A new tumor detection marker, TAGMe, and its applications

Abstract

Tumor detection marker TAGMe and its uses. Through analysis and research of a large number of clinical samples, a new DNA methylation tumor marker was isolated. Both of these markers were hypomethylated in normal tissues and hypermethylated in tumor samples. Significant differences in methylation status were observed in tumor patients, and these differences were statistically significant. The use of these markers in combination for detection is more effective. This tumor marker may be used as a marker for clinical tumor diagnosis, screening, classification, detection, and prognosis, or as a new molecule to aid in the clinical diagnosis and prognosis of tumors. It may also be used in the design of diagnostic reagents and kits.

Description

[Technical Field]

[0001] This application was filed on September 22, 2022, and claims priority to the invention entitled "A new type of tumor detection marker TAGMe and its use" and application number CN202211157754.9, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of gene epigenetic modification, and more particularly to a novel tumor detection marker and its application. [Background technology]

[0003] Endometrial cancer (EC) is one of the most common malignant tumors of the female reproductive tract, second only to cervical cancer in incidence. With improving living conditions, the incidence of EC has been increasing year by year. By 2030, the economic and medical burden of EC worldwide is estimated to increase by 60%. While most EC occurs in postmenopausal women, the incidence of EC has recently increased significantly in women under 40 years of age, ranging from 2% to 14%. For young women of childbearing age, early diagnosis of EC, provided there is no myometrial invasion or extrauterine spread, still offers the opportunity to preserve the uterus and / or ovaries. Therefore, early diagnosis of EC is crucial, potentially reducing female mortality, improving treatment opportunities for younger patients, and preserving fertility or reproductive endocrine function.

[0004] However, there is currently a lack of reliable, effective, non-invasive early screening and diagnostic aids in clinical practice. In the early stages of endometrial cancer, many patients have no obvious associated positive symptoms; according to numerous studies, 90% of endometrial cancer patients experience various types of vaginal bleeding, but only 5-10% of women who experience abnormal vaginal bleeding are diagnosed with endometrial cancer or precancerous lesions. Furthermore, there is currently a lack of reliable, effective, non-invasive early screening and diagnostic aids in clinical practice. Existing conventional testing methods include: (1) Ultrasound: Transvaginal ultrasound (TVS) is often the initial test for postmenopausal bleeding patients and the most commonly used non-invasive auxiliary testing method, but due to its high false positive rate, it cannot reliably distinguish between benign and malignant endometrium. At the same time, EC also occurs in women whose endometrium does not thicken; (2) Imaging tests: Imaging tests including magnetic resonance (pelvic MRI), CT, PET-CT, etc. can reveal information such as the size and specific location of the lesion, but it is difficult to diagnose early EC and precancerous lesions, so it is generally used for phenotyping; (3) Cytological tests: As a non-invasive testing method, it has high specificity but low sensitivity. The positive rate of vaginal exfoliation cytological tests is low, but cancer cells in cervical exfoliation are easily dissolved and degenerated, making them difficult to visualize after staining. (4) Serum tumor markers: There are no specific and sensitive diagnostic markers for endometrial cancer. Some patients have abnormalities in CA125, CA19-9, CA153, or HE4. These abnormalities correlate with factors such as histological type, depth of myometrial invasion, and extrauterine involvement, but have little correlation with the benign or malignant nature of the endometrium. Due to the lack of more minimally invasive diagnostic methods, all patients with abnormal vaginal bleeding must undergo pathological examination of endometrial tissue through invasive testing (uterine curettage and / or endoscopic biopsy) to confirm the diagnosis of endometrial cancer. Many endometrial cancers develop from endometrial hyperplasia to atypical hyperplasia (precancerous lesions), and this progression continues for many years. Over the long course of the disease, these patients with abnormal vaginal bleeding often undergo multiple invasive endometrial biopsies, which causes triple stress—physiological, psychological, and economic. Therefore, new methods for early cancer detection are needed. Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a novel DNA methylation tumor marker set, consisting of TAGMe-1 ​​and TAGMe-2, and their combination, which is hypomethylated in normal tissues and hypermethylated in endometrial cancer samples, and can be used to detect endometrial cancer in sample types including, but not limited to, tissue, cervical sloughs, uterine cavity scrapings, and vaginal secretions. [Means for solving the problem]

[0006] In a first aspect of the present invention, there is provided use of an isolated nucleic acid or a nucleic acid converted therefrom in the preparation of a reagent or kit for detecting (including screening, diagnosing, detecting, or prognostic evaluation) a tumor, wherein the nucleic acid is (1): a nucleic acid or nucleic acid combination of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5; or (2): a nucleic acid or nucleic acid combination that is sequence complementary to the nucleic acid of (1); wherein the nucleic acid converted from the isolated nucleic acid is a nucleic acid corresponding to (1) or (2), in which unmodified cytosines are converted to T or U, but cytosine C in modified CpG sites remains unchanged.

[0007] In one or more embodiments, SEQ ID NO:1 or SEQ ID NO:5 further includes sequence variants or homologous sequences thereof. Preferably, the sequence variants or homologous sequences have 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 sequence identity to the sequence set forth in SEQ ID NO:1 or SEQ ID NO:5. Also included are polynucleotides that are converted from the sequence variants or homologous sequences (in which unmodified cytosines are converted to T or U, and cytosine C at modified CpG sites remains unchanged).

[0008] In one or more embodiments, the nucleic acid complementary to the nucleic acid (1) has the nucleotide sequence set forth in SEQ ID NO:2 or SEQ ID NO:6, respectively. The present invention also includes sequence variants or homologous sequences of SEQ ID NO:2 or SEQ ID NO:6, which have 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 sequence identity to the sequence set forth in SEQ ID NO:2 or SEQ ID NO:6. Similarly, the present invention also includes polynucleotides that are converted from the sequence variants or homologous sequences (unmodified cytosines are converted to T or U, and cytosine C at modified CpG sites remains unchanged).

[0009] In one or more embodiments, the tumor comprises endometrial cancer, cervical cancer (cervical squamous cell carcinoma and cervical adenocarcinoma), pancreatic cancer, head and neck tumors, colon cancer, rectal cancer, esophageal cancer, lung cancer (lung squamous cell carcinoma, lung adenocarcinoma), and bile duct cancer.

[0010] In one or more embodiments, the subject of the tumor detection is a tumor (e.g., endometrial cancer) or a precancerous lesion thereof (e.g., endometrial atypical proliferation).

[0011] In one or more embodiments, samples targeted for tumor detection include, but are not limited to, blood samples, tissue samples (e.g., paraffin-embedded samples), cervical samples, uterine cavity samples, pleural fluid collection samples, bronchoalveolar lavage fluid samples, ascites samples, ascites lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, and cell samples; preferably, the samples include, but are not limited to, cervical smear or scraping samples, cervical swabs, cervical tissue (scrapes), cervical exfoliated cells, uterine cavity scrapes, uterine cavity washings, and vaginal secretions.

[0012] In another aspect of the present invention, there is provided a method for detecting the methylation level of a sample to be detected, comprising extracting nucleic acid from the sample to be detected and detecting the CpG site modification status of a target sequence or a fragment thereof of the extracted nucleic acid, wherein the target sequence is a nucleic acid or a combination of nucleic acids converted from the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5, and corresponds to the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5, in which unmodified cytosines are converted to T or U, but cytosine C at modified CpG sites remains unchanged.

[0013] In one or more embodiments, the method for detecting the CpG site modification status of the target sequence of the extracted nucleic acid includes pyrosequencing, bisulfite sequencing, methylation chip, methylation-specific PCR, methylation-sensitive restriction endonuclease enzyme cleavage, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, Reduced Representation Bisulfite Sequencing (RRBS) technology, HPLC, MassArray, or a combination thereof.

[0014] In one or more embodiments, the method for detecting the CpG site modification status of a target sequence of extracted nucleic acid includes: (i) treating the extracted nucleic acid to convert unmodified cytosines therein to uracils.

[0015] In one or more embodiments, the modification comprises a 5-methylation modification (5mC), a 5-hydroxymethylation modification (5hmC), a 5-formylation modification (5-fC), or a 5-carboxylation modification (5-caC).

[0016] In one or more embodiments, the nucleic acid described in step (i) is treated with bisulfite; and (ii): the modification status of the target sequence of the nucleic acid treated in (i) is analyzed.

[0017] In one or more embodiments, the nucleic acid is a combination of "SEQ ID NO:1 or its reverse complement" and "SEQ ID NO:5 or its reverse complement."

[0018] In one or more embodiments, an abnormal methylation profile means that the C in the CpG of the nucleic acid is highly methylated.

[0019] In one or more embodiments, other methylation detection methods and newly developed methylation detection methods in the future are also applicable to the present invention.

[0020] In one or more embodiments, the methylation profiling method is not a diagnostic method, ie, it is not intended to directly result in a diagnosis of disease.

[0021] In one or more embodiments, the method for detecting the chilling profile of the sample is an in vitro method.

[0022] In one or more embodiments, the methylation-sensitive restriction endonuclease is an endonuclease that is sensitive to methylated bases contained in its recognition site, including, but not limited to, one or a combination of HpaII, AciI, Bsu15I, Hin1I, Hin6I, HpyCH4IV, NarI, and the like.

[0023] In another aspect of the present invention, there is provided a method for preparing a reagent for use in tumor detection (including screening, diagnosis, detection, or prognosis evaluation), the method comprising: providing a nucleic acid having the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:5; and designing a detection reagent that uses the full length or a fragment of the nucleic acid as a target sequence to specifically detect the CpG site modification status of the target sequence.

[0024] In one or more embodiments, the detection reagent includes, but is not limited to, a primer, a probe, a chip, or a test strip.

[0025] In one or more embodiments, one or more sets of reagents can be prepared for full-length or fragments of SEQ ID NO:1.

[0026] In one or more embodiments, one or more sets of reagents can be prepared for full-length or fragments of SEQ ID NO:5.

[0027] In one or more embodiments, the detection reagent is incorporated into the chip.

[0028] In another aspect of the present invention, a reagent or a combination of reagents is provided, which specifically detects the CpG site modification status of a target sequence, wherein the target sequence is a nucleic acid or a nucleic acid combination converted from the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5.

[0029] In one or more embodiments, the reagent or combination of reagents is directed to a gene sequence that includes the target sequence.

[0030] In one or more embodiments, the gene array comprises a gene panel or group of genes.

[0031] In one or more embodiments, preferably, the reagent or combination of reagents is selected from one or more of the following: primers having the sequences shown in SEQ ID NO:9 and SEQ ID NO:10; primers having the sequences shown in SEQ ID NO:11 and SEQ ID NO:12; primers having the sequences shown in SEQ ID NO:13 and SEQ ID NO:14; or primers having the sequences shown in SEQ ID NO:15 and SEQ ID NO:16.

[0032] In another aspect of the present invention, there is provided a use of the reagent or combination of reagents for the preparation of a kit for detecting (including screening, diagnosing, detecting or prognostic evaluation of) tumors; preferably, the tumors include endometrial cancer, cervical cancer, colorectal cancer and lung cancer.

[0033] In another aspect of the present invention, there is provided a kit for use in detecting (including screening, diagnosing, detecting or prognostic evaluation of) a tumor, said kit comprising the reagent or combination of reagents described above.

[0034] In one or more embodiments, the kit further includes, but is not limited to, DNA purification reagents, DNA extraction reagents, bisulfite, and PCR amplification reagents.

[0035] In one or more embodiments, the kit also includes instructions describing the detection procedure steps and result criteria.

[0036] In another aspect of the present invention, there is provided an isolated nucleic acid or a nucleic acid converted therefrom, wherein the nucleic acid is (1): a nucleic acid or nucleic acid combination of the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:5; or (2): a nucleic acid or nucleic acid combination that is sequence-complementary to the nucleic acid of (1); wherein the nucleic acid converted therefrom is a nucleic acid corresponding to (1) or (2), in which unmodified cytosines are converted to T or U, but cytosine C at modified CpG sites remains unchanged.

[0037] In one or more embodiments, the modified CpG site includes a CpG site where a 5-formylation modification, a 5-hydroxymethylation modification, a 5-methylation modification, or a 5-carboxylation modification occurs.

[0038] Other aspects of the present invention will be apparent to those skilled in the art based on the disclosure herein. [Brief explanation of the drawings]

[0039] [Figure 1] Figure 1 is a heat map of candidate markers for diagnosing endometrial cancer and precancerous lesions. [Figure 2] Figures 2A-D show the performance of TAGMe-1 ​​and TAGMe-2 as biomarkers in other cancer types in the TCGA database. [Figure 3] 3A-D show the results of tissue target NGS in Example 3. [Figure 4] 4A-D show the results of TAGMe-1 ​​analysis in Example 4. [Figure 5] 5A-D show the results of Me-qPCR of cervical cells in Example 5. [Figure 6] 6A-D show the results of Me-qPCR of uterine luminal cells in Example 6. [Figure 7] FIG. 7 shows the sequence information of the TAGMe-1 ​​methylated tumor marker. [Figure 8] FIG. 8 shows the sequence information of the TAGMe-2 methylated tumor marker. DETAILED DESCRIPTION OF THE INVENTION

[0040] According to the present invention, DNA methylation mutations play an important role in the development and progression of endometrial cancer and can be used as a marker for applications such as early screening, diagnostic support, treatment efficacy evaluation, and recurrence monitoring of endometrial cancer. In the present invention, a new DNA methylation tumor marker was isolated through analysis and research of a large number of clinical samples. These markers are hypomethylated in normal tissues and hypermethylated in tumor (particularly endometrial cancer) samples, demonstrating significant differences in methylation status between tumor patients, which are statistically significant. The use of these markers in combination for detection is more effective. Therefore, the tumor markers of the present invention can be used as markers for clinical tumor diagnosis, screening, classification, detection, and prognosis, as new molecules to aid in the clinical diagnosis and prognosis of tumors, and can be used in the design of diagnostic reagents and kits.

[0041] In the present invention, the term "sample" or "specimen" includes any substance suitable for detecting DNA methylation status obtained from any individual (preferably human) or isolated tissue, cell, or body fluid (such as plasma). For example, the sample includes, but is not limited to, a blood sample, a tissue sample (e.g., a paraffin-embedded sample), a cervical sample, a uterine cavity sample, a pleural fluid collection sample, a bronchoalveolar lavage fluid sample, an ascites sample, an ascites lavage fluid sample, a bile sample, a fecal sample, a urine sample, a saliva sample, a cerebrospinal fluid sample, a cell smear sample, and a cell sample; preferably, the sample includes, but is not limited to, a cervical smear or scraping sample, a cervical swab, a cervical tissue (scrape), a cervical exfoliated cell, a uterine cavity scrape, a uterine cavity lavage fluid, and a vaginal secretion.

[0042] In the present invention, the term "hypermethylation" refers to the presence of highly methylated, hydroxymethylated, formylated, or carboxylated CpG modifications in a gene sequence. For example, in the case 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 being tested is in a hypermethylated state. For example, in the case of real-time quantitative methylation-specific PCR, the determination of the hypermethylation state can be analyzed for statistical differences based on the relative methylation state of a control sample.

[0043] In the present invention, the term "tumor" refers to tumors in whose genome the segment of SEQ ID NO: 1 or SEQ ID NO: 5 exhibits a hypermethylated state as described in the present invention.

[0044] As used herein, the term "detection" includes screening, diagnosis, detection or prognostic evaluation.

[0045] As used in the present invention, the "endometrial cancer" broadly includes "endometrial cancer and its precancerous lesions," the latter also being called "atypical endometrial proliferation" or "early endometrial cancer." However, in certain cases, for example, in some embodiments, the "endometrial cancer" is distinct from "precancerous endometrial lesions," and in this case, it can be understood that "endometrial cancer" refers to cancer that has passed the precancerous lesion stage, for example, cancer at an advanced stage.

[0046] This study demonstrates that several significant epigenetic changes are important factors in the development of endometrial cancer. Compared with DNA mutations, specific regions of the genome exhibit hypermethylation, which occurs earlier and more stably in early tumor diagnosis. Abnormal DNA methylation patterns can be used to predict the risk of atypical proliferation leading to endometrial cancer metastasis. The DNA hypermethylation state permeates the entire process of normal cells transforming into tumor cells, and occurs very early in tumor development, preceding gene mutations. Therefore, tumor detection based on DNA hypermethylation can be applied to very early tumor screening, diagnostic assistance, treatment efficacy evaluation, and recurrence monitoring, covering the entire process of tumor diagnosis.

[0047] Based on this, the present invention provides a methylation marker set consisting of TAGMe-1 ​​and TAGMe-2 as molecular detection markers for endometrial cancer. These markers are hypomethylated in normal and peritumoural endometrial tissues, but hypermethylated in endometrial cancer and precancerous lesion (atypical hyperplasia) tissues. Using exfoliated cells from the cervix and uterine cavity, this set can provide noninvasive auxiliary diagnosis of early-stage endometrial cancer and precancerous lesions, filling the gaps in existing endometrial cancer screening technologies. Further development could lead to the realization of a relatively simple, inexpensive, reliable, and high-throughput clinical test.

[0048] In the present invention, the methylation status of the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:5 or a partial region (fragment) thereof is significantly different between tumor tissues and non-tumor tissues, and if an abnormal hypermethylation status is detected in the gene sequence region, the subject is considered to be suffering from tumor or in a high-risk group for tumors. Such significant differences in methylation status represented by the gene sequence shown in SEQ ID NO:1 or SEQ ID NO:5 or a partial region thereof are very prominent in endometrial cancer (including early-stage cancer); at the same time, such significant differences in methylation status are also present in several other tumors, including cervical cancer (cervical squamous cell carcinoma and cervical adenocarcinoma), pancreatic cancer, head and neck tumors, colon cancer, rectal cancer, esophageal cancer, lung cancer (lung squamous cell carcinoma and lung adenocarcinoma), and bile duct cancer.

[0049] The present invention also includes "conservative variants" of SEQ ID NO:1 (or its reverse complementary sequence) or SEQ ID NO:5 (or its reverse complementary sequence), which are conserved or have high sequence identity with the nucleotide sequence shown in SEQ ID NO:1 (or its reverse complementary sequence) or SEQ ID NO:5 (or its reverse complementary sequence). The "high sequence identity" means, for example, more than 90%, more than 92%, more than 95%, more than 98%, more than 99%, etc. Individual sequence sites may differ between different organisms (e.g., meaningless SNPs may exist), but it should be understood that this does not affect the detection in the overall technical solution based on the present invention.

[0050] Therefore, the present invention provides nucleic acids derived from a specific region of the human genome, which have the gene sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5 or a partial region thereof, as well as antisense strands thereof. In tumor cells, 5-methylcytosine (5mC) is generated at the C position of 5'-CpG-3' bases at many sites in the nucleic acid sequence.

[0051] The present invention may detect one or more CpGs. The present invention also includes nucleic acid fragments of the above nucleotide sequences, which contain at least one methylated CpG site. The at least one may include 2 to 53, more specifically, for example, 3, 5, 8, 10, 12, 15, 18, 20, 30, 40, or 50, CpGs from SEQ ID NO:1 or its reverse complement. The at least one may include 2 to 52, more specifically, for example, 3, 5, 8, 10, 12, 15, 18, 20, 30, 40, or 50, CpGs from SEQ ID NO:5 or its reverse complement. Those skilled in the art will understand that, after the present invention provides CpG numbers based on one DNA strand, the numbers corresponding to the sense strand for each CpG site in the complementary DNA strand can be easily obtained from the information provided by the present invention.

[0052] Based on the information of the specific fragments in the human genome provided by the present invention, those skilled in the art can easily obtain and apply the CpG sites. In the examples of the present invention, a series of sequence fragments containing CpG sites are provided as some examples of preferred embodiments, but it should be understood that, according to the information provided by the present invention, people can, for example, select longer sequences that include the sequences of the present invention, or select sequences that intersect with the corresponding sequences of the present invention in a regional manner.

[0053] The present invention also includes a gene panel or group of genes having the nucleotide sequence or sequence fragment shown in SEQ ID NO:1 or SEQ ID NO:5 or its complementary sequence (SEQ ID NO:2 or SEQ ID NO:6), for which detection of DNA methylation status can also be used to characterize normal and tumor cells.

[0054] It should be understood that various techniques available 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 are important regions for analyzing methylation status in the genome, and their methylation status can be analyzed by various techniques known in the art to analyze the development and progression of tumors.

[0055] In the nucleic acids of the present invention set forth in SEQ ID NO:1 or SEQ ID NO:5, or fragments thereof, or sequences complementary thereto, unmethylated cytosines are converted to uracil, while methylated cytosines are retained after bisulfite treatment. Therefore, the present invention also provides nucleic acids obtained by bisulfite treatment of the nucleic acids (including their complementary strands (antisense strands)), including nucleic acids or nucleic acid fragments of the nucleotide sequence set forth in SEQ ID NO:3 or SEQ ID NO:7, or, for example, nucleic acids or nucleic acid fragments of the nucleotide sequence set forth in SEQ ID NO:4 or SEQ ID NO:8. These nucleic acids can be more directly targeted for designing detection reagents or kits.

[0056] The nucleic acid of the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:5 of the present invention and / or its complementary nucleic acid and / or one or more fragments thereof may be integrated into one or more wholes (e.g., one or more groups of nucleic acids) and used by those skilled in the art, for example, to select one or more nucleic acids or nucleic acid fragments from the group of nucleic acids and design a target analysis reagent. The designed target analysis reagent may be integrated into one or more wholes, for example, one or more kits.

[0057] The nucleic acids of the present invention having the nucleotide sequence set forth in SEQ ID NO:1 or SEQ ID NO:5 and / or their complementary nucleic acids and / or one or more fragments thereof converted (e.g., by bisulfite conversion) can be integrated into one or more wholes (e.g., one or more groups of nucleic acids) and used by those skilled in the art, for example, to select one or more nucleic acids or nucleic acid fragments from the group of nucleic acids and design a target analysis reagent. The designed target analysis reagent can be integrated into one or more wholes, for example, one or more kits or one or more chips.

[0058] In a preferred embodiment of the present invention, a nucleic acid derived from the nucleotide sequence set forth in SEQ ID NO:1 and / or its complementary nucleic acid and / or one or more fragments thereof and a nucleic acid derived from the nucleotide sequence set forth in SEQ ID NO:5 and / or its complementary nucleic acid and / or one or more fragments thereof are used in combination for detection.

[0059] Based on the target genes and their epigenetic characteristics provided by the present invention, these techniques, as well as several techniques yet to be developed, known in the art can be applied to the present invention to detect methylation levels. The methylation profile of nucleic acids can be measured by existing technologies (e.g., methylation-specific PCR (MSP) or real-time quantitative methylation-specific PCR, Methylight), or other technologies currently under development. For example, quantitative methylation-specific PCR (QMSP) can be used to detect methylation levels. This method is based on continuous optical monitoring based on fluorescent PCR and is more sensitive than the MSP method. It has high throughput and avoids the use of electrophoresis for analyzing the results. Other techniques that can be used include conventional methods in the art, such as qPCR (Me-qPCR), next-generation sequencing, pyrosequencing, Sanger sequencing, bisulfite sequencing, whole-genome methylation sequencing, DNA enrichment detection, reduced representation bisulfite sequencing (RRBS), HPLC, and combined gene group detection. Although the embodiments of the present invention provide some preferred methods, the overall technical idea of ​​the present invention is not limited thereto.

[0060] In a preferred embodiment of the present invention, a method for detecting the methylation profile of nucleic acid in a sample in vitro is also provided. The method is based on the following principle: bisulfite can convert unmethylated cytosine to uracil, which is then converted to thymine in the subsequent PCR amplification process, while methylated cytosine remains unchanged; therefore, after treating nucleic acid with bisulfite, a nucleic acid polymorphism (SNP) such as C / T is generated at the methylated site. Detecting the methylation profile of nucleic acid in a sample based on the above principle can effectively distinguish between methylated and unmethylated cytosine.

[0061] The method described in the present invention includes: first, providing a sample and extracting genomic DNA; then, treating the genomic DNA with bisulfite to convert unmethylated cytosine in the genomic DNA to uracil; and then analyzing the presence or absence of methylation profile abnormalities in the treated genomic DNA.

[0062] The method of the present invention may be used to detect a test subject's sample to assess whether the test subject has a tumor or to distinguish between high-risk groups for tumors. The method may be used in cases where the purpose is not to diagnose the final outcome of a disease, such as human regional analysis studies, scientific research, and censuses, and the like, where the purpose is not to obtain a direct disease diagnosis result.

[0063] In a preferred embodiment of the present invention, DNA methylation is detected by PCR amplification and pyrosequencing, but the actual application is not limited to this method and other DNA methylation detection methods known or improved in the art are also possible. When performing PCR amplification, the primers used are not limited to those provided in the examples, and primers that target the nucleic acids or corresponding CpG sites indicated in the present invention but have different sequences from the primers provided in the examples of the present invention can also be obtained.

[0064] A preferred embodiment of the present invention also provides an in vitro method for detecting the methylation status of nucleic acids in a sample, which is a methylation-sensitive restriction endonuclease (MSRE) enzyme cleavage method. Methylation-sensitive restriction endonucleases cannot cleave DNA if the cleavage site contains a methylated base. The MSRE method is based on the fundamental principle that methylation-sensitive type II restriction endonucleases cannot cleave sequences containing one or more methylation cleavage sites. Fragments containing one or more methylated CpG sequences are cleaved with methyl-sensitive type II endonucleases and their isozymes (which are insensitive to methylation) and then analyzed by DNA blotting. The advantages of this method are that it does not require detailed knowledge of the primary structure of the target DNA, provides a direct assessment of the methylation status of CpG islands, and provides quantitative analytical information on the methylation of the test gene.

[0065] Other detection methods and reagents known to those skilled in the art for determining the sequence of a genome, its mutations and methylation status, centered on the marker nucleic acids provided herein, are encompassed by the present invention.

[0066] The present invention provides a method for preparing a tumor detection reagent, which includes: providing the above-mentioned nucleic acid; using the full length or a fragment of the nucleic acid as a target sequence; and designing a detection reagent that specifically detects the target sequence, provided that the target sequence contains at least one methylated CpG site. The detection reagent can include, but is not limited to, a chip, a primer, a probe, etc.; after obtaining the marker, the selection of the detection reagent can be performed by those skilled in the art.

[0067] Given the sequence of the nucleic acid, primer design is well known to those skilled in the art, with two primers flanking a specific sequence of the target gene to be amplified (including CpG sequences, where the primer complementary to the CpG targets the originally methylated gene region, and the primer complementary to the TpG targets the originally demethylated gene region). In a preferred embodiment of the present invention, the reagent is a primer, preferably one listed in Tables 2 and 6. Diagnostic or detection reagents other than primers can also be prepared, including, but not limited to, probes, chips, etc.

[0068] The reagents may be a combination of reagents, such as a combination of primers, for example, the combination may include multiple pairs of primers, each capable of amplifying the multiple nucleic acids.

[0069] The present invention also provides a kit for detecting the methylation profile of a nucleic acid in a sample in vitro, the kit comprising a container and the above-described primer pair located in the container.

[0070] The kit may further include various reagents necessary for DNA extraction, DNA purification, PCR amplification, etc., and other reagents, such as sample processing reagents. Furthermore, the kit may include an instruction manual showing the detection operation steps and result criteria for the convenience of those skilled in the art.

[0071] The method and reagent of the present invention are highly accurate when used in clinical tumor diagnosis, as demonstrated by the detection of various tumor clinical samples in the examples of the present invention. The present invention can be applied to fields such as early tumor screening, evaluation of treatment efficacy, diagnostic assistance, and prognostic monitoring, as well as to cases where the purpose is not to obtain direct disease diagnostic results, as mentioned above.

[0072] The tumor markers for endometrial cancer and other cancers provided by the present invention have very high specificity and sensitivity when used alone, but when two markers are used in combination, the sensitivity and specificity are further improved. Therefore, the markers of the present invention are highly valuable in fields such as tumor diagnosis assistance, treatment efficacy assessment, and prognosis monitoring.

[0073] The present invention provides a tumor marker for endometrial cancer and the like, and can be applied to the detection of cervical exfoliated cells and the like, and allows for easy and non-invasive sample acquisition, which significantly reduces the subject's pain, improves compliance, and is easier for clinicians to operate than the clinical practice of surgically obtaining tissue samples. Obviously, this represents a significant advance.

[0074] The present invention will be further described below with reference to specific examples. It should be understood that these examples are merely illustrative of the present invention and do not limit the scope of the present invention. Experimental methods in the following examples that do not specify specific conditions are usually carried out according to the standard conditions described in J. Sambrook et al., Guide to Molecular Cloning, Third Edition, Science Press, or according to the conditions recommended by the manufacturer. [Example]

[0075] Example 1: Screening for endometrial cancer detection markers A total of 43 frozen tissue samples were collected from clinical sites, including normal endometrium, typical proliferative endometrium, atypical proliferative endometrium (precancerous lesions), endometrial cancer, and five pairs of tissue samples surrounding paired tumors. Using Reduced Representation Bisulfite Sequencing (RRBS), their genome-wide methylation data were analyzed to screen for specific DNA methylation markers for endometrial cancer and precancerous lesions.

[0076] 1. Obtaining clinical samples A total of 43 frozen tissue samples were collected from five pairs of tissues: normal endometrium, typical hyperplastic endometrium, atypical hyperplastic endometrium (precancerous lesions), endometrial cancer, and tissues surrounding paired tumors. Each sample was the size of a soybean.

[0077] 2. DNA extraction Genomic DNA was extracted from the samples using an Epiprobe genomic DNA extraction kit (Epiprobe Biotech, K-21) (however, the nucleic acid extraction technique of the present invention is not limited to this method).

[0078] 3. Construction of RRBS sequence library The extracted gDNA samples were enzymatically digested using MspI endonuclease, and then fragments of the appropriate size were selected using magnetic beads, followed by end-filling, addition of dA, addition of linkers, and other steps.

[0079] 4. Bisulfite treatment The above library samples were modified with bisulfite, and in this experiment, the procedure was carried out using ZYMO Research's EZ DNA Methylation-Gold Kit, catalog number D5006, strictly following the instructions in the manual (although the present invention is not limited to this kit).

[0080] 5. PCR amplification and product purification Each library sample was amplified with different molecularly labeled primers, and the amplified products were selected and purified with magnetic beads, their concentrations were measured, and then they were sequenced.

[0081] 6. RRBS Sequencing Genomic RBS libraries from tissue samples were sequenced using an Illumina Hiseq 2000. After quality control and alignment, the offline data was analyzed. Table 1 shows basic information, such as the number of valid sequences, number of detected CpG sites, and coverage, for each sample. The total offline data volume for the RBS library reached 198.31 M. The average sequence length obtained from the normal endometrium group (Group A, NE), typical proliferation group (Group B, EH), atypical proliferation group (Group C, AH), endometrial cancer group (Group D, EC), and peritumo- nal tissue (Group E, PC) was 16,686,194 M, 16,847,536 M, 14,942,502 M, 14,729,170 M, and 14,637,777 M, respectively. The total number of CpG sites with a coverage of 5 or more is 4,055,958 M, 4,169,395 M, 4,369,673 M, 3,984,048 M, and 4,271,290 M, respectively. Statistical data and preliminary experimental analysis show that this RRBS sequencing is of good overall quality, with good sequencing depth and CpG coverage, a uniform distribution of data volume in each group, and highly reliable results.

[0082] [Table 1]

[0083] 7. Marker screening By analyzing the methylation sequencing data across different stages and combining it with experimental analysis, we screened for specific methylation markers present in atypical endometrial proliferation (precancerous lesions) and / or cancer stages. Figure 1 shows a heat map of some of the candidate markers for diagnosing endometrial cancer and precancerous lesions. We obtained two sets of candidate methylation markers for endometrial cancer detection: Precancer-DMRs (precancerous lesions) and Cancer-DMRs (endometrial cancer). These DMRs show significantly high methylation in the AH and EC only or EC only stages, and low methylation in other stages (including peritumor tissue).

[0084] (1) TAGMe-1 The resulting nucleotide sequence of the TAGMe-1 ​​methylation tumor marker is shown in SEQ ID NO:1, see FIG. 7, where the shaded sites are CG sites (53 sites) that may be methylated.

[0085] The reverse complement of SEQ ID NO:1 is shown in SEQ ID NO:2, see FIG. 7, where the shaded sites are CG sites that may be methylated.

[0086] The bisulfite-converted sequence of SEQ ID NO:1 is shown in SEQ ID NO:3, see FIG. 7, where the shaded sites are potential CG sites for methylation, and Y represents C or T.

[0087] The reverse complement of SEQ ID NO:3, converted with bisulfite, is shown in SEQ ID NO:4, see Figure 7, where the shaded sites are potential methylation CG sites and Y represents C or T.

[0088] (2) TAGMe-2 The resulting nucleotide sequence of the TAGMe-2 methylation tumor marker is shown in SEQ ID NO:5, see FIG. 8, where the shaded sites are CG sites (52 sites) that may be methylated.

[0089] The reverse complement of SEQ ID NO:5 is shown in SEQ ID NO:6, with the shaded sites being CG sites that may be methylated.

[0090] The bisulfite-converted sequence of SEQ ID NO:5 is shown in SEQ ID NO:7, see Figure 8, where the shaded sites are potential methylation CG sites and Y represents C or T.

[0091] The reverse complement of SEQ ID NO:7, converted with bisulfite, is shown in SEQ ID NO:8, see Figure 8, where the shaded sites are potential methylation CG sites and Y represents C or T.

[0092] 8. Clinical validation of DNA methylation biomarker candidates using pyrosequencing To further verify whether the marker candidates screened above are worthy of clinical application, we performed a comparative experiment using pyrosequencing technology on clinical tissue samples collected separately, comparing the markers of this invention with other precancer DMRs analyzed to be significantly related to methylation differences (those ranked highest in the heat map of precancer DMRs in Figure 1). Forty-five clinical tissue samples were collected from clinical sites, including tissues from 14 subjects with normal endometrium (NE), 11 subjects with hyperplasia (without atypical hyperplasia, EH), 5 subjects with atypical hyperplasia (AH), and 15 subjects with endometrial cancer (EC).

[0093] The amplification primers for pyrosequencing are shown in Table 2.

[0094] [Table 2]

[0095] The diagnosis of endometrial precancerous lesions by candidate methylation markers in clinical tissue samples and the above performance indicators are shown in Table 3.

[0096] [Table 3]

[0097] From the above, the AUCs of the two DMR sites TAGMe-1 ​​and TAGMe-2 were 0.938 and 0.925, respectively, both higher than 0.9. This high AUC could be achieved for patients with early stage endometrial cancer (precancer). Surprisingly, this proved to be an excellent performance as an early screening methylation marker for endometrial cancer.

[0098] Example 2: Performance verification of TAGMe-1 ​​and TAGMe-2 The TCGA database is a recognized tumor database containing data on various tumor types, including a series of expression data, miRNA expression data, methylation data, mutation data, and copy number data for each tumor. Therefore, we used this database to validate the performance of TAGMe-1 ​​and TAGMe-2.

[0099] Downloading methylation data for multiple cancer types in the TCGA database: We downloaded the methylation 450K chip data for all tumor samples in the TCGA database, including 11,087 tumor and control normal samples. The relevant 35 tumor types and sample volumes are listed in Table 4.

[0100] [Table 4]

[0101] Calculation of TAGMe-1 ​​and TAGMe-2 methylation values: First, the average methylation values ​​of TAGMe-1 ​​and TAGMe-2 in different types of tumor tissue samples and control normal tissue samples were calculated, and then the difference between the average methylation values ​​of TAGMe-1 ​​and TAGMe-2 in the tumor samples and control samples of that type was calculated.

[0102] Diagnostic performance analysis of TAGMe-1 ​​and TAGMe-2 in other cancers: The differences in methylation levels of TAGMe-1 ​​and TAGMe-2 between tumor and control samples of different cancer types are shown in Figures 2A and 2C. Two-tailed Mann-Whitney t-test analysis revealed that TAGMe-1 ​​showed statistically significant differences in methylation levels between tumor and control groups in many tumor types, including endometrial cancer, cervical cancer, bile duct cancer, rectal cancer (colon cancer, rectal cancer), esophageal cancer, lung cancer (squamous cell carcinoma, lung adenocarcinoma), head and neck tumors, and pancreatic cancer. Meanwhile, TAGMe-2 showed statistically significant differences in methylation levels between tumor and control groups in rectal cancer, endometrial cancer, and cervical cancer. The corresponding diagnostic AUCs and performances are shown in Figures 2B and 2D and Table 5.

[0103] [Table 5]

[0104] Therefore, TAGMe-1 ​​and TAGMe-2 have diagnostic value in multiple tumor types.

[0105] Example 3: Endometrial cancer - DNA methylation detection in peritumor tissue samples: targeted NGS method In this example, we used the targeted NGS method to analyze DNA methylation in endometrial cancer peritumoral tissue samples. The steps were as follows: 1. Obtaining clinical samples Twenty samples of normal endometrial tissue / peritumoral tissue, six samples of endometrial atypical proliferation, and twenty samples of endometrial cancer tissue were obtained from clinical trials. The normal / peritumoral samples served as the control group, and the atypical proliferation and cancer tissue samples served as the tumor detection experimental groups.

[0106] 2. DNA extraction DNA was extracted from the experimental and control groups, respectively; in this experiment, DNA extraction was performed using an Epiprobe genomic DNA extraction kit (Epiprobe Biotech, K-21) (although the present invention is not limited to this method).

[0107] 3. Bisulfite treatment The extracted DNA was modified with bisulfite. In this experiment, the procedure was carried out using ZYMO Research's EZ DNA Methylation-Gold Kit, catalog number D5006, strictly following the instructions in the manual (although the present invention is not limited to this kit).

[0108] 4. PCR amplification and library construction After the modification was completed, the target region after bisulfite treatment was first amplified and enriched using PCR primers designed for amplification. The primer sequences are shown in Table 6.

[0109] [Table 6]

[0110] The products were then purified and sequencing linkers were attached to construct an NGS sequencing library.

[0111] 5. NGS sequencing The test was performed using an Illumia HiSeq X high-throughput sequencer, and the operation was carried out in strict accordance with the instructions (although the present invention is not limited to this model of high-throughput sequencer).

[0112] 6. Calculation of methylation values ​​of TAGMe-1 ​​and TAGMe-2 The offline data from Target NGS sequencing includes the methylation status of every single CpG site within the target region. The methylation level of TAGMe-1 ​​and TAGMe-2 is calculated as follows: single-site methylation level = number of methylated reads for that site / total number of reads; target region methylation level = average methylation level of all sites.

[0113] 7. Single marker result analysis The results of tissue Target NGS are shown in Figure 3. The differences in TAGMe-1 ​​and TAGMe-2 methylation levels between the peritumor / normal control group and the tumor experimental group were compared and ROC analysis was performed. The results for TAGMe-1 ​​are shown in Figure 3A. In endometrial cancer clinical samples, the TAGMe-1 ​​methylation levels in atypical hyperplasia and cancer tissue samples were significantly higher than in the control group. The results of TAGMe-1 ​​ROC analysis are shown in Figure 3B. The AUC for distinguishing endometrial cancer from control was 0.995, and the AUC for distinguishing atypical hyperplasia from control was 0.983, demonstrating good discrimination performance. Using the maximum Youden index as the threshold, the sensitivity and specificity of TAGMe-1 ​​for distinguishing between endometrial cancer and control groups were 95% and 100%, respectively; the sensitivity and specificity for distinguishing between atypical proliferation and control groups were 83.33% and 90%, respectively.

[0114] The results of TAGMe-2 are shown in Figure 3C. In clinical endometrial cancer samples, methylation levels in both atypical proliferation and cancer tissue samples were significantly higher than in the control group. The ROC analysis results for TAGMe-2 are shown in Figure 3D. The AUC for TAGMe-2 was 0.990 for distinguishing endometrial cancer from control, and 0.967 for distinguishing atypical proliferation from control, demonstrating excellent performance. Using the maximum Youden index as the threshold, the sensitivity and specificity of TAGMe-2 for distinguishing endometrial cancer from control were 95% and 100%, respectively; the sensitivity and specificity for distinguishing atypical proliferation from control were 83.33% and 100%, respectively.

[0115] 8. Marker combination analysis Combined detection was performed using TAGMe-1 ​​and TAGMe-2, and the combined performance is shown in Table 7.

[0116] [Table 7]

[0117] The results showed that the combination of the two has a synergistic effect, further increasing the sensitivity of detecting endometrial cancer and precancerous lesions using methylation markers.

[0118] Example 4: Endometrial cancer - DNA methylation detection in peritumor tissue samples: pyrosequencing In this example, pyrosequencing was used to detect DNA methylation in endometrial cancer peritumoral tissue samples and analyze markers. The steps were as follows: 1. Obtaining clinical samples Twenty-five normal cervical decidual cell samples, five cervical decidual cell samples from patients with atypical endometrial hyperplasia, and 15 cervical decidual cell samples from patients with endometrial cancer were obtained from clinical trials. The normal cervical decidual cell samples served as the control group, and the cervical decidual cell samples from patients with atypical hyperplasia and endometrial cancer served as the tumor detection experimental groups.

[0119] 2. DNA extraction DNA was extracted from clinical samples; in this experiment, DNA extraction was performed using an Epiprobe genomic DNA extraction kit (Epiprobe Biotech, K-21) (although the present invention is not limited to this method).

[0120] 3. Bisulfite treatment The extracted DNA was modified with bisulfite. In this experiment, the procedure was carried out using ZYMO Research's EZ DNA Methylation-Gold Kit, catalog number D5006, strictly following the instructions in the manual (although the present invention is not limited to this method).

[0121] 4. Primer design Based on the TAGMe-1 ​​and TAGMe-2 sequences (SEQ ID NO:1 and SEQ ID NO:5), amplification primers and pyrosequencing primers were designed, respectively; the methylation levels of the corresponding CpG sites in the target sequences were detected and used as a representative of the methylation levels. The PCR primer amplification sequences, pyrosequencing primer sequences, pyrosequencing machine detection sequences, and detection sites were the same as in Example 1.

[0122] 5. PCR Amplification and Agarose Gel Electrophoresis The bisulfite-treated samples were used as templates for PCR amplification, and the specificity of the PCR amplification was determined by agarose gel electrophoresis.

[0123] 6. Pyrosequencing Detection was performed using a QIAGEN PyroMark Q96 ID pyrosequencer, and the procedure was operated in strict accordance with the instructions.

[0124] 7. Calculation of methylation value Pyrosequencing can independently detect the methylation status of single CpG sites within the target region, and the average methylation value of all CpG sites was calculated as the methylation value of TAGMe-1 ​​and TAGMe-2 in the sample.

[0125] 8. Analysis of results The differences in TAGMe-1 ​​and TAGMe-2 methylation levels between the peritumoral / normal control group and the tumor experimental group were compared, and ROC analysis was performed.

[0126] The results of the TAGMe-1 ​​analysis are shown in Figure 4A. In clinical endometrial cancer samples, the methylation levels of TAGMe-1 ​​in atypical proliferation and cancer tissue samples were significantly higher than in the control group. The results of the receiver operating characteristic (ROC) analysis are shown in Figure 4B. The AUC for distinguishing endometrial cancer from control groups using TAGMe-1 ​​was 0.955, and the AUC for distinguishing atypical proliferation from control groups was 0.952, demonstrating good performance. Using the maximum Youden index as the threshold, the sensitivity and specificity for distinguishing endometrial cancer from control groups using TAGMe-1 ​​were 100% and 84%, respectively; the sensitivity and specificity for distinguishing atypical proliferation from control groups were 100% and 84%, respectively.

[0127] The results of the TAGMe-2 analysis are shown in Figure 4C. In the endometrial cancer clinical samples, the TAGMe-2 methylation levels in both atypical proliferation and cancer tissue samples were significantly higher than in the control group. The results of the TAGMe-2 receiver operating characteristic (ROC) analysis are shown in Figure 4D. The AUC for distinguishing endometrial cancer from control was 0.943, and the AUC for distinguishing atypical proliferation from control was 0.928, demonstrating excellent performance. Using the maximum Youden index as the threshold, the sensitivity and specificity of TAGMe-2 for distinguishing endometrial cancer from control were 86.67% and 88%, respectively; the sensitivity and specificity for distinguishing atypical proliferation from control were 100% and 80%, respectively.

[0128] 9. Combined analysis of TAGMe-1 ​​and TAGMe-2 The combined detection of TAGMe-1 ​​and TAGMe-2 was performed to further analyze the sensitivity of methylation markers for detecting endometrial cancer and precancerous lesions, and the combined performance is shown in Table 8 .

[0129] [Table 8]

[0130] Peritumoral samples cannot simply be considered normal samples, and when collected clinically, peritumoral samples are prone to contamination with some cancer tissue or to certain epigenetic mutations due to the influence of cancerous lesions, which can reduce specificity. Therefore, the specificity of some samples in the table may vary compared to the examples above. However, the combined detection of two markers can significantly improve the specificity, overall accuracy rate, and positive predictive value in detecting atypical endometrial hyperplasia.

[0131] Example 5: Endometrial cancer - DNA methylation detection in cervical exfoliated cell samples In this example, DNA methylation detection and marker analysis were performed in endometrial cancer cervical exfoliated cell samples using Me-qPCR. The steps were as follows: 1. Obtaining clinical samples: 38 normal cervical decidual cell samples, 8 cervical decidual cell samples from patients with atypical endometrial hyperplasia, and 33 cervical decidual cell samples from patients with endometrial cancer were obtained from clinical trials. The normal cervical decidual cell samples served as the control group, and the cervical decidual cell samples from patients with atypical hyperplasia and endometrial cancer served as the tumor detection experimental groups.

[0132] 2. DNA extraction: DNA was extracted from clinical samples. In this experiment, DNA was extracted using an Epiprobe genomic DNA extraction kit (Epiprobe Biotech, K-21) (however, the present invention is not limited to this method).

[0133] 3. Enzymatic cleavage reaction: DNA is enzymatically cleaved using a methylation-sensitive restriction endonuclease, and unmethylated enzyme cleavage sites are cleaved. In this experiment, the enzymatic cleavage reaction was performed using HpaII enzyme (NEB, R0171) (however, the present invention is not limited to this method).

[0134] 4. Primer design: Based on the TAGMe-1 ​​and TAGMe-2 sequences, corresponding amplification primers were designed to amplify the cleavage products of methylation-sensitive restriction endonucleases and detect the abundance of DNA fragments after cleavage. The GAPDH gene was used as an endogenous reference gene to represent the methylation levels of TAGMe-1 ​​and TAGMe-2, respectively.

[0135] 5. qPCR amplification: The enzyme-cleaved sample was used as a template for qPCR to perform qPCR amplification, and the results were detected using Thermo's ABI 7500 qPCR device (however, the present invention is not limited to this method).

[0136] 6. Calculation of methylation value: The DNA methylation level of each sample was evaluated using the following formula: ΔCt_ 本発明マーカー =Ct_ 本発明マーカー -Ct_ GAPDH A smaller ΔCT indicates a higher level of methylation.

[0137] 7. Results analysis: The differences in TAGMe-1 ​​and TAGMe-2 methylation levels between the control group and the tumor experimental group were compared, and ROC analysis was performed.

[0138] The results of Me-qPCR for cervical cells are shown in Figure 5. The results for TAGMe-1 ​​are shown in Figure 5A. In clinical endometrial cancer samples, the methylation levels of TAGMe-1 ​​in atypical hyperplasia and cancer tissue samples were significantly higher than in the control group. The ROC analysis results for TAGMe-1 ​​are shown in Figure 5B. The AUC for distinguishing endometrial cancer from control groups using TAGMe-1 ​​was 0.916, and the AUC for distinguishing atypical hyperplasia from control groups was 0.870, demonstrating good discrimination performance. Using the maximum Youden index as the threshold, the sensitivity and specificity of TAGMe-1 ​​for distinguishing endometrial cancer from control groups were 84.85% (68.1%-94.89%) and 89.47% (75.2%-97.06%), respectively; the sensitivity and specificity for distinguishing atypical proliferation from control groups were 87.5% (47.35%-99.68%) and 76.32% (59.76%-88.56%), respectively. The results for TAGMe-2 are shown in Figure 5C. In clinical endometrial cancer samples, the methylation levels of TAGMe-2 in atypical proliferation and cancer tissue samples were significantly higher than in the control group. The ROC analysis results for TAGMe-2 are shown in Figure 5D. The AUC for distinguishing endometrial cancer from control groups was 0.921, and the AUC for distinguishing atypical proliferation from control groups was 0.845, demonstrating excellent performance. Using the maximum Youden index as the threshold, the sensitivity and specificity for distinguishing endometrial cancer from control groups by TAGMe-2 were 87.88% and 86.84%, respectively; the sensitivity and specificity for distinguishing atypical proliferation from control groups were 100% and 57.89%, respectively.

[0139] 8. Combined analysis of TAGMe-1 ​​and TAGMe-2: The combined detection of TAGMe-1 ​​and TAGMe-2 was performed to further analyze the sensitivity of methylation markers in detecting endometrial cancer and precancerous lesions. The performance of the combined model is shown in Table 9.

[0140] [Table 9]

[0141] In conclusion, the combined detection of two markers can obviously improve the overall accuracy in detecting atypical endometrial proliferation, and the sensitivity and accuracy in detecting endometrial cancer.

[0142] Example 6: Endometrial cancer - Detection of DNA methylation in cervical exfoliated cell samples (Me-qPCR method) Nineteen normal cervical decidual cell samples, six cervical decidual cell samples from patients with atypical endometrial hyperplasia, and 21 cervical decidual cell samples from patients with endometrial cancer were obtained from clinical trials. The normal cervical decidual cell samples served as the control group, and the cervical decidual cell samples from patients with atypical endometrial hyperplasia and endometrial cancer served as the tumor detection experimental group. According to the Me-qPCR detection steps in Example 5, the differences in the methylation levels of TAGMe-1 ​​and TAGMe-2 between the control group and the tumor experimental group were compared, and ROC analysis was performed.

[0143] The results of Me-qPCR for uterine luminal cells are shown in Figure 6. The results for TAGMe-1 ​​are shown in Figure 6A. In clinical endometrial cancer samples, the methylation levels of TAGMe-1 ​​in atypical proliferation and cancer tissue samples were significantly higher than in the control group. The ROC analysis results are shown in Figure 6B. The AUC for distinguishing endometrial cancer from control groups using TAGMe-1 ​​was 0.988, and the AUC for distinguishing atypical proliferation from control groups was 0.921, demonstrating good discrimination performance. Using the maximum Youden index as the threshold, the sensitivity and specificity for distinguishing endometrial cancer from control groups using TAGMe-1 ​​were 90.48% and 100%, respectively; the sensitivity and specificity for distinguishing atypical proliferation from control groups were 83.33% and 94.74%, respectively.

[0144] The TAGMe-2 results are shown in Figure 6C. In the endometrial cancer clinical samples, the TAGMe-2 methylation levels in both atypical proliferation and cancer tissue samples were significantly higher than in the control group. The ROC analysis results are shown in Figure 6D. The AUC for TAGMe-2 was 0.985 for distinguishing endometrial cancer from control, and 0.973 for distinguishing atypical proliferation from control, demonstrating good performance. Using the maximum Youden index as the threshold, the sensitivity and specificity of TAGMe-2 for distinguishing endometrial cancer from control were 95.24% and 94.74%, respectively; the sensitivity and specificity for distinguishing atypical proliferation from control were 100% and 84.21%, respectively.

[0145] The combined detection of TAGMe-1 ​​and TAGMe-2 was performed to further analyze the sensitivity of methylation markers for detecting endometrial cancer and precancerous lesions. The performance of the combined model is shown in Table 10.

[0146] JPEG2025531418000010.jpg47170

[0147] In conclusion, the combined detection of two markers can obviously improve the overall accuracy rate in detecting atypical endometrial proliferation.

[0148] The above-described examples illustrate some embodiments of the present invention, and although the descriptions are more specific and detailed, this does not imply any limitation on the patent scope of the present invention. Those skilled in the art can make some modifications and improvements without departing from the concept of the present invention, and these fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent of the present invention shall be governed by the appended claims. All references mentioned in this application are incorporated herein by reference as if they were individually incorporated by reference.

Claims

1. 1. Use of the isolated nucleic acid or a nucleic acid converted therefrom in the preparation of a reagent or kit for detecting tumors, comprising: However, the nucleic acid is (1) a nucleic acid or a combination of nucleic acids having the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; or (2) a nucleic acid or a combination of nucleic acids that is complementary in sequence to the nucleic acid of (1); However, the nucleic acid converted from the isolated nucleic acid corresponds to (1) or (2), and the unmodified cytosine is converted to T or U, but the cytosine C at the modified CpG site remains unchanged; 1. A use characterized by:

2. The use according to claim 1, characterized in that the nucleic acid sequence complementary to the nucleic acid (1) is a nucleic acid having the nucleotide sequence shown in SEQ ID NO: 2 or SEQ ID NO: 6, respectively.

3. 2. The use according to claim 1, wherein the tumor comprises endometrial cancer, cervical cancer, pancreatic cancer, head and neck tumors, colon cancer, colorectal cancer, esophageal cancer, lung cancer, bile duct cancer.

4. The use according to claim 1, characterized in that the target of the tumor detection is a tumor or a precancerous lesion thereof.

5. The use according to claim 1, characterized in that the samples to be used for tumor detection include blood samples, tissue samples, cervical samples, uterine cavity samples, pleural fluid collection samples, bronchoalveolar lavage fluid samples, ascites samples, ascites lavage fluid samples, bile samples, fecal samples, urine samples, saliva samples, cerebrospinal fluid samples, cell smear samples, and cell samples; preferably, the samples include cervical smear or scraping samples, cervical swabs, cervical tissue, cervical exfoliated cells, uterine cavity scrapings, uterine cavity lavage fluid, and vaginal secretions.

6. Extracting the nucleic acid of the sample to be detected; and detecting the modification status of CpG sites of a target sequence of the extracted nucleic acid or a fragment thereof, wherein the target sequence is a nucleic acid or a combination of nucleic acids converted from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5, and corresponds to the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5, in which unmodified cytosines are converted to T or U, but cytosine C in modified CpG sites remains unchanged; A method for detecting the methylation level of a sample to be detected, comprising:

7. 7. The method of claim 6, wherein the method for detecting the CpG site modification status of the target sequence of the extracted nucleic acid comprises pyrosequencing, bisulfite sequencing, methylation chip, methylation-specific PCR, methylation-sensitive restriction endonuclease enzyme cleavage, qPCR, digital PCR, next-generation sequencing, third-generation sequencing, whole-genome methylation sequencing, DNA enrichment detection, Reduced Representation Bisulfite Sequencing (RRBS) technology, HPLC, Mass Array, or a combination thereof.

8. The method for detecting the modification status of a CpG site in a target sequence of an extracted nucleic acid comprises: (i) treating the extracted nucleic acid so as to convert unmodified cytosine therein to uracil; preferably, the modification comprises a 5-methylation modification, a 5-hydroxymethylation modification, a 5-formylation modification, or a 5-carboxylation modification.

9. The method of claim 8, further comprising treating the nucleic acid described in step (i) with bisulfite; and (ii): analyzing the modification status of the target sequence of the nucleic acid treated in step (i).

10. 1. A method of preparing a reagent, the reagent being used in the detection of a tumor, the method comprising: Providing a nucleic acid having a nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; and designing a detection reagent that specifically detects the CpG site modification status of the target sequence using the full length or a fragment of the nucleic acid as a target sequence. A method for preparing a reagent comprising:

11. 11. The method of claim 10, wherein the detection reagent includes, but is not limited to, a primer, a probe, a chip, or a test strip.

12. A reagent or a combination of reagents for specifically detecting the modification status of a CpG site of a target sequence, wherein the target sequence is a nucleic acid or a combination of nucleic acids converted from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; preferably, the reagent or the combination of reagents targets a gene sequence including the target sequence, and preferably, the gene sequence includes a gene panel or a gene group; preferably, the reagent or the combination of reagents is Primers having the sequences shown in SEQ ID NO:9 and SEQ ID NO:10; Primers having the sequences shown in SEQ ID NO:11 and SEQ ID NO:12; Primers having the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 14; or Primers having the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 16; Selected from.

13. A reagent or reagent combination according to claim 12 for preparing a kit for tumor detection. Preferably, the tumors include endometrial cancer, cervical cancer, pancreatic cancer, head and neck tumors, colon cancer, colorectal cancer, esophageal cancer, lung cancer, and bile duct cancer.

14. A kit for use in tumor detection, comprising the reagent or reagent combination according to claim 12.

15. An isolated nucleic acid or a nucleic acid obtained by transformation thereof, said nucleic acid comprising: (1): a nucleic acid or nucleic acid combination of the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 5; or (2): A nucleic acid or nucleic acid combination that is complementary in sequence to the nucleic acid of (1); is; However, the nucleic acid converted from the nucleic acid corresponds to (1) or (2), and the unmodified cytosine is converted to T or U, but the cytosine C in the modified CpG site remains unchanged; A nucleic acid characterized by:

Images