Use of a reagent for detecting the methylation level of a target region in the preparation of a diagnostic product for endometrial cancer

Abstract

The present invention relates to the use of a reagent for detecting the methylation level of a target region in the preparation of a diagnostic product for endometrial cancer; the target region includes region 1 or a partial region of region 1, and / or region 2 or a partial region of region 2; with GRCh38.p14 as the reference, region 1 is the positive strand of Chr6:27679636-27680274, and region 2 is the positive strand of Chr6:10390489-10390886. In this application, these regions are used as the target regions, and by detecting the methylation level of these target regions or their partial regions, healthy people, patients with benign endometrial lesions, and patients with endometrial cancer can be effectively distinguished. Moreover, the detection rate of this application for other uterine malignancies (such as cervical cancer) is not high. It is applicable to cervical exfoliated cell samples, can achieve simple, rapid, minimally invasive detection of endometrial cancer, with high sensitivity and strong specificity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more particularly, to the use of a reagent for detecting the methylation level of a target region in the preparation of a diagnostic product for endometrial cancer. Background Art

[0002] Endometrial cancer is one of the most common gynecological malignancies. In recent years, the incidence rate of endometrial cancer has been showing an increasing trend globally year by year, and the age of patients is getting younger and younger. According to statistics, in 2018, there were more than 380,000 new cases of endometrial cancer globally and 89,929 death cases. The increase in the incidence rate of endometrial cancer is partly attributed to the rising prevalence of risk factors related to its occurrence and development, such as obesity, diabetes, hypertension, etc.

[0003] Endometrial cancer is usually divided into two types, namely type I and type II. Type I is the most common form, and the proportion of type I cancer patients among all endometrial cancer patients is about 70%. The occurrence of type I endometrial cancer is related to the continuous stimulation of estrogen without progesterone antagonism. Type I cancer is also called endometrial adenocarcinoma, and this type of cancer is usually low-grade. Type II cancer is usually high-grade, mainly including papillary serous carcinoma and clear cell carcinoma. The prognosis of type II cancer is poor, and the risks of recurrence and metastasis are relatively high. The proportion of type II endometrial cancer patients among all endometrial cancer patients is about 10%, but type II cancer is related to 40% of the endometrial cancer death cases. Endometrial hyperplasia is a precancerous lesion of endometrial cancer. 1% - 3% of endometrial hyperplasia patients have the risk of canceration. Among them, atypical hyperplasia patients have a higher risk of canceration than simple hyperplasia or complex hyperplasia patients. About 30% - 40% of atypical endometrial hyperplasia patients are accompanied by endometrial adenocarcinoma.

[0004] Generally, patients with endometrial cancer will present abnormal vaginal bleeding symptoms before diagnosis, which provides a valuable opportunity for the early diagnosis and curative treatment of the cancer. However, many gynecological diseases, such as vaginitis, pelvic inflammatory disease, endometrial polyps, uterine fibroids, adenomyosis, endometrial atrophy, etc., can also cause abnormal vaginal bleeding. Therefore, during the diagnostic process before diagnosis, these patients need to undergo more examinations and bear great psychological pressure. The commonly used method for diagnosing endometrial cancer is vaginal ultrasound, but under the conditions of adenomyosis, multiple uterine fibroids, endometrial polyps or previous uterine surgeries in patients, the performance of vaginal ultrasound diagnosis will be greatly reduced, that is, conventional imaging methods are difficult to distinguish between benign uterine diseases and endometrial cancer. At this time, endometrial biopsy is usually performed on patients to determine whether they are endometrial cancer patients, but endometrial biopsy is greatly limited by sampling and is an invasive operation.

[0005] Therefore, it is necessary to develop a molecular diagnostic reagent that can effectively distinguish endometrial cancer patients from patients with benign uterine lesions, so as to properly diagnose patients with abnormal vaginal bleeding, relieve the psychological pressure of patients, and avoid over-treatment of patients with benign diseases.

[0006] In view of this, the present application is specifically proposed. Summary of the Invention

[0007] One of the purposes of the embodiments of the present application is to use a reagent for detecting the methylation level of a target region (Chr6: 27679636-27680274, Chr6: 10390489-10390886, or partial fragments thereof) to prepare an endometrial cancer diagnostic product, so as to effectively distinguish endometrial cancer patients from patients with benign uterine lesions and achieve minimally invasive diagnosis of endometrial cancer.

[0008] In the first aspect of the present application, there is provided an application of a reagent for detecting the methylation level of a target region in the preparation of an endometrial cancer diagnostic product;

[0009] The target region includes: region 1 or a partial region of region 1, and / or, region 2 or a partial region of region 2;

[0010] Taking GRCh38.p14 as a reference,

[0011] Region 1 is the positive strand of Chr6: 27679636-27680274,

[0012] Region 2 is the positive strand of Chr6: 10390489-10390886.

[0013] In some embodiments, the partial region of region 1 is selected from one or more of region 1-1, region 1-2, and region 1-3 defined as follows: region 1-1 is Chr6: 27679695-27679811, region 1-2 is Chr6: 27679919-27680065, and region 1-3 is Chr6: 27680099-27680254;

[0014] Or / and,

[0015] The partial region of region 2 is selected from one or more of region 2-1, region 2-2, and region 2-3 defined as follows: region 2-1 is Chr6: 10390551-10390673, region 2- is Chr6: 10390668-10390804, and region 2-3 is Chr6: 10390758-10390876.

[0016] In some of these embodiments, the reagent detects the methylation level of the target region by one or more of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome bisulfite sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve analysis, methylation-sensitive restriction enzyme method, and methylation-specific quantitative fluorescence PCR method.

[0017] In some of these embodiments, the reagent comprises a nucleic acid combination for detecting the methylation level of the target region.

[0018] In some of these embodiments, the nucleic acid combination detects the methylation level of the target region by the bisulfite sequencing method, and the nucleic acid combination comprises a primer pair for detecting the methylation level of the target region.

[0019] In some of these embodiments, the nucleic acid combination for detecting the methylation level of Region 1 comprises a primer pair with sequences shown in SEQ ID No.7 and SEQ ID No.8, and / or the nucleic acid combination for detecting the methylation level of Region 2 comprises a primer pair with sequences shown in SEQ ID No.9 and SEQ ID No.10.

[0020] In some of these embodiments, the nucleic acid combination detects the methylation level of the target region by the methylation-specific quantitative fluorescence PCR method, and the nucleic acid combination comprises a primer pair for detecting the methylation level of the target region and a detection probe.

[0021] In some of these embodiments, the nucleic acid combination for detecting the methylation level of region 1-1 includes a detection primer pair with sequences as shown in SEQ ID No.11 and SEQ ID No.12 and a detection probe with a sequence as shown in SEQ ID No.13; the nucleic acid combination for detecting the methylation level of region 1-2 includes a detection primer pair with sequences as shown in SEQ ID No.14 and SEQ ID No.15 and a detection probe with a sequence as shown in SEQ ID No.16; the nucleic acid combination for detecting the methylation level of region 1-3 includes a detection primer pair with sequences as shown in SEQ ID No.17 and SEQ ID No.18 and a detection probe with a sequence as shown in SEQ ID No.19; the nucleic acid combination for detecting the methylation level of region 2-1 includes a detection primer pair with sequences as shown in SEQ ID No.20 and SEQ ID No.21 and a detection probe with a sequence as shown in SEQ ID No.22; the nucleic acid combination for detecting the methylation level of region 2-2 includes a detection primer pair with sequences as shown in SEQ ID No.23 and SEQ ID No.24 and a detection probe with a sequence as shown in SEQ ID No.25; or / and, the nucleic acid combination for detecting the methylation level of region 2-3 includes a detection primer pair with sequences as shown in SEQ ID No.26 and SEQ ID No.27 and a detection probe with a sequence as shown in SEQ ID No.28.

[0022] In some of these embodiments, the sample types for detection include cell samples, blood samples or tissue samples.

[0023] In a second aspect of the present application, there is provided an endometrial cancer diagnostic kit, comprising the reagent defined in the first aspect.

[0024] In some of these embodiments, the diagnostic kit further includes one or more of sequencing reagents, amplification reagents, quality control products, reagents for converting unmethylated cytosine bases into uracil, DNA extraction reagents and DNA purification reagents.

[0025] In some of these embodiments, the amplification reagents include one or more of amplification buffers, dNTPs, DNA polymerases and Mg 2+ among others.

[0026] Compared with the prior art, the beneficial effects of the present application include:

[0027] The inventors of the present application found that the methylation status of the regions Chr6:27679636-27680274 and Chr6:10390489-10390886 in benign endometrial diseases and endometrial cancer is completely different. Based on this discovery, the inventors targeted these regions and effectively distinguished healthy people, patients with benign endometrial lesions, and patients with endometrial cancer by detecting the methylation level of these target regions or partial regions thereof. Moreover, the present application has a low detection rate for other uterine malignancies (such as cervical cancer), is applicable to cervical exfoliated cell samples, and can achieve simple, rapid, minimally invasive detection of endometrial cancer with high sensitivity and strong specificity. Detailed implementation manners

[0028] The present invention will be further described in detail below in conjunction with the implementation manners and examples. It should be understood that these implementation manners and examples are only used to illustrate the present invention and not to limit the scope of the present invention. The purpose of providing these implementation manners and examples is to make the understanding of the disclosed content of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the implementation manners and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention herein are only for the purpose of describing the implementation manners and examples and are not intended to limit the present invention.

[0030] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0031] As used in this application, the selection scope of the terms "and / or" and "or / and" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and / or" and "or / and", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, B, C, and / or D" includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0032] In this application, terms such as "multiple", "multiple kinds", "multiple times", "multiple elements", etc., unless otherwise specifically defined, refer to a quantity greater than 2 or equal to 2. For example, "one or more kinds" means one kind or more than or equal to two kinds. "Above" includes the base number. For example, "two or more" includes two, three, or more kinds.

[0033] In this application, "at least one" and "at least one kind" mean that it can be any one of the listed items, or a combination of any two or more of them.

[0034] In this application, "its combination", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or any two or more of the listed items.

[0035] In this application, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0036] In this application, "preferred", "better", "more preferable", "it is advisable" are only used to describe the implementation modes or embodiments with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0037] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only for descriptive purposes, and cannot be construed as indicating or implying relative importance or quantity, nor can it be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumerative description, and it should be understood that they do not constitute a closed limitation on quantity.

[0038] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0039] In this application, the term "diagnosis" includes aspects such as auxiliary diagnosis, recurrence risk assessment, canceration risk and canceration degree assessment, prognosis judgment, etc.

[0040] The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and usually more than ten. The exact size will depend on many factors, and these factors in turn depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced in any way, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. The typical deoxyribonucleotides of DNA are thymine, adenine, cytosine, and guanine. The typical ribonucleotides of RNA are uracil, adenine, cytosine, and guanine.

[0041] The term "methylation" is a form of DNA chemical modification that can change genetic expression without changing the DNA sequence. DNA methylation refers to the covalent binding of a methyl group to the 5th carbon position of cytosine in genomic CpG dinucleotides under the action of DNA methyltransferase. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way of DNA-protein interaction, thereby controlling gene expression.

[0042] The term "methylation level" refers to whether cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency / ratio / percentage of methylation, representing both qualitative and quantitative concepts. In practical applications, different detection indicators can be used to compare DNA methylation levels according to actual situations. For example, in some cases, comparison can be made based on the Ct values detected in samples; in some cases, the proportion of gene methylation in the sample can be calculated, that is, the number of methylated molecules / (the number of methylated molecules + the number of unmethylated molecules) × 100, and then comparison can be made; in some cases, statistical analysis and integration of various indicators are also required to obtain the final determination indicator. It can be understood that the target region of the gene to be detected in this article is a DNA sequence including at least one CpG dinucleotide (CG).

[0043] The term "primer" refers to an oligonucleotide that can be used in an amplification method (such as polymerase chain reaction PCR) to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a part of its region. Usually, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence-specific for the polynucleotide sequence. The exact length of the primer depends on many factors, including temperature, primer source, and the method used, etc. For example, for diagnostic and prognostic applications, depending on the complexity of the target sequence, oligonucleotide primers usually contain at least 10, 15, 20, 25 or more nucleotides, but can also contain fewer nucleotides. In the present disclosure, the term "primer" refers to a pair of primers that can hybridize with the double strand of a target DNA molecule or can hybridize with the regions flanking the nucleotide sequence to be amplified in the target DNA molecule.

[0044] The term "TaqMan probe" refers to an oligonucleotide sequence containing a 5' fluorescent group and a 3' quenching group. When the probe binds to the corresponding site on the DNA, the probe does not emit fluorescence because the quenching group is near the fluorescent group. During the amplification process, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of DNA polymerase (such as Taq enzyme) will digest the probe, and the fluorescent group will be far from the quenching group, and its energy will not be absorbed, that is, a fluorescent signal is generated. For each PCR cycle, the fluorescent signal, like the target fragment, has a synchronous exponential growth process.

[0045] DNA methylation is the transfer of a methyl group to the 5th carbon atom of the cytosine base under the action of DNA methyltransferase. DNA methylation in the promoter region of tumor suppressor genes is an important event in the carcinogenesis process. Abnormal DNA methylation usually occurs in the early stage of cancer and exists stably. Abnormal DNA methylation usually leads to the inactivation of tumor suppressor genes and the activation of oncogenes. In view of this, genes with methylation changes can be used as molecular markers for diagnosing canceration or precancerous lesions, having certain diagnostic value. In the process of determining the positivity or negativity of cancer, it is very crucial to screen for appropriate molecular markers and detect their methylation.

[0046] The first aspect of the present application

[0047] The application of a reagent for detecting the methylation level of a target region in the preparation of an endometrial cancer diagnostic product;

[0048] The target region includes: region 1 or a partial region of region 1, and / or, region 2 or a partial region of region 2;

[0049] Taking GRCh38.p14 as a reference,

[0050] Region 1 is the positive strand of Chr6:27679636 - 27680274,

[0051] Region 2 is the positive strand of Chr6:10390489 - 10390886.

[0052] Optionally, the target region is region 1 or a combination of region 1 and region 2. Further, the target region is a combination of region 1 and region 2, and the combined diagnosis of early endometrial cancer by region 1 and region 2 has high sensitivity.

[0053] Optionally, the partial region of region 1 is selected from one or more of the following defined regions 1 - 1, 1 - 2, and 1 - 3: region 1 - 1 is Chr6:27679695 - 27679811, region 1 - 2 is Chr6:27679919 - 27680065, and region 1 - 3 is Chr6:27680099 - 27680254; optionally, the partial region of region 1 is region 1 - 2 and region 1 - 3, and further, the partial region of region 1 is region 1 - 2;

[0054] or / and,

[0055] Part of the region 2 is selected from one or more of the following defined regions: region 2-1, region 2-2, and region 2-3. Region 2-1 is Chr6:10390551-10390673, region 2-2 is Chr6:10390668-10390804, and region 2-3 is Chr6:10390758-10390876. Optionally, part of the region 2 is region 2-2 and region 2-3. Further optionally, part of the region 2 is region 2-2.

[0056] Optionally, the target region is a combination of region 1-1 and region 2-1, a combination of region 1-1 and region 2-2, a combination of region 1-1 and region 2-3, a combination of region 1-2 and region 2-1, a combination of region 1-2 and region 2-2, a combination of region 1-2 and region 2-3, a combination of region 1-3 and region 2-1, a combination of region 1-3 and region 2-2, or a combination of region 1-3 and region 2-3. Optionally, the target region is a combination of region 1-2 and region 2-1, a combination of region 1-2 and region 2-2, a combination of region 1-2 and region 2-3, a combination of region 1-3 and region 2-2, or a combination of region 1-3 and region 2-3.

[0057] Optionally, the reagent detects the methylation level of the target region by one or more of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome bisulfite sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve analysis, methylation-sensitive restriction enzyme method, and methylation-specific quantitative fluorescence PCR.

[0058] Optionally, the reagent includes a nucleic acid combination for detecting the methylation level of the target region.

[0059] Optionally, the nucleic acid combination detects the methylation level of the target region by the bisulfite sequencing method, and the nucleic acid combination includes a pair of detection primers for detecting the methylation level of the target region.

[0060] Optionally, the nucleic acid combination for detecting the methylation level of region 1 includes a pair of detection primers with sequences as shown in SEQ ID No.7 and SEQ ID No.8, and / or the nucleic acid combination for detecting the methylation level of region 2 includes a pair of detection primers with sequences as shown in SEQ ID No.9 and SEQ ID No.10.

[0061] Optionally, the nucleic acid combination detects the methylation level of the target region by the methylation-specific fluorescence quantitative PCR method. The nucleic acid combination includes a detection primer pair for detecting the methylation level of the target region and a detection probe.

[0062] Optionally, the nucleic acid combination for detecting the methylation level of Region 1-1 includes a detection primer pair with sequences shown in SEQ ID No.11 and SEQ ID No.12 and a detection probe with a sequence shown in SEQ ID No.13; the nucleic acid combination for detecting the methylation level of Region 1-2 includes a detection primer pair with sequences shown in SEQ ID No.14 and SEQ ID No.15 and a detection probe with a sequence shown in SEQ ID No.16; the nucleic acid combination for detecting the methylation level of Region 1-3 includes a detection primer pair with sequences shown in SEQ ID No.17 and SEQ ID No.18 and a detection probe with a sequence shown in SEQ ID No.19; the nucleic acid combination for detecting the methylation level of Region 2-1 includes a detection primer pair with sequences shown in SEQ ID No.20 and SEQ ID No.21 and a detection probe with a sequence shown in SEQ ID No.22; the nucleic acid combination for detecting the methylation level of Region 2-2 includes a detection primer pair with sequences shown in SEQ ID No.23 and SEQ ID No.24 and a detection probe with a sequence shown in SEQ ID No.25; and / or the nucleic acid combination for detecting the methylation level of Region 2-3 includes a detection primer pair with sequences shown in SEQ ID No.26 and SEQ ID No.27 and a detection probe with a sequence shown in SEQ ID No.28.

[0063] Optionally, the sample types for detection include cell samples, blood samples, or tissue samples.

[0064] The second aspect of the present application

[0065] This application provides an endometrial cancer diagnostic kit, which includes the reagents defined in the first aspect.

[0066] Optionally, the diagnostic kit further includes one or more of sequencing reagents, amplification reagents, quality control products, reagents for converting unmethylated cytosine bases into uracil, DNA extraction reagents, and DNA purification reagents.

[0067] Optionally, the amplification reagents include one or more of amplification buffers, dNTPs, DNA polymerases, and Mg 2+ in them. Specific embodiments

[0069] The implementation scheme of the present invention will be described in detail below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, the guidance given in the present invention is preferentially referred to. It can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0070] In the following specific embodiments, for the measurement parameters of raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0071] Example 1: Detection of methylation level of target region in tissue samples by bisulfite sequencing

[0072] In this example, bisulfite sequencing was used to analyze the methylation status of target region 1 and region 2 in endometrial cancer tissue samples and endometrial benign disease tissue samples. It was found that the methylation status of the target region was completely different in the cases of endometrial benign diseases and cancers. Furthermore, it was considered that endometrial benign and malignant diseases could be distinguished according to the methylation status of target region 1 and target region 2 in the sample to be tested. The specific experimental process is as follows:

[0073] 1. Sample collection

[0074] In this example, cancer tissue samples and paired adjacent normal tissue samples of patients diagnosed with endometrial cancer by histopathological biopsy were collected, including 76 endometrial cancer tissue samples and 46 endometrial cancer adjacent normal tissue samples, and the patients had not undergone any cancer-related treatments before diagnosis; in addition, 98 endometrial tissue samples of patients diagnosed with benign diseases by histopathological biopsy were collected in this example. These benign diseases included: endometrial polyps, uterine fibroids, adenomyosis, etc. All tissue samples were formalin-fixed and paraffin-embedded tissue samples. The collection process of all tissue samples was approved by the ethics committee, all volunteers signed informed consent forms, and all tissue samples were anonymized.

[0075] 2. Extraction of template DNA in the sample

[0076] For tissue samples, QIAamp DNA FFPE Tissue Kit (56404) was used to extract DNA, and the specific operation was carried out according to the kit instructions.

[0077] 3. Bisulfite conversion

[0078] The extracted sample genomic DNA is subjected to bisulfite conversion and purification after conversion. The nucleic acid conversion kit used is the nucleic acid conversion reagent of Wuhan AmySen Life Science Co., Ltd. (E Han Medical Equipment Preparation 20200843). For specific experimental operations, refer to the kit instructions.

[0079] 4. PCR Amplification and Sequencing

[0080] Using Region 1 (Chr6: 27679636 - 27680274) and Region 2 (Chr6: 10390489 - 10390886) as target regions respectively, degenerate primer pairs are designed based on the converted DNA sequences. The degenerate primer pairs do not contain CG dinucleotide sites. Therefore, the bisulfite treatment does not affect the amplification of the template by the degenerate primer pairs, which can amplify using either methylated fragments or non-methylated fragments as templates. The original DNA sequences, fully methylated and bisulfite-converted sequences, and fully unmethylated and bisulfite-converted sequences of Region 1 and Region 2 are shown in Table 1.

[0081] The sequences of the degenerate primer pairs for Region 1 and Region 2 are shown in Table 2.

[0082] After artificially synthesizing the degenerate primer pairs, the PCR reaction system is configured according to the formula in Table 3. The PCR amplification program is shown in Table 4. After the PCR amplification is completed, Sanger sequencing is performed on the amplification products using the degenerate primer pairs. The sequencing is sent to the company for processing, and sequencing is performed from both the 5' end and the 3' end simultaneously.

[0083] Table 1. Nucleotide Sequences of Region 1 and Region 2

[0084]

[0085]

[0086] Table 2. Degenerate Primer Pairs for Amplifying Region 1 and Region 2

[0087]

[0088]

[0089] Table 3. PCR Reaction System with Degenerate Primers Added

[0090] Component Dosage (μL) <![CDATA[10×Taq buffer (Mg 2+ Free)]]> 5 <![CDATA[25mM Mg 2+ > 4 dNTP Mix (10 mM each) 1 Forward primer (10 μM) 1 Reverse primer (10 μM) 1 Hot start Taq DNA polymerase 0.5 Template DNA 10 Ultra-pure water Make up to 50

[0091] Table 4. PCR Reaction Program

[0092]

[0093] 5. Result Analysis

[0094] Analysis of methylation level:

[0095] Based on the sequencing peak map, the methylation status of differential CpG sites in each amplicon of each sample was analyzed. The methylation status of cytosine in a CpG dinucleotide is divided into two types, namely unmethylated and methylated, and the methylation is further divided into fully methylated and partially methylated. If the sequencing result of cytosine in a CpG dinucleotide is thymine, it is unmethylated. If the sequencing result of cytosine in a CpG dinucleotide is still cytosine, it is fully methylated. If the sequencing result of cytosine in a CpG dinucleotide has both cytosine and thymine (double peaks), it is partially methylated. If more than 95% of the cytosines in the CpG dinucleotides in an amplicon are methylated, then this sample is considered to be methylated positive in this gene region.

[0096] Sample determination criteria:

[0097] 1) Under the condition that region 1 is the target region, if the tissue sample to be tested is methylated positive in this region, then this tissue sample is a positive sample for endometrial cancer; if the tissue sample to be tested is methylated negative in region 1, then this tissue sample is a negative sample for endometrial cancer.

[0098] 2) Under the condition that region 2 is the target region, if the tissue sample to be tested is methylated positive in this region, then this tissue sample is a positive sample for endometrial cancer; if the tissue sample to be tested is methylated negative in region 2, then this sample is a negative sample for endometrial cancer.

[0099] 3) Under the condition that region 1 and region 2 are used as the target regions, if the tissue sample to be tested is methylated positive in at least one of region 1 and region 2, then this tissue sample is a positive sample for endometrial cancer; if the tissue sample to be tested is methylated negative in both region 1 and region 2, then this tissue sample is a negative sample for endometrial cancer.

[0100] Statistics were used to analyze the methylation levels of region 1 and region 2 by bisulfite sequencing method and then determine the sensitivity and specificity of various tissue samples, as shown in Table 5.

[0101] Sensitivity is the proportion of samples with positive pathological results that are determined to be positive by this method, and specificity is the proportion of samples with negative pathological results that are determined to be negative by this method.

[0102] Table 5. Performance of bisulfite sequencing method in analyzing the methylation levels of target regions and then determining various tissue samples

[0103]

[0104] According to statistics, the 5-year survival rate of patients with early-stage (stage I and II) endometrial cancer can reach 70%, while the 5-year survival rates of patients with stage III and IV endometrial cancer are 40% - 50% and 15% - 20% respectively. Therefore, it is necessary to diagnose and treat endometrial cancer patients at an early stage. As can be seen from Table 5, the performance of diagnosing whether the tissue sample to be tested is an endometrial cancer sample by analyzing the methylation levels of Region 1 and / or Region 2 through bisulfite sequencing is very good. Specifically, the sensitivity of Region 1 in diagnosing early endometrial cancer is 80.00%, the sensitivity of Region 2 in diagnosing early endometrial cancer is 68.89%, and the combined sensitivity of Region 1 and Region 2 in diagnosing early endometrial cancer is as high as 93.33%; the sensitivities of Region 1 and / or Region 2 in diagnosing mid- and late-stage endometrial cancer are all relatively high, ranging from 90.32% to 100%. In addition, the specificities of Region 1, Region 2, and Region 1 + Region 2 in diagnosing normal tissues adjacent to endometrial cancer are 91.30%, 95.65%, and 91.30% respectively.

[0105] Considering that the applicable population of this technical solution is pregnant women and menopausal women with abnormal vaginal bleeding, it is necessary to analyze the performance of this technical solution in differentiating benign uterine diseases and endometrial cancer. In Table 5, the specificities of separately detecting Region 1, separately detecting Region 2, and simultaneously detecting Region 1 + Region 2 in diagnosing benign uterine diseases such as endometrial polyps, uterine fibroids, and adenomyosis are 94.90%, 97.96%, and 92.86% respectively. It can be seen that the target detection region can effectively differentiate between benign and malignant uterine diseases.

[0106] Although the sensitivity of separately detecting Region 1 or Region 2 in diagnosing endometrial cancer is relatively high and the specificity is very good, simultaneously detecting Region 1 and Region 2 can significantly improve the detection rate of early endometrial cancer, which is more conducive to the early diagnosis and treatment of endometrial cancer. Considering comprehensively, the methylation levels of Region 1 and Region 2 can be analyzed simultaneously to diagnose endometrial cancer.

[0107] Example 2. Detection of the methylation level of the target region in tissue samples by methylation-specific fluorescence quantitative PCR

[0108] Considering clinical applications, in this example, the methylation levels of Region 1 and / or Region 2 were further detected by methylation-specific fluorescence quantitative PCR, and then the performance of this method in diagnosing endometrial cancer was analyzed. The specific experimental procedures are as follows:

[0109] 1. The collection of clinical samples, extraction, transformation, and purification of template DNA were the same as in Example 1.

[0110] 2. Methylation-specific fluorescence quantitative PCR reaction

[0111] The bisulfite-converted DNA was subjected to methylation fluorescence quantitative PCR reactions respectively to detect the methylation status of the target regions in each sample. To improve the amplification efficiency of PCR, Region 1 and Region 2 were each divided into 3 short fragment regions (not exceeding 160 bp), namely Region 1-1, Region 1-2, Region 1-3, Region 2-1, Region 2-2, and Region 2-3, and multiple pairs of methylation detection primer pairs and detection probes were designed for each short fragment region. The performance of each pair of detection primers for each target region was analyzed using the SYBR fluorescence quantitative PCR system, requiring that the amplification curve had an obvious exponential growth phase, the amplification efficiency was in the range of 90% - 110%, and there was no non-specific amplification. After obtaining the methylation primer pairs for each short fragment region that met all the above requirements, corresponding detection probes were designed for each pair of primer pairs. All the detection probes were TaqMan probes, with a fluorescent group at the 5' end of the probe and a fluorescence quenching group at the 3' end of the probe, and it was required that there was no non-specific binding between the detection primer pairs and the detection probes, and between the detection probes and the target regions. Finally, the combined effect of the methylation primer pairs and the detection probes was verified in the fluorescence quantitative PCR system, and the primer pairs and probes with an exponential amplification phase were retained as the final detection products.

[0112] Through the above method, the finally screened methylation detection primer pairs and detection probes are shown in Table 6. The methylated cytosine sites that can be recognized by the detection primer pairs and probes for each short fragment region are shown in Table 7.

[0113] Table 6. Detection primer pairs and detection probes for each short fragment region

[0114]

[0115] Table 7. Methylated cytosine sites that can be recognized by the methylation primer pairs and detection probes

[0116]

[0117]

[0118] Using the converted tissue sample DNA as a template, a fluorescence quantitative PCR reaction is carried out using the detection primer pairs and detection probes provided in Table 6 to amplify the 6 different short fragment target regions in each sample. If only one target region is detected, in the PCR reaction system, the methylation detection primer pair and detection probe specific to the target region, as well as the detection primer pair and detection probe for the internal reference gene ACTB, need to be added. If two target regions are detected simultaneously, in the PCR reaction system, 2 methylation detection primer pairs and detection probes specific to the target regions, as well as the detection primer pair and detection probe for the internal reference gene ACTB, need to be added simultaneously. The internal reference gene ACTB is used to evaluate the content and quality of DNA in the sample. The upstream primer sequence for amplifying the ACTB gene fragment used in this example is: 5'-AAGGTGGTTGGGTGGTTGTTTTG-3' (SEQ ID No.29), the downstream primer sequence for amplifying the ACTB gene fragment is: 5'-AATAACACCCCCACCCTGC-3' (SEQ ID No.30), and the corresponding detection probe sequence is: 5'-VIC-GGAGTGGTTTTTGGGTTTG-BHQ1-3' (SEQ ID No.31). In each PCR reaction system, the 5'-end fluorescent groups of the detection probes for the target regions are FAM and / or ROX respectively, and the 3'-end fluorescence quenching groups are BHQ1 and / or MGB. Specifically, configure the PCR amplification system according to the formula provided in Table 8 (Table 8 is the amplification system under the condition of detecting two target regions simultaneously. If only one target region is detected, the detection primer pair and detection probe for the other target region are not added, and the total volume is made up to 25 μL with ultrapure water correspondingly). The components such as DNA polymerase and buffer used in the formula are all purchased from Invitrogen (Cat: 14966005). In addition, when detecting each target region in the tissue sample, a quality control experiment needs to be set up simultaneously, that is, positive control PCR tubes and negative control PCR tubes need to be set up. For a certain target region, the configuration systems of the positive control PCR tube and the negative control PCR tube are the same as those of the experimental test PCR tube, but the template of the positive control PCR tube is a 10 3 copy / μL plasmid containing the converted target region and 10 3 copy / μL plasmid containing the converted ACTB gene fragment mixed in equal volume. The template of the negative control PCR tube is TE buffer. After the system configurations of the positive control PCR tube, the experimental test PCR tube, and the negative control PCR tube are completed, carry out the reaction on a fluorescence quantitative PCR instrument according to the program provided in Table 9.

[0119] Table 8. qPCR reaction system

[0120] Component Specification Volume (μL) Platinum II PCR buffer 5× 5 dNTPs Each 2.5 mM 3 Upstream primer for one region 10 μM 0.5 Downstream primer for one region 10 μM 0.5 Detection probe for one region 10 μM 0.5 Upstream primer for another region 10 μM 0.5 Downstream primer for another region 10 μM 0.5 Detection probe for another region 10 μM 0.5 ACTB forward primer 10 μM 0.5 ACTB reverse primer 10 μM 0.5 ACTB detection probe 10 μM 0.5 DNA Polymerase / 0.5 DNA of the sample to be tested / 5 Ultra-pure water / Make up to 25

[0121] Table 9. qPCR reaction procedure

[0122]

[0123]

[0124] After the qPCR reaction, adjust the baseline and set the threshold. The threshold must be within the exponential amplification phase. The straight line parallel to the X-axis that crosses the threshold is called the threshold line, and the cycle number corresponding to the intersection of the threshold line and the amplification curve is the Ct value. Analyze the results of the qPCR reaction, requirements: ① The negative control PCR tube has no amplification (i.e., no amplification curve); ② The positive control PCR tube has an obvious exponential growth phase, and the Ct value of the target gene in the positive control PCR tube is between 26 and 30; ③ The Ct value of the internal reference gene of the sample to be tested is less than or equal to 33. If the positive control, negative control, and internal reference gene all meet the above requirements, then the test results of the sample to be tested can be analyzed and the results can be interpreted. Otherwise, the current experiment is invalid and must be retested.

[0125] 3. Result analysis

[0126] Methylation level analysis:

[0127] Analyze the methylation level of the sample to be tested according to the Ct value of qPCR. For a certain endometrial tissue sample in this embodiment, if the Ct value of amplifying a certain short fragment target region ≤ 38, then this region in the tissue sample is considered methylation positive. If the Ct value of amplifying a certain short fragment target region > 38, then this region in the tissue sample is considered methylation negative.

[0128] Sample determination criteria:

[0129] 1) Under the condition that one short fragment in Region 1 or Region 2 is used as the target region, if the tissue sample to be tested is methylation positive in this region, then this tissue sample to be tested is a positive sample for endometrial cancer; if the tissue sample to be tested is methylation negative in this region, then this tissue sample to be tested is a negative sample for endometrial cancer.

[0130] 2) Under the condition that one short fragment in Region 1 and one short fragment in Region 2 are used as the target regions, if the tissue sample to be tested is methylation positive in at least one short fragment target region, then this tissue sample to be tested is a positive sample for endometrial cancer; if the tissue sample to be tested is methylation negative in both short fragment target regions, then this tissue sample to be tested is a negative sample for endometrial cancer.

[0131] Taking one short fragment in Region 1 or Region 2 as the target region, and taking one short fragment in Region 1 and one short fragment in Region 2 as the target regions, and then the performance of diagnosing endometrial tissue samples by analyzing the methylation status of the target regions is shown in Table 10.

[0132] Table 10. Performance of diagnosing various tissue samples by analyzing methylation levels of target regions using qPCR

[0133]

[0134]

[0135] As can be seen from Table 10, using a short fragment in Region 1 or Region 2 as the target region, and using a short fragment in Region 1 and a short fragment in Region 2 as the target region both have a certain detection rate for endometrial cancer tissue samples. Specifically, when using a short fragment in Region 1 or Region 2 as the target region, the sensitivity range for detecting early-stage endometrial cancer tissue samples is 62.22% - 77.78%, the sensitivity range for detecting mid-late stage endometrial cancer tissue samples is 83.87% - 93.55%, and the total sensitivity range for detecting endometrial cancer tissue samples is 71.05% - 84.21%; when using a short fragment in Region 1 and a short fragment in Region 2 as the target region, the sensitivity range for detecting early-stage endometrial cancer tissue samples is 75.56% - 91.11%, the sensitivity range for detecting mid-late stage endometrial cancer tissue samples is 93.55% - 100%, and the total sensitivity range for detecting endometrial cancer tissue samples is 84.21% - 94.74%. In addition, the target regions listed in Table 10 have high detection specificity for normal tissues adjacent to endometrial cancer and uterine benign disease tissue samples. The specificity for detecting normal tissues adjacent to endometrial cancer is higher than 84%, and the specificity for detecting uterine benign disease tissue samples is higher than 91%. Generally speaking, using a short fragment in Region 1 and a short fragment in Region 2 as the target region has a higher detection sensitivity for tissue samples than using a short fragment in Region 1 or Region 2 as the target region, and the detection specificity of the former is not significantly reduced compared to the latter.

[0136] Example 3. Detection of methylation levels of target regions in cell samples by methylation-specific fluorescence quantitative PCR

[0137] 1. Sample collection

[0138] In this example, 120 cervical exfoliated cell samples were collected from patients diagnosed with endometrial cancer by histopathological biopsy, 78 cervical exfoliated cell samples were collected from patients diagnosed with cervical cancer by histopathological biopsy, 145 cervical exfoliated cell samples were collected from patients diagnosed with benign uterine diseases (endometrial polyps, uterine fibroids, adenomyosis, etc.) by histopathological biopsy, and 130 cervical exfoliated cell samples were collected from healthy women who underwent physical examinations. The collection process of all cell samples was approved by the ethics committee, all volunteers signed informed consent forms, and all samples were anonymized.

[0139] 2. Extraction of Template DNA from Exfoliated Cell Samples

[0140] For cervical exfoliated cells, genomic DNA was extracted using the blood / cell / tissue genomic DNA extraction kit (Cat: DP304) from Tiangen Biochemical Technology Co., Ltd., and the specific operations were performed according to the instructions.

[0141] 3. The transformation and purification of sample DNA were the same as in Example 1.

[0142] 4. Methylation-specific fluorescent quantitative PCR reaction

[0143] Considering that the detection performance of using a short fragment in region 1 and a short fragment in region 2 as the target region is better than using a short fragment in region 1 or region 2 alone as the target region, this embodiment only uses the former as the target region, that is, in one PCR reaction system, the detection primer pairs and detection probes of the two target regions are added at the same time. According to the method provided in Example 2, using the exfoliated cell sample DNA converted and purified by bisulfite as a template, using the detection primer pairs and detection probes in Table 6, a methylation-specific fluorescent quantitative PCR reaction was performed to detect the methylation level of each cell sample to be tested. The control settings and quality control of the qPCR reaction are the same as those in Example 2.

[0144] 5. The sample judgment criteria are the same as in Example 2.

[0145] Taking a short fragment in region 1 and a short fragment in region 2 as target regions, the performance of diagnosing cervical exfoliated cell samples by analyzing the methylation status of the target regions by qPCR is shown in Table 11.

[0146] Table 11. Performance of qPCR in analyzing methylation levels in target regions and diagnosing cervical exfoliated cell samples

[0147]

[0148]

[0149] As can be seen from Table 11, taking a short fragment in Region 1 and a short fragment in Region 2 as the target regions, and detecting the methylation levels of the target regions in cervical exfoliated cell samples by methylation-specific fluorescence quantitative PCR can effectively distinguish endometrial cancer patients from non-endometrial cancer patients. Specifically, the sensitivity range of detecting cervical exfoliated cell samples of early endometrial cancer patients in the target regions is 73.53% - 88.24%, the sensitivity range of detecting cervical exfoliated cell samples of middle and late endometrial cancer patients in the target regions is 92.31% - 100%, and the overall sensitivity range of detecting cervical exfoliated cell samples of endometrial cancer patients in the target regions is 81.67% - 93.33%; the specificity range of detecting cervical exfoliated cell samples of patients with uterine benign diseases in the target regions is 91.03 - 95.17%, and the specificity range of detecting cervical exfoliated cell samples of healthy women in the target regions is 93.08% - 98.46%. In addition, the detection rate of the target regions for cervical cancer patient cell samples is not high. Therefore, although the detected samples are cervical exfoliated cells, misdiagnosis will not occur.

[0150] In summary, the effect of diagnosing endometrial cancer by analyzing the methylation levels of the target regions is very good, and it has a high detection rate for early cancer patients, so as to facilitate the treatment of patients in the early stage and is beneficial to improving the quality of life and life cycle of endometrial cancer patients.

[0151] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0152] The above-described embodiments only represent several implementation manners of the present invention, which are convenient for understanding the technical solutions of the present invention specifically and in detail, but should not be construed as limiting the protection scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided by the present invention are all within the protection scope of the appended claims of the present invention. Therefore, the protection scope of this invention patent should be determined by the content of the appended claims, and the specification can be used to explain the content of the claims.

Claims

1. Use of a reagent for detecting the methylation level of a target region in the preparation of a diagnostic product for endometrial cancer; Using GRCh38.p14 as a reference, the target region includes Region 1-2 as Chr6:27679919-27680065 and Region 2-2 as Chr6:10390668-10390804; The reagent includes: detection primer pairs with nucleotide sequences as shown in SEQ ID No.14 and SEQ ID No.15 and a detection probe with a nucleotide sequence as shown in SEQ ID No.16; and, detection primer pairs with nucleotide sequences as shown in SEQ ID No.23 and SEQ ID No.24 and a detection probe with a nucleotide sequence as shown in SEQ ID No.

25.

2. The application according to claim 1, wherein The reagent detects the methylation level of the target region by one or more of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray.

3. The application according to any one of claims 1 to 2, characterized in that, The sample types for detection include cell samples or tissue samples.

4. An endometrial cancer diagnostic kit, characterized in that, It includes the reagent defined in any one of claims 1 to 3.

5. The endometrial cancer diagnostic kit according to claim 4, characterized in that, The diagnostic kit further includes one or more of sequencing reagents, amplification reagents, quality control products, bisulfite, DNA extraction reagents or DNA purification reagents.

6. The endometrial cancer diagnostic kit according to claim 5, characterized in that, The amplification reagent includes one or more of an amplification buffer, dNTPs, a DNA polymerase, or Mg 2+ in it.

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