Primer-probe combination for early detection of endometrial cancer, methylation detection kit, and use thereof

Abstract

The present invention relates to a primer-probe combination for early detection of endometrial cancer, a methylation detection kit, and the use thereof. Provided in the present invention is a methylation gene marker for endometrial cancer detection, which specifically comprises at least one of VSX1, SYT1, ZNF132 and VWC2 genes. Measuring the methylation levels of the genes can remarkably distinguish subjects with endometrial cancer from subjects without cancer. The gene marker of the present invention can well identify subjects with endometrial cancer and subjects without cancer from each of tissue / urine / brush smear / swab / blood / lavage fluid specimens, and can achieve a good detection effect simply by means of a single index and further improve the detection efficiency by means of multi-index combination.

Description

Primer probe combination for early detection of endometrial cancer, methylation detection kit and application thereof Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a primer-probe combination for early detection of endometrial cancer, a methylation detection kit and applications thereof. Background Art

[0002] Endometrial cancer is a common epithelial malignancy arising in the endometrium, accounting for approximately 20%-30% of all gynecological malignancies. Endometrial cancer is most common in perimenopausal and postmenopausal women, with a peak age of onset between 50 and 70 years old and a median age of diagnosis of 61. In recent years, the incidence of endometrial cancer has gradually increased with the increasing prevalence of risk factors for endometrial cancer, particularly obesity and an aging population. Furthermore, the incidence of endometrial cancer is trending younger, with a significant increase in the incidence of endometrial cancer in premenopausal women. The treatment response to endometrial cancer is closely related to clinical staging, with patients with early-stage endometrial cancer generally having a better prognosis. According to statistics, the five-year survival rate for patients with early-stage (stage I and II) disease can exceed 70%, while the five-year survival rate for patients with stage III disease is approximately 40-50%, and the five-year survival rate for patients with stage IV disease is only 15-20%. Early diagnosis is crucial for endometrial cancer.

[0003] The first symptom of most endometrial cancer patients is irregular vaginal bleeding or vaginal discharge. Because this symptom is not specific, about 14% of women of childbearing age and 4%-11% of menopausal women experience this symptom, of which only 0.33%-1.04% are eventually diagnosed with endometrial cancer. Based on this symptom, after seeing a doctor, a large number of clinical examinations are often required to confirm whether endometrial cancer is present, including but not limited to imaging examinations (such as vaginal ultrasound examinations), laboratory tests (testing commonly used clinical markers such as CA125 and HE4), and endometrial biopsy (diagnostic curettage and hysteroscopic curettage, removing endometrial tissue and further performing a histopathological biopsy). The sensitivity and specificity of imaging and laboratory tests are low, and endometrial biopsy is an invasive procedure that can easily cause physical discomfort. Therefore, there is an urgent need to develop a non-invasive detection method that is convenient, fast, and can accurately identify endometrial cancer in the early stages.

[0004] DNA methylation is a type of epigenetic modification, which involves the addition of a methyl group to the 5th carbon atom of the cytosine base. This modification is often associated with gene silencing. DNA methylation is a key epigenetic regulator of gene expression and often leads to gene expression defects. Increased methylation of tumor suppressor genes is an early event in many tumors. Methylation signals are currently a widely recognized source of tumor screening markers, with advantages such as early appearance in tumor development, stable signals, and convenient detection methods. However, DNA methylation has not been effectively clinically applied in the early screening and diagnosis of endometrial cancer. Summary of the Invention

[0005] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a methylation gene marker for early detection of endometrial cancer.

[0006] The present invention obtains endometrial cancer-specific methylation early screening markers through screening, including at least one of the VSX1, SYT1, ZNF132, and VWC2 genes. The methylation of these genes is detected to distinguish endometrial cancer from non-cancer individuals. The stability and effectiveness of the markers of the present invention are verified by testing in multiple non-invasive sampling sample types.

[0007] Another object of the present invention is to provide a kit for diagnosing endometrial cancer.

[0008] The present invention has discovered sites that can eliminate background signal interference in various non-invasively collected samples and accurately distinguish between endometrial cancer and other diseased / healthy individuals, and based on them, a stable and reliable marker detection kit has been established.

[0009] Another object of the present invention is to provide a use of the above-mentioned kit in the preparation of endometrial cancer diagnostic products.

[0010] The purpose of the present invention is achieved through the following solutions:

[0011] According to a first aspect of the present invention, a methylation gene marker for detecting endometrial cancer is provided. The methylation gene marker specifically includes at least one of the VSX1, SYT1, ZNF132, and VWC2 genes. The detection method involves detecting the methylation level of at least one of the target genes VSX1, SYT1, ZNF132, and VWC2 to diagnose endometrial cancer.

[0012] According to a second aspect of the present invention, a methylation gene marker for endometrial cancer detection is provided. The methylation gene marker comprises a methylated nucleic acid sequence in at least one target region of at least one of the target genes VSX1, VWC2, ZNF132, and SYT1, wherein the target region is selected from the group consisting of at least one of the following methylation regions in at least one of the genes VSX1, VWC2, ZNF132, and SYT1:

[0013] VSX1 gene: Chr20:25058334-25058603; Chr20:25061937-25062310 or Chr20:25061874-25062088; Chr20:25062398-25062760 or Chr20:25062376-25062484; Chr20:25062681-25062788 or Chr20:25062655-25062739; Chr20:25062736-25062940 or Chr20:25062749-25062900;

[0014] SYT1 gene: Chr12:79258369-79258486 or Chr12:79258392-79258498;

[0015] ZNF132 gene: Chr19:58951204-58951524 or Chr19:58951402-58951524; Chr19:58951554-58951637; Chr19:58951672-58952006 or Chr19:58951724-58951825;

[0016] VWC2 gene: Chr7:49812992-49813086; Chr7:49813051-49813343 or Chr7:49813066-49813161; Chr7:49813455-49813811 or Chr7:49813387-49813537; Chr7:49814721-49815073 or Chr7:49814765-49814936; Chr7:49815144-49815236; Chr7:49815290-49815635 or Chr7:49815340-49815439.

[0017] The genes described in the present invention use the human genome version GRCh37 (hg19) as the reference genome.

[0018] This study identified specific methylation markers for early screening of endometrial cancer, including at least one of the genes VSX1, SYT1, ZNF132, and VWC2. Detecting the methylation levels of these genes can significantly distinguish individuals with endometrial cancer from those without the disease. The stability and effectiveness of these markers were validated by testing them in a variety of noninvasive sample types. This study provides new markers and diagnostic strategies for the diagnosis of endometrial cancer and its precancerous lesions.

[0019] Furthermore, by detecting at least one methylation region corresponding to at least one target gene in an in vitro test sample, the diagnosis of endometrial cancer can be achieved. The sample can be any body fluid, exfoliated cells, and tissue samples, such as body fluid samples such as urine and blood, and exfoliated cell collection samples such as swabs, brushes, and lavage fluids. The gene markers of the present invention can better distinguish endometrial cancer from non-cancer individuals in tissue / urine / brush / swab samples. A single indicator can achieve a good detection effect, and the combination of multiple indicators can further improve the detection efficiency.

[0020] According to a third aspect of the present invention, a detection primer for endometrial cancer detection is provided, which is used to detect the methylation status of the methylation region of the above-mentioned marker gene, and the nucleotide sequence of the detection primer is at least one of the following:

[0021] VSX1 gene:

[0022] The detection primers corresponding to Chr20:25058334-25058603 are SEQ ID NOs:1-2;

[0023] The detection primers corresponding to Chr20:25061937-25062310 are SEQ ID NOs:4-5;

[0024] The detection primers corresponding to Chr20:25061874-25062088 are SEQ ID NOs:6-7;

[0025] The detection primers corresponding to Chr20:25062398-25062760 are SEQ ID NOs:9-10;

[0026] The detection primers corresponding to Chr20:25062376-25062484 are SEQ ID NOs:12-13;

[0027] The detection primers corresponding to Chr20:25062681-25062788 are SEQ ID NOs:15-16;

[0028] The detection primers corresponding to Chr20:25062655-25062739 are SEQ ID NOs:18-19;

[0029] The detection primers corresponding to Chr20:25062736-25062940 are SEQ ID NOs:21-22;

[0030] The detection primers corresponding to Chr20:25062749-25062900 are SEQ ID NOs:23-24;

[0031] SYT1 gene:

[0032] The detection primers corresponding to Chr12:79258369-79258486 are SEQ ID NOs:26-27;

[0033] The detection primers corresponding to Chr12:79258392-79258498 are SEQ ID NOs:29-30;

[0034] ZNF132 gene:

[0035] The detection primers corresponding to Chr19:58951204-58951524 are SEQ ID NO:32 and SEQ ID NO:34;

[0036] The detection primers corresponding to Chr19:58951402-58951524 are SEQ ID NO:33 and SEQ ID NO:34;

[0037] The detection primers corresponding to Chr19:58951554-58951637 are SEQ ID NOs:36-37;

[0038] The detection primers corresponding to Chr19:58951672-58952006 are SEQ ID NOs:39-40;

[0039] The detection primers corresponding to Chr19:58951724-58951825 are SEQ ID NOs:41-42;

[0040] VWC2 gene:

[0041] The detection primers corresponding to Chr7:49812992-49813086 are SEQ ID NOs:44-45;

[0042] The detection primers corresponding to Chr7:49813051-49813343 are SEQ ID NOs:47-48;

[0043] The detection primers corresponding to Chr7:49813066-49813161 are SEQ ID NOs:49-50;

[0044] The detection primers corresponding to Chr7:49813455-49813811 are SEQ ID NOs:52-53;

[0045] The detection primers corresponding to Chr7:49813387-49813537 are SEQ ID NOs:54-55;

[0046] The detection primers corresponding to Chr7:49814721-49815073 are SEQ ID NOs:57-58;

[0047] The detection primers corresponding to Chr7:49814765-49814936 are SEQ ID NOs:59-60;

[0048] The detection primers corresponding to Chr7:49815144-49815236 are SEQ ID NOs:62-63;

[0049] The detection primers corresponding to Chr7:49815290-49815635 are SEQ ID NOs:65-66;

[0050] The detection primers corresponding to Chr7:49815340-49815439 are SEQ ID NOs:67-68.

[0051] According to a fourth aspect of the present invention, a detection probe for early screening and diagnosis of endometrial cancer is provided, which is used to detect the methylation status of the methylation region of the above-mentioned marker gene, and the nucleotide sequence of the probe is one of the following:

[0052] VSX1 gene:

[0053] The detection probe corresponding to Chr20:25058334-25058603 is SEQ ID NO:3;

[0054] The detection probe corresponding to Chr20:25061937-25062310 is SEQ ID NO:8;

[0055] The detection probe corresponding to Chr20:25061874-25062088 is SEQ ID NO:8;

[0056] The detection probe corresponding to Chr20:25062398-25062760 is SEQ ID NO:11;

[0057] The detection probe corresponding to Chr20:25062376-25062484 is SEQ ID NO:14;

[0058] The detection probe corresponding to Chr20:25062681-25062788 is SEQ ID NO:17;

[0059] The detection probe corresponding to Chr20:25062655-25062739 is SEQ ID NO:20;

[0060] The detection probe corresponding to Chr20:25062736-25062940 is SEQ ID NO:25;

[0061] The detection probe corresponding to Chr20:25062749-25062900 is SEQ ID NO:25;

[0062] SYT1 gene:

[0063] The detection probe corresponding to Chr12:79258369-79258486 is SEQ ID NO:28;

[0064] The detection probe corresponding to Chr12:79258392-79258498 is SEQ ID NO:31;

[0065] ZNF132 gene:

[0066] The detection probe corresponding to Chr19:58951204-58951524 is SEQ ID NO:35;

[0067] The detection probe corresponding to Chr19:58951402-58951524 is SEQ ID NO:35;

[0068] The detection probe corresponding to Chr19:58951554-58951637 is SEQ ID NO:38;

[0069] The detection probe corresponding to Chr19:58951672-58952006 is SEQ ID NO:43;

[0070] The detection probe corresponding to Chr19:58951724-58951825 is SEQ ID NO:43;

[0071] VWC2 gene:

[0072] The detection probe corresponding to Chr7:49812992-49813086 is SEQ ID NO:46;

[0073] The detection probe corresponding to Chr7:49813051-49813343 is SEQ ID NO:51;

[0074] The detection probe corresponding to Chr7:49813066-49813161 is SEQ ID NO:51;

[0075] The detection probe corresponding to Chr7:49813455-49813811 is SEQ ID NO:56;

[0076] The detection probe corresponding to Chr7:49813387-49813537 is SEQ ID NO:56;

[0077] The detection probe corresponding to Chr7:49814721-49815073 is SEQ ID NO:61;

[0078] The detection probe corresponding to Chr7:49814765-49814936 is SEQ ID NO:61;

[0079] The detection probe corresponding to Chr7:49815144-49815236 is SEQ ID NO:64;

[0080] The detection probe corresponding to Chr7:49815290-49815635 is SEQ ID NO:69;

[0081] The detection probe corresponding to Chr7:49815340-49815439 is SEQ ID NO:69.

[0082] According to a fifth aspect of the present invention, the present invention proposes the use of a reagent for detecting methylation of a target gene in the preparation of a detection kit or device, wherein the detection kit or device is used to detect, screen or diagnose endometrial cancer; the target gene is selected from at least one of the following genes: VSX1, SYT1, ZNF132, VWC2.

[0083] The reagents include at least one of an antibody, probe, primer, and mass spectrometry detection reagent specifically for detecting the target gene. Specifically, the reagents include a product specifically detecting the target gene, which can be at least one of an antibody, probe, primer, and mass spectrometry detection reagent. The primers include the detection primers described in claim 3, and can also be other reagents with similar functions. Furthermore, the kit can be similar to existing products, and the equipment can be sequence detection equipment. Both primers and probes can be selected, and combinations can be used as needed.

[0084] More specifically, the primers include at least one of the above-mentioned detection primers for endometrial cancer detection.

[0085] More specifically, the probe includes at least one of the above-mentioned detection probes for early screening and diagnosis of endometrial cancer.

[0086] Furthermore, the test samples of the detection kit or device can be any body fluids, exfoliated cells, and tissue samples, etc. For example, body fluid samples such as urine and blood, and exfoliated cell collection samples such as swabs, brushes, and lavage fluids.

[0087] According to a sixth aspect of the present invention, a kit for diagnosing endometrial cancer is provided, comprising a reagent for detecting methylation of a target gene; the target gene is selected from at least one of the following genes: VSX1, SYT1, ZNF132, VWC2.

[0088] The reagents include at least one of an antibody, a probe, a primer, and a mass spectrometry detection reagent specifically targeting the target gene, wherein the primer includes at least one of the detection primers for endometrial cancer detection described above. The probe includes at least one of the detection probes for early screening and diagnosis of endometrial cancer described above.

[0089] Furthermore, the above-mentioned reagents can detect the methylation level of the target gene by at least one of the following methods: fluorescent quantitative methylation-specific PCR, methylation-specific PCR method, whole-genome methylation sequencing method, digital PCR method, bisulfite sequencing method, pyrophosphate sequencing method, methylation-specific high-resolution melting curve method, methylation-specific microarray method, methylation-specific high-performance liquid chromatography method, methylation-sensitive restriction endonuclease method or fluorescence quantitative method, simplified methylation genome sequencing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, etc.

[0090] It should be noted that methods for detecting gene methylation are well known to those skilled in the art, including, but not limited to, the methods listed above. These methods can all be used to detect the methylation level of the target gene. Therefore, reagents suitable for the above methods can be prepared into a kit for diagnosing endometrial cancer.

[0091] Furthermore, the kit also includes a primer pair and a probe for an internal reference gene, wherein the internal reference gene is the ACTB gene. The nucleotide sequence of the primer pair for the internal reference gene is shown below, and the nucleotide sequence of the probe is shown below:

[0092] Forward primer: 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' (SEQ ID NO: 70)

[0093] Reverse primer: 5'-AACCAATAAAACCTACTCCTCCCTTAA-3' (SEQ ID NO: 71)

[0094] Probe: 5'-ACCACCACCCAACACACAATAACAAACACA-3' (SEQ ID NO: 72).

[0095] Furthermore, the reporter fluorescent group at the 5' end of the probe specific for the target region and the internal reference gene is one or more of FAM, VIC, HEX, NED, ROX, Red, TET, Texas, JOE, TAMRA, CY3, and CY5; the quencher fluorescent group at the 3' end of the probe specific for the target region and the internal reference gene is one or more of MGB, BHQ-1, BHQ-2, BHQ-3, IBRQ, MGB-NFQ, DABCYL, and ECLIPSE.

[0096] Furthermore, the kit also includes a negative control, a positive control, and a no-template control. The no-template control is ddH2O. The positive control is a plasmid DNA with fully methylated target regions. The negative control is a plasmid DNA with completely unmethylated target regions.

[0097] The present invention also proposes the use of the above gene markers in the preparation of endometrial cancer diagnosis products.

[0098] The present invention also proposes the use of the above kit in preparing a diagnostic product for endometrial cancer.

[0099] Furthermore, the diagnostic product may include any one of a kit, a preparation, and a chip. The gene markers of the present invention can be used to prepare various types of diagnostic products, which can be flexibly selected according to actual conditions.

[0100] The present invention has obtained endometrial cancer-specific methylation early screening markers through screening, including at least one gene among VSX1, SYT1, ZNF132, and VWC2. By detecting the methylation levels of these genes, endometrial cancer can be significantly distinguished from non-cancer individuals. Endometrial cancer can be diagnosed by in vitro detection of at least one methylated region corresponding to any one of the target genes in the sample. The gene markers of the present invention can effectively distinguish endometrial cancer from non-cancer individuals in samples such as tissue, urine, swabs, blood, and lavage fluid. A single indicator can achieve good detection results, and the combination of multiple indicators can further improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0102] FIG1 is a schematic diagram of the process of endometrial cancer gene marker screening and system construction according to the present invention.

[0103] Figures 2 and 3 are diagrams showing the methylation levels of the gene markers of the present invention detected by real-time fluorescence quantitative PCR amplification.

[0104] FIG4 is a diagram showing the M-index distribution of the methylation levels of the four gene markers of the present invention in urine samples.

[0105] FIG5 is a diagram showing the M-index distribution of the methylation levels of the four gene markers of the present invention in urine samples from multiple centers.

[0106] FIG6 is a diagram showing the M-index distribution of the methylation levels of the four gene markers of the present invention in urine samples from patients with endometrial cancer and benign gynecological diseases in multiple centers.

[0107] FIG7 is a diagram showing the M-index distribution of the methylation levels of the four gene markers of the present invention in cervical swab samples.

[0108] FIG8 is a diagram showing the M-index distribution of the methylation levels of the four gene markers of the present invention in vaginal swab samples. DETAILED DESCRIPTION

[0109] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the materials involved in the following examples can be obtained from commercial channels. The methods described are conventional methods unless otherwise specified.

[0110] The present invention utilizes 450k open data on endometrial cancer from The Cancer Genome Atlas (TCGA) (http: / / cancergenome.nih.gov / ). Based on data modeling and analysis, the present invention identifies methylation sites strongly associated with the development of endometrial cancer. Multiple regions of four genes, including VSX1, VWC2, ZNF132, and SYT1, were screened for potential as DNA methylation biomarkers for detecting endometrial cancer. The present invention's detection method was used to test these methylation sites in cancerous and adjacent tissues from endometrial cancer patients, urine samples from endometrial cancer patients (182 cases) and non-cancer patients (334 cases), cervical swab samples from endometrial cancer patients and healthy controls, and vaginal swab samples from endometrial cancer patients and healthy controls. The primer-probe combination designed based on multiple regions of the above-mentioned four genes detected that the methylation signal of the cancer tissue / urine / cervical brush / vaginal swab sample DNA of endometrial cancer individuals was significantly higher than that of non-cancer populations, indicating that the methylation degree of these methylation sites can more sensitively and specifically reflect the occurrence of endometrial cancer. Further, the urine samples collected by the present invention include some patients with benign gynecological diseases (including uterine fibroids, ovarian chocolate cysts, uterine polyps, endometritis, etc.), and the above-mentioned sites can also distinguish these benign gynecological disease patients from endometrial cancer patients, indicating that the methylation site combination selected by the present invention has a higher signal related to endometrial cancer in urine DNA, which has a better effect in distinguishing endometrial cancer from benign tumors that need differential diagnosis, and has superior sensitivity to the detection of endometrial cancer. At the same time, using urine as a test sample is a non-invasive method, which can greatly reduce the burden on patients and increase the compliance of patient detection. Therefore, the methylation markers of the four genes of the present invention can be used as non-invasive detection markers for early diagnosis of endometrial cancer.

[0111] Example 1: Screening of methylation gene markers for endometrial cancer

[0112] To identify DNA methylation markers that can uniquely distinguish endometrial cancer patients from non-cancer groups, the present invention first obtained 450K methylation chip data from 425 cases of endometrial cancer tissue and 34 cases of adjacent tissue from the TCGA public database, and screened for sites that were significantly hypermethylated in endometrial cancer tissue. After data quality control, 385,577 methylation sites were included in the analysis. The CHAMP.DMP method was further used to screen for differential sites, with an FDR-corrected P value of <0.05 and an average methylation ratio of cancer tissue minus the average methylation ratio of adjacent tissue >0.35 as the screening criteria. A total of 8,730 differential sites were screened.

[0113] In addition to collecting cells or DNA from tumors, sampling methods such as urine, cervical swabs, vaginal swabs, and blood inevitably involve the incorporation of components such as white blood cells or urothelial cells. The incorporation of these non-target exogenous DNAs may interfere with the detection of target markers as background signals. Therefore, the interference of background signals should be carefully handled when screening markers. Taking the above into account, the present invention further incorporated 78 cases of urinary tract paracancerous tissues and 338 cases of white blood cell 450k methylation chip data from females in the TCGA and GEO databases, and performed CHAMP.DMP difference tests with 425 cases of endometrial cancer tissue data. Sites that simultaneously met the following conditions were included in the screening range: (1) P value after two difference tests with FDR correction < 0.05; (2) average methylation rate of cancer tissue minus average methylation rate of urinary tract adjacent tissue > 0; (3) average methylation rate of cancer tissue minus average methylation rate of leukocytes > 0; (4) average methylation rate of urinary tract adjacent tissue < 0.1; (5) average methylation rate of leukocytes < 0.1. Sites that met the above requirements were intersected with the differential sites in the endometrial cancer / adjacent tissue differential screening, and a total of 1464 sites were included in the next screening process.

[0114] To further evaluate the methylation levels of the above 1,464 candidate sites in actual samples, the present invention performed Illumina Epic850k chip testing on exfoliated cell samples collected from 12 endometrial cancer patients and 15 controls at the Sun Yat-sen University Cancer Center and Foshan First People's Hospital. This chip is an upgraded version of the 450K chip, retaining most of the 450K chip sites while adding nearly 400,000 new methylation sites. Ultimately, the present invention screened and obtained four genes, including VSX1, SYT1, ZNF132, and VWC2, as detection genes for endometrial cancer diagnosis. The screening process is shown in Figure 1. The methylation sites with the best diagnostic effect on endometrial cancer and their effect information are shown in Table 1.

[0115] The areas under the curve (AUCs) for the optimal sites for four genes (VSX1, SYT1, ZNF132, and VWC2) ranged from 0.906 to 0.972. VWC2 and ZNF132 achieved AUCs greater than 0.95, demonstrating excellent diagnostic performance. VSX1 and SYT1 also exhibited strong diagnostic performance, with AUCs above 0.90. Different genes exhibited varying strengths in sensitivity and specificity, with VWC2 and ZNF132 achieving 100% specificity and VSX1 achieving 91.7%. These genes demonstrated excellent discrimination between endometrial cancer and controls. Furthermore, sites with strong discriminatory power clustered around the optimal site. Within 500 bp upstream and downstream of the optimal site, these genes exhibited multiple sites with AUCs greater than 0.8. VWC2, ZNF132, and VWC2 had eight, seven, five, and two sites with AUCs above 0.8, respectively. The clustering of multiple sites further demonstrates the validity of the aforementioned gene methylation as a diagnostic marker for endometrial cancer and facilitates the development of methylation site detection methods. These results demonstrate that the four genes selected by this invention have excellent discriminatory power between endometrial cancer and controls.

[0116] Table 1 Note: A is the methylation site with the best prediction effect; B is the location of the best site; C is the number of sites with AUC>0.8 within 500bp upstream and downstream of the best site.

[0117] Example 2: Construction of a detection method system for endometrial cancer

[0118] 1. Selection of detection region: The sequence of the region where the methylation sites are located for the diagnosis of endometrial cancer is shown in Table 2:

[0119] Table 2 Note: * indicates the preferred area in this region.

[0120] Table 2 shows the specific primer and probe positions for the amplified region and the methylation sites they detect. Each primer and probe set covers 7-10 methylation sites. While the probes cover densely methylated sites, the detection scheme also includes primers near the 3' end that target highly discriminatory methylation sites, further enhancing the detection accuracy of the system.

[0121] 2. Design and selection of primer and probe sequences

[0122] Using bisulfite-treated sequences from the four gene regions described above as templates, we analyzed a series of parameters, including amplification product length, annealing temperature, primer dimer, hairpin structure, and specificity, for different detection regions. Primer-probe combinations targeting different regions were designed and screened. The sequences and their numbers are shown in Table 3. The designed primers and probes were synthesized by Ruibo Xingke Biotechnology Co., Ltd. "F" represents the forward detection primer, "R" represents the reverse detection primer, and "P" represents the detection probe.

[0123] Table 3

[0124] The probe sequences are labeled with modification groups at both ends, including 5' and 3' groups. The 5' group is selected from any one of FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3, and CY5. In this example, CY5 is used as the target gene, and FAM is used as the internal reference gene. The 3' group is selected from any one of MGB, BHQ-1, BHQ-2, BHQ-3, IBRQ, and MGB-NFQ. In this example, BHQ-2 is used as the target gene, and BHQ-1 is used as the internal reference gene.

[0125] 3. The components of the sample detection kit are as follows: PCR reaction solution: 2× Universal Probe qPCR Master Mix (NEB, catalog number: M3004E); primer-probe mixture; positive control: synthetic target gene methylation plasmid fragment (Qingke); negative control: synthetic target gene non-methylation plasmid fragment (Qingke); blank control: enzyme-free water.

[0126] 4. Sample detection: The methylation markers discovered by the present invention are specific markers for endometrial cancer and can be used to detect various biological samples, including but not limited to tissues, urine, swabs, brushes, blood, etc. The present invention will further elaborate on its application scenarios. The present invention detects DNA methylation in samples. Before detecting methylation signals, DNA extraction and bisulfite conversion of the extracted DNA are required for various samples to facilitate subsequent fluorescence quantitative PCR detection.

[0127] 4.1 Sample collection

[0128] 4.1.1 Tissue Sample Collection: After obtaining informed consent from patients undergoing total hysterectomy and bilateral oophorectomy, a mung bean-sized sample of gross postoperative cancer lesion tissue was collected as the cancer tissue sample. Tissue within 3 cm of the cancer lesion edge was collected as the para-cancer tissue sample. The samples were placed in tissue cryopreservation tubes and frozen at -80°C.

[0129] 4.1.2 Urine Sample Collection: Instruct the subject to collect the first urine of the morning urination using a 50 mL urine cup. The urine should then be poured into a urine collection tube pre-filled with Urine Conditioning Buffer (zymo, Catalog No.: D3061-1-140). A volume of 40-50 mL of urine should be collected for testing.

[0130] 4.1.3 Cervical swab sample collection: A uniformly trained medical staff, using a vaginal speculum, inserts a cervical swab into the cervix and rotates it clockwise five times to collect cervical cells. The cervical swab is then placed in a 2 mL PBS-filled exfoliated cell collection bottle for storage and transportation at low temperatures.

[0131] 4.1.4 Vaginal swab sample collection: Uniformly trained medical personnel use a sterile swab to rotate and collect samples at the 1 / 3 of the vaginal side wall, and the swab with secretions can be clearly seen attached to the secretion. The swab with secretions attached is placed in a detached cell collection tube with 2 mL of PBS added, and stored and transported at low temperature.

[0132] 4.2 Sample DNA extraction

[0133] 4.2.1 Tissue / brush / swab samples: Blood / cell / tissue genomic DNA extraction kit (TIANGEN Biochemical, Cat. No. DP304-03) was used as the tissue / brush / swab genomic DNA extraction reagent for extraction.

[0134] 4.2.2 Urine samples: using Quick-DNA TM DNA was extracted from urine samples using Urine Kit (zymo, catalog number: D3061).

[0135] 4.3 Bisulfite conversion of sample DNA

[0136] The DNA bisulfite conversion kit was purchased from Zymo. The extracted DNA was subjected to bisulfite conversion according to the kit instructions. Unmethylated cytosine (C) in the DNA was converted to uracil (U), while methylated cytosine (C) remained unchanged, thus obtaining converted bis-DNA.

[0137] 4.4 Fluorescence quantitative PCR amplification of Bis-DNA

[0138] Use different primer and probe combinations in Table 2 to perform amplification detection on the corresponding different regions. The details are as follows:

[0139] 4.4.1 Prepare the PCR reaction solution and primer-probe mixture: 2× EpiTect MethyLight Master Mix 5 μL; forward detection primer F 0.3-0.5 μmol; reverse detection primer R 0.3-0.5 μmol; detection probe P 0.1-0.3 μmol; sample DNA <100 ng; make up to 10 μL with purified water.

[0140] 4.4.2 Sample Loading: Aliquot the prepared mixture into a 96-well or 384-well plate, 8 μL per well. Add 2 μL of Bis-DNA to the sample wells, with one replicate per well. Also prepare three quality control samples: a positive quality control, a negative quality control, and a no-template control (NTC). Load the samples using the same method as above.

[0141] 4.4.3 Fluorescence quantitative PCR amplification detection: Samples were tested using a ROCHE 480 fluorescence quantitative PCR instrument. The sample amplification program was set as follows: 95°C for 60 s; then 95°C for 15 s, 55-65°C for 30 s (fluorescence signal collection), 40-50 cycles*; and finally 4°C for 30 s (*adjusted appropriately according to the specific gene and sample type).

[0142] 4.4.4 Signal collection: collect FAM and CY5 signals at 55-65°C.

[0143] 5. Interpretation of test results

[0144] 5.1 Detection System Evaluation: If the positive quality control product has a normal amplification curve, and the negative quality control product and the no-template control sample have no amplification curve, the test results are considered valid.

[0145] 5.2 Sample test result evaluation

[0146] 5.2.1 The result is valid if the internal standard channel has an S-shaped amplification curve and a Ct value ≤ 30;

[0147] 5.2.2 The unamplified Ct value was assigned as 50.

[0148] 5.2.3 The methylation levels of the four genes are expressed using M-index = 2^(-ΔCt) values:

[0149] M-index(VSX1)=2^(Ct(ACTB)-Ct(VSX1));

[0150] M-index(SYT1)=2^(Ct(ACTB)-Ct(SYT1));

[0151] M-index(ZNF132)=2^(Ct(ACTB)-Ct(ZNF132));

[0152] M-index(VWC2)=2^(Ct(ACTB)-Ct(VWC2)).

[0153] 6. Testing system evaluation

[0154] In order to evaluate the stability of the results of each test reaction plate, the kit artificially synthesized the sequence corresponding to the complete methylation of the amplified product, and constructed 2 -10 7 Standards are placed in copies / well. A standard curve is constructed by measuring the Ct values ​​of a series of standards in each reaction plate during each assay. Comparing the standard curves across different assay plates allows for evaluation of assay stability.

[0155] 7. Evaluation of detection effect

[0156] After PCR testing, the Ct value of the methylation sites of the ACTB, VSX1, VWC2, ZNF132, and SYT1 genes of each sample can be obtained. The Ct value without amplification is set to 50, and the M-index of the target gene of each sample is calculated by the above formula. In different sample types (tissue / urine / brush film / swab), the M-index of the four methylation sites detected above is used to construct a case-control Logistic model in turn, and the endometrial cancer risk score and the threshold for judging the occurrence of endometrial cancer are calculated by the fitting equation. According to the comparison of the sample score with the threshold, the sample is divided into an endometrial cancer positive group and a negative group. The grouping of the Logistic model based on M-index is compared with the clinical pathological grouping of the sample to obtain a ROC curve for judging the diagnostic performance of the methylation degree model, and the area under the curve AUC, sensitivity and specificity are calculated by the ROC curve. The detection efficiency of the reagent of the present invention can be judged according to the AUC / sensitivity / specificity indicators.

[0157] Example 3: Detection of tissue samples using primer-probe combinations

[0158] To further optimize the primer-probe combination in Example 2, clinicians collected eight pairs of cancerous and adjacent tissues from postoperative gross specimens of endometrial cancer patients for testing. The tissue specimens were extracted, transformed, and tested according to the method described in Example 2. The M-index (i.e., 2^(-ΔCt)) for different genes in each sample was obtained, and the test results are shown in Figures 2 and 3. As can be seen from the figures, compared with adjacent tissues, the methylation levels of the VSX1, SYT1, ZNF132, and VWC2 gene detection sites in the cancer tissue samples of endometrial cancer patients were significantly higher than those in the adjacent tissues, indicating a significant difference.

[0159] Example 4: Detection of exfoliated cell samples using primer-probe combinations

[0160] In this example, exfoliated cell samples from 32 endometrial cancer patients and 33 non-cancer controls were collected for testing. The results showed that each primer-probe combination achieved an AUC greater than 0.7 for differentiating between endometrial cancer and controls, demonstrating excellent diagnostic efficacy. The results are shown in Table 4. Specifically, the AUCs for the detection regions of VSX1_4_2, VSX1_5_2, VWC2_2_2, VWC2_4_2, VWC2_5_1, VWC2_6_2, ZNF132_1_1, ZNF132_2_1, ZNF132_3_2, and SYT1_1_2 reached AUCs greater than 0.8, demonstrating that multiple regions of these genes possess diagnostic value as endometrial cancer markers.

[0161] Table 4

[0162] Taking into account the evaluation indicators such as AUC, sensitivity and specificity, VSX1_4_2 (Chr20:25062655-25062739), VWC2_5_1 (Chr7:49815144-49815236), ZNF132_2_1 (Chr19:58951554-58951637) and SYT1_1_2 (Chr12:79258392-79258498) were selected as the representative regions of the above four genes for testing urine samples.

[0163] Example 5: Testing urine samples

[0164] A total of 133 urine samples were collected at the Cancer Prevention and Treatment Center of Sun Yat-sen University, including 65 preoperative urine samples from patients with endometrial cancer histopathologically confirmed, 16 preoperative urine samples from patients with benign gynecological diseases (including uterine fibroids and ovarian chocolate cysts), and 52 urine samples from healthy volunteers (physical examinations in the past year showed no abnormalities). According to whether the sample object suffers from endometrial cancer, the urine samples are divided into a case group (65 endometrial cancer patients) and a control group (68 benign gynecological disease patients and healthy volunteers). After the urine samples are extracted and transformed according to the method described in Example 2 for detection, the M-index of different genes in each sample is obtained. The test results are shown in a scatter plot in Figure 4. The gene loci found by the present invention can also achieve the effect of distinguishing the case group and the control group in the urine sample. The methylation level of the case group is significantly higher than that of the control group. Further, modeling analysis is performed according to the detection effect evaluation method described in Example 2, and the analysis results are as follows.

[0165] Through further modeling analysis, the detection effect of the single gene methylation site model is shown in the following table.

[0166] Table 5

[0167] As shown in the table, the detection method constructed in this invention demonstrated excellent discrimination between case and control groups in urine samples. The AUCs for modeled single-gene indices were all above 0.8. The VSX1 gene demonstrated the strongest comprehensive discrimination capability among the single-gene indices, with an AUC of 0.858 and a specificity of 77.9% at a sensitivity of 81.5%. Different genes exhibited varying discriminatory strengths. Methylation sites in the VSX1 gene demonstrated high sensitivity, reaching 81.5% at the optimal cutoff, indicating that it could identify more cancer cases. Methylation sites in the VWC2 gene demonstrated high specificity, reaching 98.5% at the optimal cutoff, identifying more non-cancer patients. Furthermore, combined analysis of the methylation indices of the four genes using two, three, or four indices yielded the results shown in Table 6.

[0168] Table 6

[0169] As shown in the table above, in a sample of 133 urine samples collected at the Sun Yat-sen University Cancer Center, a model constructed by combining 2-4 gene methylation markers further improved its ability to discriminate between endometrial cancer cases and controls, achieving an AUC as high as 0.932 (combining VSX1, VWC2, and SYT1). When combining only two markers, the AUC for VSX1 and VWC2 reached 0.928, with a sensitivity of 84.6% and a specificity of 88.2%. The model combining VWC2 and SYT1 achieved a sensitivity of 89.2%, while the model combining VWC2 and ZNF132 achieved a specificity of 98.5%. These results demonstrate that the various markers complement each other to some extent, and that the combined model can leverage the strengths of each, resulting in superior performance compared to single-marker models.

[0170] Example 6: Collecting urine samples from multiple centers for testing

[0171] In order to evaluate the performance stability of the method constructed by the present invention for urine sample detection, further, in other centers: the Affiliated Tumor Hospital of Guangzhou Medical University and the First People's Hospital of Foshan, a total of 336 urine samples from endometrial cancer patients and non-cancer individuals were collected for detection, including 93 samples from endometrial cancer patients (case group) and 243 samples from non-cancer patients (control group). The specific detection method is the same as in Example 2. After detection, the M-index of different genes in each sample is obtained. The test results are shown in the scatter plot in Figure 5. The gene methylation index found by the present invention can also achieve the effect of distinguishing case group and control group in urine samples collected from multiple centers. The methylation level of the case group is significantly higher than the methylation level of the control group. Further, modeling analysis is performed according to the detection effect evaluation method described in Example 2, and the analysis results are as follows.

[0172] As shown in Table 7, the detection method constructed by the present invention still has good discrimination ability for the case group and the control group in urine samples collected from multiple centers. The AUC after modeling of the single gene index is between 0.779-0.879. Different genes show different discrimination advantages. In the urine samples collected from multiple centers, the ZNF132 index has good discrimination ability, with an AUC of 0.879 and a corresponding sensitivity of 64.5% when the specificity is 94.7%. The methylation index of the VSX1 gene still has a high sensitivity, with a specificity of 78.6% and a corresponding sensitivity of up to 81.7%, indicating that it can identify more cancer cases. In summary, the single methylation marker discovered by the present invention still has a good detection effect in urine samples collected from multiple centers. Further, the methylation indexes of the four genes were subjected to a two / three / four-index joint analysis, and the results shown in Table 8 below were obtained.

[0173] Table 7

[0174] Table 8

[0175] As shown in the table above, in a multicenter sample collection of 336 urine samples, models constructed by combining 2-4 gene methylation markers improved the ability to discriminate between endometrial cancer cases and controls. With the exception of the VWC2+SYT1 model, the AUCs of all the models were above 0.8. The highest AUC reached 0.893 for the VSX1+ZNF132+SYT1 three-marker model, which achieved a sensitivity of 80.6% and a specificity of 79.8%. These results demonstrate that the aforementioned markers complement each other in urine samples collected from multiple centers, and that combining multiple markers can further enhance the detection of endometrial cancer.

[0176] Example 7: Differentiation of Endometrial Cancer from Benign Diseases

[0177] In actual clinical work, patients with endometrial cancer usually have symptoms of abnormal vaginal bleeding in the early stages, but this symptom is not specific. Most patients with benign gynecological diseases in women of childbearing age and menopause also have symptoms of abnormal vaginal bleeding. Therefore, the present invention further evaluates the ability of the detection method to distinguish between patients with endometrial cancer and patients with benign gynecological diseases. By defining urine samples from 158 endometrial cancer patients in multiple centers as the case group and urine samples from 236 patients with benign gynecological diseases as the control group, the specific conditions of benign gynecological diseases were: 13 cases of endometrial hyperplasia; 60 cases of gynecological inflammation (including cervicitis, endometritis); 103 cases of uterine polyps; 32 cases of uterine fibroids; 28 cases of others (including infertility, pelvic inflammatory disease, etc.).

[0178] The specific detection method was the same as in Example 2. After testing, the M-index of different genes in each sample was obtained. The test results are shown in the scatter plot in Figure 6. The gene methylation index discovered by the present invention showed significant differences between patients with endometrial cancer and patients with benign gynecological diseases, with the methylation level in the case group being significantly higher than that in the control group. Furthermore, modeling analysis was performed based on the detection effect evaluation method described in Example 2, and the analysis results are described below.

[0179] As shown in Table 9, the detection method constructed by the present invention has excellent discrimination ability between patients with endometrial cancer and patients with benign diseases in urine samples collected from multiple centers. The AUC of the single-gene index model ranged from 0.831 to 0.916. The VSX1 index has a good ability to distinguish patients with endometrial cancer from patients with benign gynecological diseases, with an AUC of 0.916 and a sensitivity of 88.0% at a specificity of 82.2%. The methylation index of the VWC2 gene has an even higher specificity, with a sensitivity of 63.9% at a specificity of 97.5% and an AUC of 0.831. In summary, the methylation marker discovered by the present invention can effectively distinguish patients with endometrial cancer from patients with other benign gynecological diseases in urine samples collected from multiple centers. Furthermore, the methylation indexes of the four genes were analyzed in a two-, three-, or four-index combination, resulting in the results shown in Table 10 below.

[0180] Table 9

[0181] Table 10

[0182] As shown in the table above, in 394 urine samples collected from multiple centers, models constructed by combining 2-4 gene methylation markers improved the ability to distinguish endometrial cancer cases from benign gynecological diseases. The area under the curve (AUC) for the combined model reached as high as 0.927. Both the VSX1+ZNF132 two-marker model and the VSX1+ZNF132+SYT1 three-marker model achieved an AUC of 0.927, corresponding to a specificity of 81.0% at a specificity of 90.7%. These results demonstrate that the above markers complement each other in urine samples collected from multiple centers, and that combining multiple markers can further improve the ability to distinguish endometrial cancer from benign gynecological diseases.

[0183] In summary, by collecting urine samples for testing and evaluation in this center, and further collecting urine samples from multiple centers to expand sample testing, the superior detection performance of the gene methylation indicators protected by the present invention is demonstrated. Each indicator has its own advantages, and the combination of multiple indicators can achieve complementary advantages and further improve the detection performance. In addition, the ability of the present invention to distinguish between patients with endometrial cancer and benign gynecological diseases in urine samples is further analyzed. The present invention also has good performance advantages in differential diagnosis. The above proves that the gene methylation markers discovered by the present invention can be used to detect endometrial cancer in urine samples with high accuracy.

[0184] Example 8: Detection of cervical swab samples

[0185] Furthermore, in order to evaluate the detection efficacy of the gene methylation markers discovered by the present invention in cervical swab samples, cervical swab samples of 17 patients with endometrial cancer diagnosed by histopathology and 14 healthy volunteers were collected from Foshan First People's Hospital, and the sample collection was as described in Example 2. The 17 endometrial cancer patient swab samples were defined as the case group, and the 14 healthy volunteer swab samples were defined as the control group. The specific detection method is as described in Example 2. After the test, the M-index of different genes in each sample was obtained. The test results are shown in the scatter plot in Figure 7. In the swab samples, the gene methylation index discovered by the present invention has obvious differences in endometrial cancer patients and healthy volunteers, and the methylation level of the case group is significantly higher than that of the control group. Further, modeling analysis was performed according to the detection effect evaluation method described in Example 2, and the analysis results are as follows. As shown in the table, the detection method constructed in this invention has excellent discrimination ability between endometrial cancer patients and healthy volunteers in the slide samples. The AUC of the single-gene index model ranged from 0.718 to 0.891. The ZNF132 index showed good discrimination ability, with an AUC of 0.891 and a corresponding sensitivity of 76.5% at a specificity of 92.9%. The VWC2 gene was second only to the ZNF132 gene in detection performance, with an AUC of 0.882 and a corresponding sensitivity of 88.2% at a specificity of 85.7%. SYT1 had a relatively high specificity of 92.9%.

[0186] Table 11

[0187] Furthermore, the methylation indices of the four genes were subjected to a two / three / four-indicator combined analysis, and the results shown in the following table were obtained.

[0188] Table 12

[0189] As can be seen from the above table, in the 31 slide samples, the model constructed by combining 2-4 gene methylation indicators has further improved the ability to distinguish endometrial cancer cases and controls. The AUC of the model after multi-indicator combination can be as high as 0.916 (combined with VWC2+ZNF132+SYT1 indicators). When the model specificity is 100%, the corresponding sensitivity is 76.5%. The above results show that there is a certain complementary effect between the various indicators in the slide samples. The combination of multiple indicators can complement the advantages of each indicator. The performance of the multi-indicator joint model is better than that of the single indicator model.

[0190] Example 9: Testing vaginal swab samples

[0191] As described in Example 8, vaginal swab samples were collected from the group of people who collected cervical swabs in Example 8 during the same period, and the sample collection and processing were as described in Example 2. The swab samples of 17 endometrial cancer patients were defined as the case group, and the swab samples of 14 healthy volunteers were defined as the control group. The specific detection method is as described in Example 2. After the test, the M-index of different genes in each sample was obtained. The test results are shown in the scatter plot in Figure 8. In the swab samples, the gene methylation index discovered by the present invention has obvious differences between endometrial cancer patients and healthy volunteers, and the methylation level of the case group is significantly higher than that of the control group. Further, modeling analysis was performed according to the detection effect evaluation method described in Example 2, and the analysis results are as follows. As shown in Table 13, the detection method constructed by the present invention has a good ability to distinguish endometrial cancer patients and healthy volunteers in swab samples. The AUC of the single gene indicator model is between 0.769-0.853. The ZNF132 indicator has a good discrimination ability, with an AUC of 0.853, and a corresponding sensitivity of 64.7% when the specificity is 100%. The detection performance of the VWC2 gene was second only to the ZNF132 gene, with an AUC of 0.849 and a sensitivity of 64.7% at a specificity of 100%. Furthermore, a combined analysis of the methylation indices of the four genes using two, three, or four indicators yielded the results shown in Table 14.

[0192] Table 13

[0193] Table 14

[0194] As shown in the table above, a model combining 2-4 gene methylation indices significantly improved the ability to distinguish endometrial cancer cases from controls in 31 swab samples. The AUC for the combined model reached as high as 0.966 (combining VSX1, ZNF132, and SYT1, and the four-indicator combination). The sensitivity of this model, at 100% specificity, was 88.2%. These results demonstrate that the various indices complement each other in swab samples, leveraging their strengths. The combined model outperformed single-indicator models.

[0195] In summary, the gene methylation markers discovered in the present invention can effectively distinguish endometrial cancer from non-cancer individuals in tissue / urine / swab samples. A single indicator can achieve good detection results, and the combination of multiple indicators can further improve detection efficiency.

[0196] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A methylation gene marker for endometrial cancer detection, characterized in that The methylation gene marker specifically includes at least one of VSX1, SYT1, ZNF132, and VWC2 genes.

2. A methylation gene marker for endometrial cancer detection, characterized in that The methylation gene marker comprises a methylated nucleic acid sequence in at least one target region of at least one of the target genes VSX1, VWC2, ZNF132, and SYT1, wherein the target region is selected from the methylation of at least one of the following segments in at least one of the genes VSX1, VWC2, ZNF132, and SYT1: VSX1 gene: Chr20:25058334-25058603; Chr20:25061937-25062310 or Chr20:25061874-25062088; Chr20:25062398-25062760 or Chr20:25062376-25062484; Chr20:25062681-25062788 or Chr20:25062655-25062739; Chr20:25062736-25062940 or Chr20:25062749-25062900; SYT1 gene: Chr12:79258369-79258486 or Chr12:79258392-79258498; ZNF132 gene: Chr19:58951204-58951524 or Chr19:58951402-58951524; Chr19:58951554-58951637; Chr19:58951672-58952006 or Chr19:58951724-58951825; VWC2 gene: Chr7:49812992-49813086; Chr7:49813051-49813343 or Chr7:49813066-49813161; Chr7:49813455-49813811 or Chr7:49813387-49813537; Chr7:49814721-49815073 or Chr7:49814765-49814936; Chr7:49815144-49815236; Chr7:49815290-49815635 or Chr7:49815340-49815439.

3. A detection primer for endometrial cancer detection, characterized in that For detecting the methylation status of the marker gene methylation region according to claim 2, the nucleotide sequence of the detection primer is at least one of the following: VSX1 gene: The detection primers corresponding to Chr20:25058334-25058603 are SEQ ID NOs:1-2; The detection primers corresponding to Chr20:25061937-25062310 are SEQ ID NOs:4-5; The detection primers corresponding to Chr20:25061874-25062088 are SEQ ID NOs:6-7; The detection primers corresponding to Chr20:25062398-25062760 are SEQ ID NOs:9-10; The detection primers corresponding to Chr20:25062376-25062484 are SEQ ID NOs:12-13; The detection primers corresponding to Chr20:25062681-25062788 are SEQ ID NOs:15-16; The detection primers corresponding to Chr20:25062655-25062739 are SEQ ID NOs:18-19; The detection primers corresponding to Chr20:25062736-25062940 are SEQ ID NOs:21-22; The detection primers corresponding to Chr20:25062749-25062900 are SEQ ID NOs:23-24; SYT1 gene: The detection primers corresponding to Chr12:79258369-79258486 are SEQ ID NOs:26-27; The detection primers corresponding to Chr12:79258392-79258498 are SEQ ID NOs:29-30; ZNF132 gene: The detection primers corresponding to Chr19:58951204-58951524 are SEQ ID NO:32 and SEQ ID NO:34; The detection primers corresponding to Chr19:58951402-58951524 are SEQ ID NO:33 and SEQ ID NO:34; The detection primers corresponding to Chr19:58951554-58951637 are SEQ ID NOs:36-37; The detection primers corresponding to Chr19:58951672-58952006 are SEQ ID NOs:39-40; The detection primers corresponding to Chr19:58951724-58951825 are SEQ ID NOs:41-42; VWC2 gene: The detection primers corresponding to Chr7:49812992-49813086 are SEQ ID NOs:44-45; The detection primers corresponding to Chr7:49813051-49813343 are SEQ ID NOs:47-48; The detection primers corresponding to Chr7:49813066-49813161 are SEQ ID NOs:49-50; The detection primers corresponding to Chr7:49813455-49813811 are SEQ ID NOs:52-53; The detection primers corresponding to Chr7:49813387-49813537 are SEQ ID NOs:54-55; The detection primers corresponding to Chr7:49814721-49815073 are SEQ ID NOs:57-58; The detection primers corresponding to Chr7:49814765-49814936 are SEQ ID NOs:59-60; The detection primers corresponding to Chr7:49815144-49815236 are SEQ ID NOs:62-63; The detection primers corresponding to Chr7:49815290-49815635 are SEQ ID NOs:65-66; The detection primers corresponding to Chr7:49815340-49815439 are SEQ ID NOs:67-68.

4. A detection probe for early screening and diagnosis of endometrial cancer, characterized in that For detecting the methylation status of the marker gene methylation region according to claim 2, the nucleotide sequence of the probe is at least one of the following: VSX1 gene: The detection probe corresponding to Chr20:25058334-25058603 is SEQ ID NO:3; The detection probe corresponding to Chr20:25061937-25062310 is SEQ ID NO:8; The detection probe corresponding to Chr20:25061874-25062088 is SEQ ID NO:8; The detection probe corresponding to Chr20:25062398-25062760 is SEQ ID NO:11; The detection probe corresponding to Chr20:25062376-25062484 is SEQ ID NO:14; The detection probe corresponding to Chr20:25062681-25062788 is SEQ ID NO:17; The detection probe corresponding to Chr20:25062655-25062739 is SEQ ID NO:20; The detection probe corresponding to Chr20:25062736-25062940 is SEQ ID NO:25; The detection probe corresponding to Chr20:25062749-25062900 is SEQ ID NO:25; SYT1 gene: The detection probe corresponding to Chr12:79258369-79258486 is SEQ ID NO:28; The detection probe corresponding to Chr12:79258392-79258498 is SEQ ID NO:31; ZNF132 gene: The detection probe corresponding to Chr19:58951204-58951524 is SEQ ID NO:35; The detection probe corresponding to Chr19:58951402-58951524 is SEQ ID NO:35; The detection probe corresponding to Chr19:58951554-58951637 is SEQ ID NO:38; The detection probe corresponding to Chr19:58951672-58952006 is SEQ ID NO:43; The detection probe corresponding to Chr19:58951724-58951825 is SEQ ID NO:43; VWC2 gene: The detection probe corresponding to Chr7:49812992-49813086 is SEQ ID NO:46; The detection probe corresponding to Chr7:49813051-49813343 is SEQ ID NO:51; The detection probe corresponding to Chr7:49813066-49813161 is SEQ ID NO:51; The detection probe corresponding to Chr7:49813455-49813811 is SEQ ID NO:56; The detection probe corresponding to Chr7:49813387-49813537 is SEQ ID NO:56; The detection probe corresponding to Chr7:49814721-49815073 is SEQ ID NO:61; The detection probe corresponding to Chr7:49814765-49814936 is SEQ ID NO:61; The detection probe corresponding to Chr7:49815144-49815236 is SEQ ID NO:64; The detection probe corresponding to Chr7:49815290-49815635 is SEQ ID NO:69; The detection probe corresponding to Chr7:49815340-49815439 is SEQ ID NO:

69.

5. Use of a reagent for detecting target gene methylation in the preparation of a detection kit or device, characterized in that The detection kit or device is used for detecting, screening or diagnosing endometrial cancer; the target gene is selected from at least one of the following genes: VSX1, SYT1, ZNF132, VWC2.

6. The use according to claim 5, characterized in that: The reagent includes at least one of an antibody, a probe, a primer, and a mass spectrometry detection reagent specifically for detecting the target gene; wherein the primer includes the detection primer according to claim 3.

7. A kit for diagnosing endometrial cancer, characterized in that The method comprises a reagent for detecting methylation of a target gene; the target gene is selected from at least one of the following genes: VSX1, SYT1, ZNF132, and VWC2.

8. The kit according to claim 7, characterized in that The reagent includes at least one of an antibody, a probe, a primer, and a mass spectrometry detection reagent specifically for detecting the target gene; wherein the primer includes the detection primer according to claim 3; and the probe includes the detection probe according to claim 4.

9. The kit according to claim 7 or 8, characterized in that Also included are primer pairs and probes for an internal reference gene, wherein the internal reference gene is the ACTB gene. The nucleotide sequence of the primer pair for the internal reference gene is shown below, and the nucleotide sequence of the probe is shown below: Forward primer: 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' Reverse primer: 5'-AACCAATAAAACCTACTCCTCCCTTAA-3' Probe: 5′-ACCACCACCCAACACACAATAACAAACACA-3′.

10. Use of the methylation gene marker according to any one of claims 1 to 2, or the detection primer according to claim 3, or the detection probe according to claim 4, or the kit according to claims 8 to 9 in the preparation of a diagnostic product for endometrial cancer.

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