenetic characteristics provided in this invention, techniques known in the art, as well as some techniques that are about to be developed, can all be applied to this invention to detect methylation levels. The determination of nucleic acid methylation profiles can be performed using existing techniques (such as methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or other techniques that are still under development or will be developed. For example, quantitative methylation-specific PCR (QMSP) can be used to detect methylation levels; it is based on continuous optical monitoring of fluorescent PCR and is more sensitive than the MSP method. It has high throughput and avoids the need for electrophoresis analysis. In addition, other available techniques include: qPCR (Me-qPCR), next-generation sequencing, pyrosequencing, Sanger sequencing, bisulfite conversion sequencing, whole-genome methylation sequencing, DNA enrichment detection, simplified bisulfite sequencing, HPLC, and combinatorial gene group detection, etc., which are conventional methods in this field. Although some preferred embodiments of this invention are provided, the overall scheme of this invention is not limited thereto.

[0087] As a preferred embodiment of the present invention, a method for in vitro detection of the methylation profile of nucleic acids in a sample is also provided. The method is based on the principle that bisulfite can convert unmethylated cytosine into uracil, which is then converted into thymine during subsequent PCR amplification, while methylated cytosine remains unchanged. Therefore, after nucleic acid treatment with bisulfite, the methylated sites produce a nucleic acid polymorphism (SNP) similar to a C / T ratio. Identifying the methylation profile of nucleic acids in a sample based on this principle can effectively distinguish between methylated and unmethylated cytosine.

[0088] The method described in this invention includes: first, providing a sample and extracting genomic DNA; second, treating the genomic DNA obtained in step (a) with bisulfite, thereby converting unmethylated cytosine in the genomic DNA into uracil; and third, analyzing whether there are abnormal methylation patterns in the genomic DNA treated in step (b).

[0089] The method of this invention can be used to: test subject samples to assess whether the subject has a tumor; or to identify high-risk groups for tumors. The method 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 the disease, population geographic analysis studies, scientific research, population censuses, etc.

[0090] In a preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing. However, this method is not limited to practical applications; other DNA methylation detection methods known in the art or currently being improved may also be used. The primers used in the PCR amplification are not limited to those provided in the embodiments; primers that differ in sequence from those provided in the embodiments of the present invention, but still target the nucleic acid or corresponding CpG site indicated by the present invention, may also be obtained.

[0091] As a preferred embodiment of the present invention, a method for detecting the methylation status of nucleic acids in a sample in vitro is also provided, wherein the method is methylation-sensitive restriction endonuclease (MSRE) digestion. When a methylated base is present at its cleavage site, the methylation-sensitive restriction endonuclease cannot cleave DNA. The MSRE method is based on the fundamental principle that methylation-sensitive type II restriction endonucleases cannot cleave sequences containing one or more methylated cleavage sites. Fragments containing one or more methylated CpG sequences are cleaved with a methylation-sensitive type II endonuclease and its isoenzymes (insensitive to methylation), and then analyzed by DNA blotting. The advantages of this method include: no need to know detailed information about the primary structure of the target DNA, and the ability to provide a direct evaluation of the methylation status of CpG islands, including obtaining some quantitative analytical information on the methylation of the gene being tested.

[0092] In relation to the marker nucleic acid provided by this invention, other methods and reagents known to those skilled in the art for determining the sequence of a genome, its variations, and methylation status may also be included in this invention.

[0093] This invention provides a method for preparing a tumor detection reagent, comprising: providing the aforementioned nucleic acid; using the full length or a fragment of the nucleic acid as a target sequence; and designing a detection reagent specifically for detecting the target sequence; wherein the target sequence includes at least one methylation CpG site. The detection reagent may include, but is not limited to, chips, primers, probes, etc.; after obtaining the aforementioned marker, the selection of the detection reagent is a matter that can be accomplished by those skilled in the art.

[0094] Once the sequence of a nucleic acid is known, designing primers is known to those skilled in the art. Two primers are positioned flanking a specific sequence of the target gene to be amplified (including the CpG sequence, where the primers are complementary to the CpG to target methylated gene regions, and complementary to the TpG to target demethylated gene regions). In a preferred embodiment of the invention, the reagent is a primer, preferably one listed in the examples. Besides primers, other diagnostic or detection reagents can also be prepared, including but not limited to probes, chips, etc.

[0095] The reagents may also be combinations of reagents, such as primer combinations. For example, the combination may include more than one set of primers, thereby enabling the amplification of the multiple nucleic acids mentioned above.

[0096] The present invention also provides a kit for in vitro detection of methylation profiles of nucleic acids in samples, the kit comprising: a container, and the aforementioned primer pair located in the container.

[0097] The kit may also include various reagents required for DNA extraction, DNA purification, PCR amplification, and other reagents, such as sample processing reagents. Furthermore, the kit may include an instruction manual specifying the detection procedures and result interpretation criteria to facilitate application by those skilled in the art.

[0098] The methods and reagents of this invention exhibit very high accuracy when used to diagnose clinical tumors, as demonstrated in the detection of various clinical tumor samples in the embodiments of this invention. This invention can be applied to fields such as pre-tumor screening, efficacy assessment, auxiliary diagnosis, and prognostic monitoring, or, as mentioned above, situations where the purpose is not to obtain a direct disease diagnosis.

[0099] This invention provides biomarkers for multiple pan-cancers, applicable to the detection of cervical exfoliated cells, etc. The samples are easy to obtain and are non-invasive. Compared to clinical procedures requiring surgical tissue sampling, this significantly reduces patient discomfort, improves compliance, and simplifies the procedure for clinicians. Clearly, this represents a very significant advancement.

[0100] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0101] Example 1: Determination of methylation detection targets

[0102] 1.1 Obtain the human DNA sequence SEQ ID NO:1, which is the TAGMe-3 gene sequence;

[0103]

[0104] In the above sequence (positive chain), each "CG" marked by a solid underline represents a methylated CpG site, numbered sequentially from 5' to 3' as CpG sites 1 to 48 (methylated CpG sites 1 to 48). The dashed underline corresponds to the upstream and downstream primer design regions in some embodiments; the italicized bold areas correspond to the detection target regions in some embodiments.

[0105] 1.2 Obtain the DNA sequence SEQ ID NO:2 after bisulfite transformation, where Y represents C or U(T):

[0106]

[0107]

[0108] In the above sequence (positive chain), each "YG" marked by a solid underline represents a transformed methylated CpG site, numbered sequentially from 5' to 3' as "YG" 1 to 48 (the 1st to 48th transformed methylated CpG sites). The dashed underline corresponds to the upstream and downstream primer design regions in some of the embodiments; the italicized bold areas correspond to the detection target regions in some of the embodiments.

[0109] 1.3 Determine the detection area and design primers upstream and downstream of the detection area.

[0110] Example 2: Design and Synthesis of Detection Reagents

[0111] 2.1 Design first-round PCR primers with a length of 25-35 bp and appropriate CG content, and make the amplification length 100-300 bp;

[0112] 2.2 Add a barcode (Sample-ID) to the end of the first-round primers (Table 1);

[0113] 2.3 Add a tag sequence to the end of the first round of primers for library construction;

[0114] 2.4 Primers for the first round of PCR reaction and primers for the second round of PCR reaction with Illumina adapters and indexes were synthesized (Table 1).

[0115] Table 1

[0116]

[0117] 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, of which positions 302 to 428 contain CpG sites 9 to 19.

[0118] Example 3: Validation of the detection reagent

[0119] A primer pair was synthesized for two rounds of PCR reactions using positive and negative references:

[0120] The first round of PCR reaction system is shown in Table 2.

[0121] Table 2

[0122]

[0123] The second round of PCR reaction system is shown in Table 3.

[0124] Table 3

[0125]

[0126] Example 4: Construction of the primer pool

[0127] After verifying the primer PCR efficiency, primer pools with different barcodes were synthesized, and primers were diluted and combined (Table 4). The first round of PCR used M×N (10×10) combinations of F and R primers, allowing for simultaneous detection and localization of multiple samples. In Table 4, the lowercase bases in primer F1 correspond to positions 1-21 (Illumina adapter) of the upstream primer F1 in Table 1, while the uppercase bases are sequencing tags. The tag is followed by a base complementary to the target detection sequence (positions 29-58 of SEQ ID NO:3 in subsequent examples). In primer R1, the lowercase bases correspond to positions 1-20 of the upstream primer R1 in Table 1, while the uppercase bases are sequencing tags. The tag is followed by a base complementary to the target detection sequence (positions 28-57 of SEQ ID NO:4 in subsequent examples). The second round of primers used were F2 and R2 from Table 1.

[0128] Table 4

[0129]

[0130]

[0131] Where R represents A or G.

[0132] Example 5: Differential methylation of TAGMe-3 CpG sites in tumor tissues and non-tumor cells using NGS sequencing.

[0133] 5.1 Obtaining clinical samples: Obtain adjacent / non-cancerous to cancerous tissue samples from clinical settings. The adjacent / non-cancerous samples serve as the control group, while the cancerous tissue samples serve as the tumor detection experimental group.

[0134] 5.2 DNA extraction: DNA was extracted from the experimental group and the control group respectively; this experiment used the adsorption column method for extraction (but is not limited to this method);

[0135] 5.3 Bisulfite treatment: The extracted DNA samples were treated with bisulfite, and the procedure was strictly followed. In this experiment, the ZYMO Research EZ DNA Methylation-Gold Kit, catalog number D5006, was used (but this kit is not the only option).

[0136] 5.4 Using primers from the primer pool (first-round PCR primers) and universal sequencing primers for the Illumina system (second-round PCR primers), two rounds of PCR amplification were performed using conventional methods to construct the NGS library.

[0137] 5.5 After PCR amplification, 2% agarose gel electrophoresis was used to detect the PCR fragment specificity. 5 μL of PCR product was taken from each sample, mixed, purified, and the target fragment library was recovered for NGS sequencing.

[0138] 5.6 Sequencing Results Analysis: Extracting sequencing information from the sample based on the primer barcode sequence;

[0139] 5.7 TAGMe-3 methylation value calculation: NGS sequencing can independently detect the methylation status of individual CpG sites within the target region, and calculate the median value of methylation at all CpG sites as the TAGMe-3 methylation value in the sample;

[0140] 5.8 Results Analysis: The TAGMe-3 methylation values ​​in non-tumor tissues and tumor tissues were compared, and the cutoff value was determined by ROC curve.

[0141] Example 6, TAGMe-3: Clinical Sample Validation for Lung Cancer - NGS Sequencing

[0142] Forty clinical samples were obtained, including 20 lung cancer adjacent normal samples as the control group and 20 lung cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers in the first round from Table 4, and F2 and R2 in Table 1 for the second round of primers) to construct NGS libraries of lung cancer clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing steps.

[0143] The results are as follows Figure 1The results showed that in clinical lung cancer samples, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (****P<0.0001). Both sensitivity and specificity were 100%.

[0144] Example 7, TAGMe-3: Validation of Clinical Samples from Liver Cancer - NGS Sequencing

[0145] Thirty clinical samples were obtained, including 15 adjacent normal samples of liver cancer as the control group and 15 liver cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of liver cancer clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing steps.

[0146] The results are as follows Figure 2 The results showed that in clinical samples of liver cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (P<0.0001). The sensitivity was 86.67% and the specificity was 100%.

[0147] Example 8, TAGMe-3: Clinical Sample Validation for Prostate Cancer - NGS Sequencing

[0148] Thirty-four clinical samples were obtained, including 17 adjacent normal samples of prostate cancer as the control group and 17 prostate cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers in the first round from Table 4, and F2 and R2 in Table 1 for the second round of primers) to construct NGS libraries of clinical prostate cancer samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing steps.

[0149] The results are as follows Figure 3 The results showed that in clinical samples of prostate cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (P<0.0001). The sensitivity was 76.47%, and the specificity was 94.12%.

[0150] Example 9, TAGMe-3: Validation of Cervical Cancer Clinical Samples - NGS Sequencing

[0151] Forty clinical samples were obtained, including 20 samples of cervical adenocarcinoma adjacent to the normal tissue as the control group and 20 samples of cervical cancer as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of cervical cancer clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0152] The results are as follows Figure 4The results showed that in clinical samples of cervical cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (P<0.0001). The sensitivity was 90% and the specificity was 95%.

[0153] Example 10, TAGMe-3: Clinical Sample Validation for Endometrial Cancer - NGS Sequencing

[0154] Twenty-four clinical samples were obtained, including 12 samples of adjacent normal endometrial cancer as the control group and 12 samples of endometrial cancer as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (one pair of primers was selected from Table 4 for the first round, and F2 and R2 from Table 1 for the second round). NGS libraries of clinical endometrial cancer samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing steps.

[0155] The results are as follows Figure 5 The results showed that in clinical samples of endometrial cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (P<0.0001). The sensitivity was 91.67% and the specificity was 91.67%.

[0156] Example 11, TAGMe-3: Validation of Clinical Samples of Urothelial Carcinoma - NGS Sequencing

[0157] Thirty-eight clinical samples were obtained, including 19 urothelial carcinoma adjacent tissue samples as the control group and 19 urothelial carcinoma samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of clinical urothelial carcinoma samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0158] The results are as follows Figure 6 The results showed that in clinical samples of urothelial carcinoma, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (****P<0.0001). Sensitivity and specificity were 100%.

[0159] Example 12, TAGMe-3: Clinical Sample Validation of Biliary Tract Tumors - NGS Sequencing

[0160] Thirty-six clinical samples were obtained, of which 18 samples of adjacent normal biliary tract tumors served as the control group and 18 samples of biliary tract tumors served as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (one pair of primers was selected from Table 4 for the first round, and F2 and R2 from Table 1 for the second round). NGS libraries of clinical biliary tract tumor samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing procedure.

[0161] The results are as follows Figure 7 The results showed that in clinical samples of biliary tract tumors, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (P<0.0001). The sensitivity was 83.33%, and the specificity was 83.33%.

[0162] Example 13, TAGMe-3: Validation of Gastric Cancer Clinical Samples - NGS Sequencing

[0163] Fourteen clinical samples were obtained, including seven gastric cancer adjacent normal samples as the control group and seven gastric cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct NGS libraries of gastric cancer clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing steps.

[0164] The results are as follows Figure 8 The results showed that in clinical samples of gastric cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 71.43%, and the specificity was 85.71%.

[0165] Example 14, TAGMe-3: Clinical Sample Validation for Breast Cancer - NGS Sequencing

[0166] Sixteen clinical samples were obtained, including eight breast cancer adjacent normal samples as the control group and eight breast cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers in the first round from Table 4, and F2 and R2 in Table 1 for the second round of primers) to construct NGS libraries of breast cancer clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing steps.

[0167] The results are as follows Figure 9 The results showed that in clinical samples of breast cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (***P<0.001). Sensitivity and specificity were 100%.

[0168] Example 15, TAGMe-3: Validation of Clinical Samples from Esophageal Cancer - NGS Sequencing

[0169] Twelve clinical samples were obtained, including six esophageal cancer adjacent normal samples as the control group and six esophageal cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of esophageal cancer clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0170] The results are as follows Figure 10 The results showed that in esophageal cancer clinical samples, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 83.33% and the specificity was 100%.

[0171] Example 16, TAGMe-3: Validation of Clinical Samples from Gliomas - NGS Sequencing

[0172] Twelve clinical samples were obtained, including six glioma adjacent normal samples as the control group and six glioma samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of clinical glioma samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0173] The results are as follows Figure 11 The results showed that in clinical samples of glioma, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 66.67% and the specificity was 100%.

[0174] Example 17, TAGMe-3: Validation of Clinical Samples from Colorectal Cancer - NGS Sequencing

[0175] Twelve clinical samples were obtained, including six adjacent normal colorectal cancer samples as the control group and six colorectal cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (one pair of primers was selected from Table 4 for the first round, and F2 and R2 from Table 1 for the second round). NGS libraries of colorectal cancer clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing steps.

[0176] The results are as follows Figure 12 The results showed that in clinical samples of colorectal cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 83.33%, and the specificity was 83.33%.

[0177] Example 18, TAGMe-3: Validation of Clinical Samples from Leukemia - NGS Sequencing

[0178] Twelve clinical samples were obtained, including six non-leukemia bone marrow smear samples as the control group and six leukemia bone marrow smear samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of leukemia clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0179] The results are as follows Figure 13 The results showed that in clinical leukemia 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). The sensitivity was 100%, and the specificity was 83.33%.

[0180] Example 19, TAGMe-3: Clinical Sample Validation for Pancreatic Cancer - NGS Sequencing

[0181] Twelve clinical samples were obtained, including six pancreatic cancer adjacent tissue samples as the control group and six pancreatic cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of liposarcoma clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0182] The results are as follows Figure 14 The results showed that in clinical samples of pancreatic cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). Sensitivity and specificity were both 100%.

[0183] Example 20, TAGMe-3: Clinical Sample Validation for Thyroid Cancer - NGS Sequencing

[0184] Twelve clinical samples were obtained, including six thyroid cancer adjacent tissue samples as the control group and six thyroid cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers in the first round from Table 4, and F2 and R2 in Table 1 for the second round of primers) to construct an NGS library of clinical thyroid tumor samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0185] The results are as follows Figure 15 The results showed that in clinical samples of thyroid cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). The sensitivity was 83.33%, and the specificity was 83.33%.

[0186] Example 21, TAGMe-3: Clinical Sample Validation for Melanoma - NGS Sequencing

[0187] Twelve clinical samples were obtained, including six normal skin tissue samples as the control group and six melanoma samples as the experimental group. Two rounds of PCR reactions were performed using the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round). NGS libraries of melanoma clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing steps.

[0188] The results are as follows Figure 16 The results showed that in clinical melanoma samples, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). Sensitivity and specificity were 100%.

[0189] Example 22, TAGMe-3: Validation of Nasopharyngeal Carcinoma Clinical Samples - NGS Sequencing

[0190] Twelve clinical samples were obtained, including six nasopharyngeal carcinoma adjacent normal samples as the control group and six nasopharyngeal carcinoma samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of nasopharyngeal carcinoma clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0191] The results are as follows Figure 17 The results showed that in clinical samples of nasopharyngeal carcinoma, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). The sensitivity was 100%, and the specificity was 83.33%.

[0192] Example 23, TAGMe-3: Clinical Sample Validation for Oral Cancer - NGS Sequencing

[0193] Twelve clinical samples were obtained, including six oral cancer adjacent normal samples as the control group and six oral cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers in the first round from Table 4, and F2 and R2 in Table 1 for the second round of primers) to construct an NGS library of oral cancer clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing steps.

[0194] The results are as follows Figure 18 The results showed that in clinical samples of oral cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 100%, and the specificity was 83.33%.

[0195] Example 24, TAGMe-3: Clinical Sample Validation for Laryngeal Cancer - NGS Sequencing

[0196] Twelve clinical samples were obtained, including six adjacent samples of laryngeal cancer as the control group and six laryngeal cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of clinical laryngeal cancer samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing steps.

[0197] The results are as follows Figure 19 The results showed that in clinical samples of laryngeal cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). Sensitivity and specificity were both 100%.

[0198] Example 25, TAGMe-3: Validation of Osteosarcoma Clinical Samples - NGS Sequencing

[0199] Twelve clinical samples were obtained, including six osteosarcoma adjacent normal samples as the control group and six osteosarcoma samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of osteosarcoma clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0200] The results are as follows Figure 20 The results showed that in osteosarcoma clinical samples, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 66.67% and the specificity was 83.33%.

[0201] Example 26, TAGMe-3: Validation of Lymphoma Clinical Samples - NGS Sequencing

[0202] Twelve clinical samples were obtained, including six normal lymphocyte samples as the control group and six lymphoma samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of lymphoma clinical samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0203] The results are as follows Figure 21 The results showed that in clinical lymphoma samples, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 83.33%, and the specificity was 83.33%.

[0204] Example 27, TAGMe-3: Clinical Sample Validation for Renal Cell Carcinoma - NGS Sequencing

[0205] Twelve clinical samples were obtained, including six renal cell carcinoma adjacent normal samples as the control group and six renal cell carcinoma samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (select one pair of primers for the first round from Table 4, and F2 and R2 from Table 1 for the second round of primers) to construct an NGS library of clinical renal cell carcinoma samples. The TAGMe-3 methylation level was analyzed according to the NGS sequencing procedure.

[0206] The results are as follows Figure 22 The results showed that in clinical samples of renal cell carcinoma, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (**P<0.01). Sensitivity and specificity were 100%.

[0207] Example 28, TAGMe-3: Validation of Ovarian Cancer Clinical Samples - NGS Sequencing

[0208] Twelve clinical samples were obtained, including six ovarian cancer adjacent normal samples as the control group and six ovarian cancer samples as the experimental group. Two rounds of PCR reactions were performed according to the primer combination method described in Example 4 above (one pair of primers was selected from Table 4 for the first round, and F2 and R2 from Table 1 for the second round). NGS libraries of ovarian cancer clinical samples were constructed, and TAGMe-3 methylation levels were analyzed according to the NGS sequencing steps.

[0209] The results are as follows Figure 23 The results showed that in clinical samples of ovarian cancer, the methylation value of TAGMe-3 in cancerous tissue was significantly higher than that in adjacent normal tissue (*P<0.05). The sensitivity was 83.33% and the specificity was 66.67%.

[0210] Example 29: Detection performance analysis of a single CpG site

[0211] Using the clinical samples obtained in the foregoing embodiments, the feasibility of using CpG sites 9 to 19 (CpG sites in the segment 302 to 428 of SEQ ID NO:1) as single CpG sites for cancer detection was analyzed. The methylation modification status of a single CpG site was determined by NGS sequencing.

[0212] The results are shown in Tables 5-9. It can be seen that a single CpG site already has high sensitivity and specificity, and can also be used as a target for methylation modification analysis, which is of significance for cancer diagnosis.

[0213] Table 5

[0214]

[0215] Table 6

[0216]

[0217]

[0218] Table 7

[0219]

[0220] Table 8

[0221]

[0222] Table 9

[0223]

[0224] Example 30: Differential methylation of TAGMe-3 CpG sites in tumor cells and non-tumor cells—BSP-Bisulfite Sequencing PCR.

[0225] The sequencing steps after bisulfite treatment are as follows:

[0226] 1. Genomic DNA was extracted from hematologic malignancy 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 was extracted.

[0227] 2. Genomic DNA extracted from cancer cell lines and normal cell lines was treated with bisulfite, respectively, to serve as templates for subsequent PCR amplification;

[0228] 3. Design amplification primers based on the sequence of SEQ ID NO:2, as shown in Table 10, and perform amplification.

[0229] 4. After PCR amplification, the PCR fragment specificity was detected by 2% agarose gel electrophoresis. The target fragment was excised and recovered, ligated into the insertion T vector, transformed into competent E. coli, plated, and clones were selected for sequencing the next day. More than 10 clones of each fragment were selected for Sanger sequencing.

[0230] Table 10. BSP Primers

[0231]

[0232] BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 1-8 in SEQ ID NO:1 is as follows: Figure 24 The results showed that the TAGMe-3 methylation level in cancer cells was significantly higher than that in normal cells.

[0233] BSP verification of methylation levels of cancer cells and normal control cells at methylation sites 20-48 in SEQ ID NO:1 is as follows: Figure 25 The results showed that the TAGMe-3 methylation level in cancer cells was significantly higher than that in normal cells.

[0234] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. Use of a detection reagent in the preparation of a reagent for identifying a tumor, comprising: (a) providing an isolated polynucleotide, which is a polynucleotide of the nucleotide sequence shown in positions 302-428 of SEQ ID NO: 1; (b) designing a detection reagent for specifically detecting the methylation level of a CpG site of the target sequence, with the polynucleotide of (a) as the target sequence. The tumor is lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial cancer, biliary tract tumor, gastric cancer, breast cancer, esophageal cancer, brain glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer.

2. Use of a reagent for specifically detecting the methylation level of a CpG site of a target sequence in the preparation of a kit for identifying a tumor; the tumor is lung cancer, liver cancer, prostate cancer, cervical cancer, endometrial cancer, urothelial cancer, biliary tract tumor, gastric cancer, breast cancer, esophageal cancer, brain glioma, colorectal cancer, leukemia, pancreatic cancer, thyroid cancer, melanoma, nasopharyngeal carcinoma, oral cancer, laryngeal cancer, osteosarcoma, lymphoma, renal cell carcinoma or ovarian cancer; wherein the target sequence is a polynucleotide of the nucleotide sequence shown in positions 302-428 of SEQ ID NO:

1.

3. Use according to claim 2, characterized in that, The sample for identifying the tumor includes a tissue sample and a body fluid sample.

4. The use according to claim 2, characterized in that, The sample for identifying the tumor includes a blood sample.

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