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

Abstract

The present invention belongs to the field of biomedicine technology, and particularly relates to a primer-probe combination, a methylation detection kit and its application for the early detection of endometrial cancer. The present invention provides a methylation gene marker for the detection of endometrial cancer, specifically comprising ZNF132, or a combination of ZNF132 and at least one of SYT1 and VWC2 genes. The present invention obtains endometrial cancer-specific methylation early screening markers by screening, and can significantly distinguish endometrial cancer from non-cancer individuals by detecting the methylation levels of these genes; by in vitro detection of any at least one methylation region corresponding to the target gene in the sample, the diagnosis of endometrial cancer can be achieved. The gene marker of the present invention can better distinguish endometrial cancer from non-cancer individuals in samples such as tissue / urine / swab / swab / blood / lavage fluid, and a single indicator can achieve a better detection effect, and the combination of multiple indicators can further improve the detection efficiency.

Description

[0001] This invention patent application is a divisional application of application number "202410299405.3". The application date of the original application is "March 15, 2024", the application number is "202410299405.3", and the name of the invention is "Primer probe combination, methylation detection kit and its application for early detection of endometrial cancer". Technical Field

[0002] 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

[0003] Endometrial cancer is a common epithelial malignant tumor that occurs in the endometrium, accounting for about 20%-30% of gynecological malignancies. Endometrial cancer is more common in perimenopausal and postmenopausal women, with a peak age of 50-70 years old and a median age of diagnosis of 61 years old. In recent years, with the increase in the prevalence of risk factors for endometrial cancer, especially obesity and an aging population, the incidence of endometrial cancer has gradually increased. In addition, the incidence of endometrial cancer has shown a trend of younger age, and the incidence of endometrial cancer in premenopausal women has increased significantly. The treatment effect of endometrial cancer is closely related to the clinical stage, and patients with early endometrial cancer usually have a better prognosis. According to statistics, the five-year survival rate of early (stage I, stage II) patients can reach more than 70%, the five-year survival rate of stage III patients is about 40-50%, and the five-year survival rate of stage IV patients is only 15-20%. Early diagnosis is of great significance for endometrial cancer.

[0004] 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 (detecting commonly used clinical markers such as CA125 and HE4), and endometrial biopsy (diagnostic curettage and hysteroscopic curettage, removing endometrial tissue and further performing histopathological biopsy). The sensitivity and specificity of imaging and laboratory tests are both low, and endometrial biopsy is an invasive operation that can easily cause physical discomfort. Therefore, there is an urgent need to develop a convenient, fast, and non-invasive detection method that can accurately identify endometrial cancer at an early stage.

[0005] DNA methylation is a type of epigenetic modification, which is the addition of a methyl group to the 5th carbon atom of the cytosine base. This modification is usually associated with gene silencing. DNA methylation is a key epigenetic regulator of gene expression and usually 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

[0006] 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.

[0007] 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, detects the methylation of these genes to distinguish endometrial cancer from non-cancer individuals, and verifies the stability and effectiveness of the markers of the present invention by testing them in a variety of non-invasive sampling sample types.

[0008] Another object of the present invention is to provide a kit for diagnosing endometrial cancer.

[0009] The present invention has discovered sites that can eliminate background signal interference in various types of non-invasively collected samples and accurately distinguish between endometrial cancer and other diseased / healthy individuals, and based on them, has established a stable and reliable marker detection kit.

[0010] Another object of the present invention is to provide the use of the above-mentioned kit in the preparation of endometrial cancer diagnosis products.

[0011] The purpose of the present invention is achieved through the following solutions:

[0012] According to the first aspect of the present invention, the present invention proposes a methylation gene marker for endometrial cancer detection, wherein the methylation gene marker specifically includes at least one of VSX1, SYT1, ZNF132, and VWC2 genes. The detection refers to the detection of the methylation level of at least one of the target genes VSX1, SYT1, ZNF132, and VWC2 to achieve the diagnosis of endometrial cancer.

[0013] According to the second aspect of the present invention, the present invention provides a methylation gene marker for endometrial cancer detection, wherein 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 sections in at least one of the genes VSX1, VWC2, ZNF132, and SYT1:

[0014] 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;

[0015] SYT1 gene: Chr12:79258369-79258486 or Chr12:79258392-79258498;

[0016] ZNF132 gene: Chr19:58951204-58951524 or Chr19:58951402-58951524; Chr19:58951554-58951637; Chr19:58951672-58952006 or Chr19:58951724-58951825;

[0017] 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.

[0018] The genes described in the present invention use the human genome version GRCh37 (hg19) as a reference genome.

[0019] The present invention obtains endometrial cancer-specific methylation early screening markers by screening, including at least one gene among VSX1, SYT1, ZNF132, and VWC2. By detecting the methylation levels of these genes, endometrial cancer and non-cancer individuals can be significantly distinguished, and the stability and effectiveness of the markers of the present invention are verified by testing in a variety of non-invasive sampling sample types. The present invention provides new markers and diagnostic strategies for the diagnosis of endometrial cancer and its precancerous lesions.

[0020] Furthermore, by detecting any at least one methylation region corresponding to any at least one target gene in the sample in vitro, the diagnosis of endometrial cancer can be achieved. The sample can be any body fluid, exfoliated cell, and tissue sample, 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.

[0021] According to the third aspect of the present invention, the present invention provides a detection primer for endometrial cancer detection, which is used to detect the methylation state 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:

[0022] VSX1 gene:

[0023] The detection primers corresponding to Chr20:25058334-25058603 are SEQ ID NOs:1-2;

[0024] The detection primers corresponding to Chr20:25061937-25062310 are SEQ ID NOs:4-5;

[0025] The detection primers corresponding to Chr20:25061874-25062088 are SEQ ID NOs:6-7;

[0026] The detection primers corresponding to Chr20:25062398-25062760 are SEQ ID NOs:9-10;

[0027] The detection primers corresponding to Chr20:25062376-25062484 are SEQ ID NOs:12-13;

[0028] The detection primers corresponding to Chr20:25062681-25062788 are SEQ ID NOs:15-16;

[0029] The detection primers corresponding to Chr20:25062655-25062739 are SEQ ID NOs:18-19;

[0030] The detection primers corresponding to Chr20:25062736-25062940 are SEQ ID NOs:21-22;

[0031] The detection primers corresponding to Chr20:25062749-25062900 are SEQ ID NOs:23-24;

[0032] SYT1 gene:

[0033] The detection primers corresponding to Chr12:79258369-79258486 are SEQ ID NOs:26-27;

[0034] The detection primers corresponding to Chr12:79258392-79258498 are SEQ ID NOs:29-30;

[0035] ZNF132 gene:

[0036] The detection primers corresponding to Chr19:58951204-58951524 are SEQ ID NO:32 and SEQ ID NO:34;

[0037] The detection primers corresponding to Chr19:58951402-58951524 are SEQ ID NO:33 and SEQ ID NO:34;

[0038] The detection primers corresponding to Chr19:58951554-58951637 are SEQ ID NOs:36-37;

[0039] The detection primers corresponding to Chr19:58951672-58952006 are SEQ ID NOs:39-40;

[0040] The detection primers corresponding to Chr19:58951724-58951825 are SEQ ID NOs:41-42;

[0041] VWC2 gene:

[0042] The detection primers corresponding to Chr7:49812992-49813086 are SEQ ID NOs:44-45;

[0043] The detection primers corresponding to Chr7:49813051-49813343 are SEQ ID NOs:47-48;

[0044] The detection primers corresponding to Chr7:49813066-49813161 are SEQ ID NOs:49-50;

[0045] The detection primers corresponding to Chr7:49813455-49813811 are SEQ ID NOs:52-53;

[0046] The detection primers corresponding to Chr7:49813387-49813537 are SEQ ID NOs:54-55;

[0047] The detection primers corresponding to Chr7:49814721-49815073 are SEQ ID NOs:57-58;

[0048] The detection primers corresponding to Chr7:49814765-49814936 are SEQ ID NOs:59-60;

[0049] The detection primers corresponding to Chr7:49815144-49815236 are SEQ ID NOs:62-63;

[0050] The detection primers corresponding to Chr7:49815290-49815635 are SEQ ID NOs:65-66;

[0051] The detection primers corresponding to Chr7:49815340-49815439 are SEQ ID NOs:67-68.

[0052] According to the fourth aspect of the present invention, the present invention provides a detection probe for early screening and diagnosis of endometrial cancer, which is used to detect the methylation state of the methylation region of the above-mentioned marker gene, and the nucleotide sequence of the probe is one of the following:

[0053] VSX1 gene:

[0054] The detection probe corresponding to Chr20:25058334-25058603 is SEQ ID NO:3;

[0055] The detection probe corresponding to Chr20:25061937-25062310 is SEQ ID NO:8;

[0056] The detection probe corresponding to Chr20:25061874-25062088 is SEQ ID NO:8;

[0057] The detection probe corresponding to Chr20:25062398-25062760 is SEQ ID NO:11;

[0058] The detection probe corresponding to Chr20:25062376-25062484 is SEQ ID NO:14;

[0059] The detection probe corresponding to Chr20:25062681-25062788 is SEQ ID NO:17;

[0060] The detection probe corresponding to Chr20:25062655-25062739 is SEQ ID NO:20;

[0061] The detection probe corresponding to Chr20:25062736-25062940 is SEQ ID NO:25;

[0062] The detection probe corresponding to Chr20:25062749-25062900 is SEQ ID NO:25;

[0063] SYT1 gene:

[0064] The detection probe corresponding to Chr12:79258369-79258486 is SEQ ID NO:28;

[0065] The detection probe corresponding to Chr12:79258392-79258498 is SEQ ID NO:31;

[0066] ZNF132 gene:

[0067] The detection probe corresponding to Chr19:58951204-58951524 is SEQ ID NO:35;

[0068] The detection probe corresponding to Chr19:58951402-58951524 is SEQ ID NO:35;

[0069] The detection probe corresponding to Chr19:58951554-58951637 is SEQ ID NO:38;

[0070] The detection probe corresponding to Chr19:58951672-58952006 is SEQ ID NO:43;

[0071] The detection probe corresponding to Chr19:58951724-58951825 is SEQ ID NO:43;

[0072] VWC2 gene:

[0073] The detection probe corresponding to Chr7:49812992-49813086 is SEQ ID NO:46;

[0074] The detection probe corresponding to Chr7:49813051-49813343 is SEQ ID NO:51;

[0075] The detection probe corresponding to Chr7:49813066-49813161 is SEQ ID NO:51;

[0076] The detection probe corresponding to Chr7:49813455-49813811 is SEQ ID NO:56;

[0077] The detection probe corresponding to Chr7:49813387-49813537 is SEQ ID NO:56;

[0078] The detection probe corresponding to Chr7:49814721-49815073 is SEQ ID NO:61;

[0079] The detection probe corresponding to Chr7:49814765-49814936 is SEQ ID NO:61;

[0080] The detection probe corresponding to Chr7:49815144-49815236 is SEQ ID NO:64;

[0081] The detection probe corresponding to Chr7:49815290-49815635 is SEQ ID NO:69;

[0082] The detection probe corresponding to Chr7:49815340-49815439 is SEQ ID NO:69.

[0083] 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.

[0084] The reagent includes at least one of an antibody, a probe, a primer and a mass spectrometry detection reagent specifically for the target gene detection. Specifically, the reagent includes a product for specific detection of the target gene, and the product can be at least any one of an antibody, a probe, a primer and a mass spectrometry detection reagent; wherein the primer includes the detection primer described in claim 3, and can also be other reagents with similar functions. In addition, the kit can be in the form of some kits similar to existing products, and the equipment can be some sequence detection equipment. Both the primer and the probe can be selected from any one of them, and can also be used in combination as needed.

[0085] More specifically, the primers include at least one of the above-mentioned detection primers for endometrial cancer detection.

[0086] More specifically, the probe includes at least one of the above-mentioned detection probes for early screening and diagnosis of endometrial cancer.

[0087] Furthermore, the test sample of the test kit or device can be any body fluid, 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.

[0088] 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.

[0089] The reagents include at least one of antibodies, probes, primers and mass spectrometry detection reagents specifically for the target gene detection, wherein the primers include at least one of the detection primers for endometrial cancer detection. The probes include at least one of the detection probes for early screening and diagnosis of endometrial cancer.

[0090] Furthermore, the above 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.

[0091] It should be noted that the detection methods for gene methylation are well known to those skilled in the art, including but not limited to the methods listed above, etc., which can detect the methylation level of the target gene. Based on this, the reagents suitable for the above methods can be prepared into a kit for diagnosing endometrial cancer.

[0092] 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, and the nucleotide sequence of the primer pair for the internal reference gene is as follows, and the nucleotide sequence of the probe is as follows:

[0093] Forward primer: 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' (SEQ ID NO: 70)

[0094] Reverse primer: 5'-AACCAATAAAACCTACTCCTCCCTTAA-3' (SEQ ID NO: 71)

[0095] Probe: 5'-ACCACCACCCAACACACAATAACAAACACA-3' (SEQ ID NO: 72).

[0096] 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 quenching 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.

[0097] Furthermore, the kit also includes a negative quality control, a positive quality control and a template-free control. The template-free control is ddH 2 O. The positive quality control product is a plasmid DNA with full methylation in the target region. The negative quality control product is a plasmid DNA with completely non-methylation in the target region.

[0098] The present invention also proposes the use of the above gene markers in the preparation of endometrial cancer diagnosis products.

[0099] The present invention also proposes the use of the above-mentioned kit in preparing endometrial cancer diagnosis products.

[0100] 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.

[0101] The present invention obtains endometrial cancer-specific methylation early screening markers by screening, including at least one gene among VSX1, SYT1, ZNF132, and VWC2. By detecting the methylation levels of these genes, endometrial cancer and non-cancer individuals can be significantly distinguished; by detecting at least one methylation region corresponding to any at least one target gene in the sample in vitro, endometrial cancer can be diagnosed. The gene markers of the present invention can better distinguish endometrial cancer from non-cancer individuals in samples such as tissue / urine / swabs / swabs / blood / lavage fluids, and a single indicator can achieve a better detection effect, and the combination of multiple indicators can further improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0102] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0103] Figure 1 The figure is a schematic diagram of the process of endometrial cancer gene marker screening and system construction of the present invention.

[0104] Figure 2-Figure 3 This is a graph of the methylation level of the gene marker of the present invention detected by real-time fluorescence quantitative PCR amplification.

[0105] Figure 4 It is the M-index distribution diagram of the methylation level of the four gene markers of the present invention in urine samples.

[0106] Figure 5 It is the M-index distribution diagram of the methylation levels of the four gene markers of the present invention in urine samples from multiple centers.

[0107] Figure 6 It is the M-index distribution diagram of the methylation levels of the four gene markers of the present invention in patients with endometrial cancer and benign gynecological diseases in urine samples from multiple centers.

[0108] Figure 7 It is the M-index distribution diagram of the methylation levels of the four gene markers of the present invention in the cervical swab samples.

[0109] Figure 8 It is the M-index distribution diagram of the methylation level of the four gene markers of the present invention in the vaginal swab sample. DETAILED DESCRIPTION

[0110] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. The materials involved in the following examples can be obtained from commercial channels unless otherwise specified. The methods described are conventional methods unless otherwise specified.

[0111] The present invention uses the 450k open data of endometrial cancer in the Cancer Genome Atlas database (TCGA) (http: / / cancergenome.nih.gov / ), and finds methylation sites that are strongly correlated with the occurrence of endometrial cancer based on data modeling analysis, and screens multiple regions of 4 genes with the potential to be used as DNA methylation biomarkers for detecting endometrial cancer, including VSX1, VWC2, ZNF132 and SYT1. The detection method of the present invention is used to detect the above methylation sites in cancer tissues and paracancerous tissues of endometrial cancer population, urine samples of endometrial cancer population (182 cases) and non-cancer population (334 cases), cervical swab samples of endometrial cancer population and healthy population, and vaginal swab samples of endometrial cancer population and healthy population. The primer probe combination detection based on multiple regions of the above 4 genes shows that the methylation signal of the cancer tissue / urine / cervical brush / vaginal swab sample DNA of endometrial cancer individuals is significantly higher than that of non-cancer populations, indicating that the methylation degree of these methylation sites can reflect the occurrence of endometrial cancer more sensitively and specifically. 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 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, and has a better effect in distinguishing endometrial cancer and 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 of patients and increase the compliance of patient detection. Therefore, the methylation markers of the 4 genes of the present invention can be used as non-invasive detection markers for early diagnosis of endometrial cancer.

[0112] Example 1: Screening of methylation gene markers for endometrial cancer

[0113] In order to screen out DNA methylation markers that can characteristically distinguish endometrial cancer patients from non-cancer groups, the present invention first obtained 450K methylation chip data of 425 cancer tissues and 34 paracancerous tissues of endometrial cancer from the TCGA public database, and screened for sites with significant high methylation in endometrial cancer tissues. After data quality control, 385,577 methylation sites were included in the analysis. The CHAMP.DMP method was further used to screen differential sites, with the FDR-corrected P value <0.05 and the average methylation rate of cancer tissue-average methylation rate of paracancerous tissue>0.35 as the screening criteria, and a total of 8730 differential sites were screened.

[0114] In addition to collecting tumor-derived cells or DNA, sampling methods such as urine, cervical swabs, vaginal swabs, and blood will 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 consideration, the present invention further incorporates 78 cases of urinary tract cancer paracancerous tissues and 338 cases of white blood cell 450k methylation chip data from females in the TCGA and GEO databases, and performs CHAMP.DMP difference tests with 425 cases of endometrial cancer tissue data. Sites that simultaneously met the following conditions were included in the screening scope: (1) P value after FDR correction of two difference tests < 0.05; (2) average methylation rate of cancer tissue minus average methylation rate of para-urinary tract cancer tissue > 0; (3) average methylation rate of cancer tissue minus average methylation rate of leukocytes > 0; (4) average methylation rate of para-urinary tract cancer tissue < 0.1; (5) average methylation rate of leukocytes < 0.1. The sites that met the above requirements were intersected with the differential sites of endometrial cancer / para-cancer differential screening, and a total of 1464 sites were included in the next screening process.

[0115] In order to further evaluate the methylation levels of the above 1464 candidate sites in actual samples, the present invention performed Illumina Epic850k chip detection 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, which retains most of the 450K chip sites and adds nearly 400,000 new methylation sites. Finally, the present invention screened out 4 genes including VSX1, SYT1, ZNF132 and VWC2 as detection genes for the diagnosis of endometrial cancer. See the screening process. Figure 1 The methylation sites of the above genes with the best diagnostic effect on endometrial cancer and their effect information are shown in Table 1.

[0116] The areas under the curve of the best sites of the four genes VSX1, SYT1, ZNF132 and VWC2 were between 0.906 and 0.972. Among them, the AUC of VWC2 and ZNF132 was greater than 0.95, showing excellent diagnostic efficacy; the AUC of VSX1 and SYT1 was above 0.90, also showing good diagnostic efficacy. Different genes showed different advantages in detection sensitivity and specificity, among which the specificity of VWC2 and ZNF132 genes could reach 100%, and the specificity of VSX1 gene could reach 91.7%. The above genes showed excellent discrimination ability between endometrial cancer and controls. In addition, the sites with strong discrimination ability appeared in clusters around the optimal site. Within 500bp upstream and downstream of the optimal site, the above genes had multiple sites with AUC greater than 0.8, among which VWC2, ZNF132 and VWC2 genes had 8, 7, 5 and 2 sites with AUC higher than 0.8, respectively. The clustering of multiple sites further demonstrates the certainty of the above gene methylation as a diagnostic marker for endometrial cancer and is conducive to the development of methylation site detection methods. The above results show that the four genes selected by the present invention have excellent discrimination ability between endometrial cancer and control populations.

[0117] Table 1

[0118] Gene A B(hg19) Sensitivity (%) Specificity (%) AUC C VWC2 cg18206027 CHR7:49813486 83.3 100.0 0.972 8 ZNF132 cg24366702 CHR19:58951778 83.3 100.0 0.950 7 VSX1 cg14763548 CHR20:25062447 91.7 80.0 0.911 5 SYT1 cg03205584 CHR12:79258407 75.0 93.3 0.906 2

[0119] 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.

[0120] Example 2: Construction of endometrial cancer detection method system

[0121] 1. Selection of detection region: The sequence of the region where the methylation site is located for the diagnosis of endometrial cancer is shown in Table 2:

[0122] Table 2

[0123]

[0124]

[0125]

[0126] Note: * indicates the preferred area in this region.

[0127] Table 2 shows the specific primers and probe positions of the amplified region and the methylation sites they detect. Each set of primers and probes covers 7-10 methylation sites. While the detection scheme covers densely methylated sites with probes, the primers are designed near the 3' end to be located at high-discrimination methylation sites, further improving the detection accuracy of the detection system.

[0128] 2. Design and selection of primer and probe sequences

[0129] The sequences of the above four gene regions after bisulfite treatment were used as templates to analyze a series of parameters such as the length of the amplified product, annealing temperature, primer dimer, hairpin structure, specificity, etc. in different detection regions, and the primer probe combinations for different regions were designed and screened. The sequences and their numbers are shown in Table 3. The designed primer 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.

[0130] Table 3

[0131]

[0132]

[0133]

[0134] Wherein, both ends of the above-mentioned probe sequence are labeled with modification groups, including a 5' group and a 3' group, wherein the 5' group is selected from any one of FAM, VIC, HEX, NED, ROX, TET, JOE, TAMRA, CY3, and CY5. In this embodiment, CY5 is selected as the target gene, and FAM is selected 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 embodiment, BHQ-2 is selected as the target gene, and BHQ-1 is selected as the internal reference gene.

[0135] 3. The components of the sample detection kit are as follows: PCR reaction solution: Universal Probe qPCR Master Mix (NEB, catalog number: M3004E); primer-probe mixture; positive control: synthetic target gene methylated plasmid fragment (Qingke); negative control: synthetic target gene non-methylated plasmid fragment (Qingke); blank control: enzyme-free water.

[0136] 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 sample DNA methylation. Before detecting the methylation signal, DNA extraction of various samples and bisulfite conversion of the extracted DNA are required for subsequent fluorescent quantitative PCR detection.

[0137] 4.1 Sample collection

[0138] 4.1.1 Tissue sample collection: After obtaining informed consent from patients undergoing total hysterectomy and bilateral oophorectomy, a mung bean-sized sample of postoperative gross cancer lesion tissue was taken as the cancer tissue sample, and tissue within 3 cm from the edge of the cancer lesion was taken as the paracancerous tissue sample. After sampling, the samples were placed in tissue cryopreservation tubes and frozen in a -80°C refrigerator;

[0139] 4.1.2 Urine sample collection: Instruct the subject to use a 50 mL urine cup to collect the first urine of the morning urination, and then pour the urine into a urine collection tube pre-added with Urine Conditioning Buffer (zymo, catalog number: D3061-1-140), and collect 40-50 mL of urine for testing;

[0140] 4.1.3 Cervical brush sample collection: Medical personnel who have received unified training will insert the cervical brush into the cervix with the help of a vaginal speculum, rotate it clockwise for 5 circles to collect cervical cells, and then place the cervical brush into a collection bottle of exfoliated cells with 2 mL of PBS added, and store and transport it at low temperature;

[0141] 4.1.4 Vaginal swab sample collection: Medical personnel who have received uniform training use a sterile swab to rotate and collect samples at 1 / 3 of the vaginal side wall, and the swab with secretions attached should be clearly visible. The swab with secretions attached should be placed in an exfoliated cell collection tube that has been added with 2 mL of PBS and stored and transported at low temperature.

[0142] 4.2 Sample DNA extraction

[0143] 4.2.1 Tissue / brush / swab samples: Blood / cell / tissue genomic DNA extraction kit (TIANGEN Biochemical (TIANGEN), catalog number: DP304-03) was used as tissue / brush / swab genomic DNA extraction reagent for extraction.

[0144] 4.2.2 Urine samples: using Quick-DNA TM Urine Kit (zymo, catalog number: D3061) was used to extract DNA from urine samples.

[0145] 4.3 Bisulfite conversion of sample DNA

[0146] The DNA bisulfite conversion kit was purchased from Zymo Company. The extracted DNA was subjected to bisulfite conversion according to the kit instructions. The unmethylated cytosine (C) in the DNA was converted to uracil (U), while the methylated cytosine (C) remained unchanged, thus obtaining the converted bis-DNA.

[0147] 4.4Bis-DNA fluorescence quantitative PCR amplification

[0148] Use different primer and probe combinations in Table 2 to perform amplification detection on the corresponding different regions. The details are as follows:

[0149] 4.4.1 Prepare PCR reaction solution and primer-probe mixture: 2×EpiTect MethyLight Master Mix 5μL; forward detection primer F 0.3-0.5μmol; reverse detection primer R 0.3-0.5μmol; detection probe P 0.1-0.3μmol; sample DNA <100ng; add purified water to 10μL.

[0150] 4.4.2 Sample loading: Dispense the prepared mixed solution into 96-well plate / 384-well plate, 8μL per well, and then add Bis-DNA into the sample wells, 2μL per sample well, and 1 replicate well for each sample. At the same time, add 3 quality control samples: positive quality control, negative quality control, and no template control (NTC), and the sample loading method is the same as above.

[0151] 4.4.3 Fluorescence quantitative PCR amplification detection: The sample was tested using the fluorescence quantitative PCR instrument ROCHE 480, and 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).

[0152] 4.4.4 Signal collection: collect FAM and CY5 signals at 55-65°C.

[0153] 5. Interpretation of test results

[0154] 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 result is considered valid.

[0155] 5.2 Evaluation of sample test results

[0156] 5.2.1 The result is valid if the internal standard channel has an S-shaped amplification curve and the Ct value is ≤30;

[0157] 5.2.2 The unamplified Ct value was assigned as 50.

[0158] 5.2. The methylation levels of the 34 genes are expressed by M-index = 2^(-ΔCt) values:

[0159] M-index(VSX1)=2^(Ct(ACTB)-Ct(VSX1));

[0160] M-index(SYT1)=2^(Ct(ACTB)-Ct(SYT1));

[0161] M-index(ZNF132)=2^(Ct(ACTB)-Ct(ZNF132));

[0162] M-index(VWC2)=2^(Ct(ACTB)-Ct(VWC2)).

[0163] 6. Testing system evaluation

[0164] 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 a 2 -10 7 Standards per copy / well. By testing the Ct values ​​of a series of standards in each reaction plate at each test, a standard curve can be constructed. By comparing the standard curves of different test plates, the stability of the test can be evaluated.

[0165] 7. Evaluation of detection effect

[0166] After PCR detection, the Ct value of the methylation sites of 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 of each sample 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 index.

[0167] Example 3: Detection of tissue samples by primer-probe combination

[0168] To further optimize the primer-probe combination in Example 2, clinicians took 8 pairs of cancerous tissues and adjacent tissues from postoperative gross specimens of endometrial cancer patients for testing. After the tissue specimens were extracted and transformed according to the method described in Example 2, the M-index (i.e., 2^(-ΔCt)) of different genes in each sample was obtained. The test results are as follows: Figure 2-Figure 3As shown in the figure, compared with adjacent tissues, the methylation levels of VSX1, SYT1, ZNF132, and VWC2 gene detection sites in cancer tissue samples of endometrial cancer patients were significantly higher than those in adjacent tissues, with significant differences.

[0169] Example 4: Detection of exfoliated cell samples using primer-probe combinations

[0170] In this embodiment, 32 exfoliated cell samples of endometrial cancer and 33 non-cancer controls were collected for detection. The results showed that the AUC of each primer probe combination in distinguishing endometrial cancer and control populations was greater than 0.7, showing a better diagnostic effect, and the results are shown in Table 4. Among them, the AUC of the detection areas such as 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 can reach more than 0.8, proving that multiple regions of the above genes have diagnostic value as diagnostic markers for endometrial cancer.

[0171] Table 4

[0172]

[0173] 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.

[0174] Example 5: Testing urine samples

[0175] A total of 133 urine samples were collected at the Cancer Center of Sun Yat-sen University, including 65 preoperative urine samples from patients with histopathologically confirmed endometrial cancer, 16 preoperative urine samples from patients with benign gynecological diseases (including uterine fibroids and ovarian chocolate cysts), and 52 urine samples from healthy volunteers (no abnormalities were found in physical examinations in the past year). The urine samples were divided into a case group (65 endometrial cancer patients) and a control group (68 patients with benign gynecological diseases and healthy volunteers) according to whether the sample subjects had endometrial cancer. After the urine samples were extracted, transformed and tested according to the method described in Example 2, the M-index of different genes in each sample was obtained. The test results are shown in the following table. Figure 4As shown in the scatter plot, the gene loci discovered by the present invention can also achieve the effect of distinguishing the case group and the control group in urine samples, 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.

[0176] Through further modeling analysis, the detection effect of the single gene methylation site model is shown in the following table.

[0177] Table 5

[0178] Single gene index AUC Sensitivity (%) Specificity (%) Youden Index VSX1(chr20:25062655-25062739) 0.858 81.5 77.9 0.594 VWC2(chr7:49815144-49815236) 0.827 70.8 98.5 0.693 ZNF132(chr19:58951554-58951637) 0.808 67.7 86.8 0.545 SYT1(chr12:79258392-79258498) 0.840 76.9 82.4 0.593

[0179] As shown in the table, the detection method constructed by the present invention has a good ability to distinguish the case group and the control group in urine samples. The AUC after the single gene index modeling is above 0.8. The VSX1 gene has the strongest comprehensive identification ability among the single gene indicators, and its AUC is 0.858. When the sensitivity is 81.5%, the corresponding specificity is 77.9%. Different genes show different differentiation advantages. The methylation site of the VSX1 gene has a higher sensitivity, and the corresponding sensitivity under its optimal cutoff value is as high as 81.5%, indicating that it can identify more cancer cases; while the methylation site of the VWC2 gene has a higher specificity, and the corresponding sensitivity under its optimal cutoff value is as high as 98.5%, which can identify more non-cancer patients. Further, the methylation indexes of the four genes are subjected to two / three / four index joint detection and analysis, and the results shown in Table 6 can be obtained.

[0180] Table 6

[0181]

[0182] As can be seen from the above table, in the 133 urine samples collected by the Cancer Prevention and Treatment Center of Sun Yat-sen University, 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 0.932 (combined with VSX1+VWC2+SYT1 indicators). When only two indicators are combined, the AUC of the combination of VSX1 and VWC2 can reach 0.928, with a sensitivity of 84.6% and a specificity of 88.2%. The sensitivity of the model combined with VWC2 and SYT1 can reach 89.2%; the specificity of the model combined with VWC2 and ZNF132 can reach 98.5%. The above results show that there is a certain complementary effect between the indicators. The combination of multiple indicators can complement the advantages of each indicator, and the performance of the multi-indicator joint model is better than that of the single indicator model.

[0183] Example 6: Collecting urine samples from multiple centers for testing

[0184] In order to evaluate the performance stability of the method constructed by the present invention for urine sample detection, a total of 336 urine samples from endometrial cancer patients and non-cancer individuals were collected for detection in other centers: Guangzhou Medical University Affiliated Tumor Hospital and Foshan First People's Hospital, 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 detection results are shown in the figure. Figure 5 As shown in the scatter plot, the gene methylation index discovered by the present invention can also achieve the effect of distinguishing the case group and the control group in urine samples collected from multiple centers, 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.

[0185] As shown in Table 7, the detection method constructed by the present invention still has a good ability to distinguish the case group and the control group in the urine samples collected from multiple centers. The AUC after the single gene index modeling is between 0.779-0.879. Different genes show different advantages in differentiation. The ZNF132 index has a good ability to distinguish in the urine samples collected from multiple centers, and its AUC is 0.879. When the specificity is 94.7%, the corresponding sensitivity is 64.5%. The methylation index of the VSX1 gene still has a high sensitivity, and the corresponding sensitivity is as high as 81.7% when its specificity is 78.6%, indicating that it can identify more cancer cases. In summary, the single methylation marker index 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 are subjected to a two / three / four-indicator joint analysis, and the results shown in Table 8 are obtained.

[0186] Table 7

[0187] Single gene index AUC Sensitivity (%) Specificity (%) Youden Index VSX1(chr20:25062655-25062739) 0.862 81.7 78.6 0.603 VWC2(chr7:49815144-49815236) 0.779 48.4 99.6 0.480 ZNF132(chr19:58951554-58951637) 0.879 64.5 94.7 0.592 SYT1(chr12:79258392-79258498) 0.863 78.5 83.1 0.616

[0188] Table 8

[0189]

[0190]

[0191] As can be seen from the table above, in the 336 urine samples collected from multiple centers, the model constructed by combining 2-4 gene methylation indicators has improved the ability to distinguish endometrial cancer cases and controls. The AUC of the multi-indicator combined model is above 0.8 except for the VWC2+SYT1 model combination. The highest AUC is 0.893, which is the VSX1+ZNF132+SYT1 three-indicator combined model. The specificity of this model is 79.8% when the sensitivity is 80.6%. The above results show that in the urine samples collected from multiple centers, the above indicators have a certain complementary effect, and the combination of multiple indicators can further improve the detection efficiency of endometrial cancer.

[0192] Example 7: Differentiation of endometrial cancer from benign diseases

[0193] In actual clinical work, patients with endometrial cancer usually have symptoms of abnormal vaginal bleeding in the early stage, 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 of 158 patients with endometrial cancer from multiple centers as the case group and urine samples of 236 patients with benign gynecological diseases as the control group, the specific conditions of benign gynecological diseases are: 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.).

[0194] 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 detection results are as follows: Figure 6 As shown in the scatter plot, the gene methylation index found by the present invention has obvious differences in patients with endometrial cancer and benign gynecological diseases, 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.

[0195] As shown in Table 9, the detection method constructed by the present invention has a good ability to distinguish between patients with endometrial cancer and patients with benign diseases in urine samples collected from multiple centers. The AUC of the single gene indicator model is between 0.831-0.916. The VSX1 indicator has a good ability to distinguish between patients with endometrial cancer and patients with benign gynecological diseases, with an AUC of 0.916 and a corresponding sensitivity of 88.0% when the specificity is 82.2%. The methylation index of the VWC2 gene still has a high specificity, with a corresponding sensitivity of 63.9% when its specificity is 97.5%, and an AUC of 0.831. In summary, the single methylation marker discovered by the present invention can well distinguish patients with endometrial cancer from patients with other benign gynecological diseases in urine samples collected from multiple centers. Further, the methylation indicators of the four genes were subjected to a two / three / four-indicator joint analysis, and the results shown in Table 10 were obtained.

[0196] Table 9

[0197] Single gene index AUC Sensitivity (%) Specificity (%) Youden Index VSX1(chr20:25062655-25062739) 0.916 88.0 82.2 0.702 VWC2(chr7:49815144-49815236) 0.831 63.9 97.5 0.614 ZNF132(chr19:58951554-58951637) 0.885 73.4 90.7 0.641 SYT1(chr12:79258392-79258498) 0.880 69.0 94.1 0.631

[0198] Table 10

[0199]

[0200]

[0201] As shown in the table above, in the 394 urine samples collected from multiple centers, the model constructed by combining 2-4 gene methylation indicators has improved the ability to distinguish endometrial cancer cases from benign gynecological diseases. The AUC of the model after multi-indicator combination can reach up to 0.927. The AUC in the VSX1+ZNF132 two-indicator joint model and the VSX1+ZNF132+SYT1 three-indicator joint model are both 0.927, and the corresponding specificity is 81.0% when the specificity is 90.7%. The above results show that in the urine samples collected from multiple centers, the above indicators have a certain complementary effect, and the combination of multiple indicators can further improve the efficiency of distinguishing endometrial cancer from benign gynecological diseases.

[0202] 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 reflected. 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, and 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 detect endometrial cancer in urine samples with high accuracy.

[0203] Example 8: Testing cervical swab samples

[0204] Furthermore, in order to evaluate the detection efficiency of the gene methylation markers discovered by the present invention in cervical swab samples, cervical swab samples of 17 patients with endometrial cancer confirmed 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 detection, the M-index of different genes in each sample was obtained, and the detection results are shown in Figure 7 As shown in the scatter plot, in the swipe samples, the gene methylation index found 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 by the present invention has a good ability to distinguish endometrial cancer patients and healthy volunteers in the swipe samples. The AUC of the single gene indicator model is between 0.718-0.891, and the ZNF132 indicator has a good ability to distinguish, with an AUC of 0.891, and a corresponding sensitivity of 76.5% when the specificity is 92.9%. The detection effect of the VWC2 gene is second only to the ZNF132 gene, with an AUC of 0.882, and a corresponding sensitivity of 88.2% when the specificity is 85.7%. SYT1 has a high specificity of 92.9%.

[0205] Table 11

[0206] Single gene index AUC Sensitivity (%) Specificity (%) Youden Index VSX1(chr20:25062655-25062739) 0.807 82.4 71.4 0.538 VWC2(chr7:49815144-49815236) 0.882 88.2 85.7 0.739 ZNF132(chr19:58951554-58951637) 0.891 76.5 92.9 0.694 SYT1(chr12:79258392-79258498) 0.718 52.9 92.9 0.458

[0207] Furthermore, the methylation indices of the four genes were subjected to two / three / four-indicator combined analysis, and the results shown in the following table were obtained.

[0208] Table 12

[0209]

[0210] It can be seen from the above table that 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 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.

[0211] Example 9: Testing vaginal swab samples

[0212] 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 patients with endometrial cancer were defined as the case group, and the swab samples of 14 healthy volunteers were defined as the control group. The specific detection method was as described in Example 2, and the M-index of different genes of each sample was obtained after the detection. The detection results are shown in Figure 8 As shown in the scatter plot, in the swab samples, the gene methylation index found 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 index model is between 0.769-0.853, and the ZNF132 index has a good ability to distinguish, with an AUC of 0.853, and a corresponding sensitivity of 64.7% when the specificity is 100%. The detection effect of the VWC2 gene is second only to the ZNF132 gene, with an AUC of 0.849, and a corresponding sensitivity of 64.7% when the specificity is 100%. Further, the methylation indexes of the four genes are subjected to a two / three / four-index joint analysis, and the results shown in Table 14 below can be obtained.

[0213] Table 13

[0214] Single gene index AUC Sensitivity (%) Specificity (%) Youden Index VSX1(chr20:25062655-25062739) 0.790 76.5 85.7 0.622 VWC2(chr7:49815144-49815236) 0.849 64.7 100.0 0.647 ZNF132(chr19:58951554-58951637) 0.853 64.7 100.0 0.647 SYT1(chr12:79258392-79258498) 0.769 76.5 64.3 0.408

[0215] Table 14

[0216]

[0217] As can be seen from the table above, in 31 swab samples, the model constructed by combining 2-4 gene methylation indicators has greatly improved the ability to distinguish endometrial cancer cases and controls. The AUC of the model after multi-indicator combination can be as high as 0.966 (combined with VSX1+ZNF132+SYT1 indicators and four indicators combined), and the corresponding sensitivity is 88.2% when the model specificity is 100%. The above results show that there is a certain complementary effect between the indicators in the swab samples. The combination of multiple indicators can complement the advantages of each indicator, and the performance of the multi-indicator joint model is better than that of the single indicator model.

[0218] In summary, the gene methylation markers discovered in the present invention can well distinguish endometrial cancer from non-cancerous individuals in tissue / urine / swab samples. A single indicator can achieve a good detection effect, and the combination of multiple indicators can further improve the detection efficiency.

[0219] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. 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 VWC2, or a combination of a methylated nucleic acid sequence in at least one target region of VWC2 and a methylated nucleic acid sequence in at least one target region of at least one gene of ZNF132 and SYT1, wherein the target region is selected from at least one of the following methylation regions: 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; The genes are based on the human genome version GRCh37 (hg19) as the reference genome.

2. A detection primer and probe combination for early screening and diagnosis of endometrial cancer, characterized in that For detecting the methylation status of the methylation gene marker according to claim 1, the nucleotide sequence of the probe is as follows: 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; The nucleotide sequence of the detection primer is as follows: 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.

3. Use of a reagent for detecting the methylated gene marker according to claim 1 in preparing a detection kit or device, characterized in that The detection kit or device is used for detecting, screening or diagnosing endometrial cancer.

4. The use according to claim 3, characterized in that: The reagent includes a probe primer specifically for detecting the methylation gene marker; wherein the probe primer is as described in claim 2.

5. A kit for diagnosing endometrial cancer, characterized in that It comprises a reagent for detecting the methylation gene marker according to claim 1.

6. The kit according to claim 5, characterized in that The reagents include probe primers specifically for detecting the target gene; wherein the probe primers are as described in claim 2.

7. The kit according to claim 6, 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 as follows, and the nucleotide sequence of the probe is as follows: Forward primer: 5'-TGGTGATGGAGGAGGTTTAGTAAGT-3' Reverse primer: 5'-AACCAATAAAACCTACTCCTCCCTTAA-3' Probe: 5′-ACCACCACCCAACACACAATAACAAACACA-3′.

8. Use of the methylation gene marker according to claim 1, or the detection primer and probe combination according to claim 2, or the kit according to any one of claims 5 to 7 in the preparation of a diagnostic product for endometrial cancer.

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