Application of substance for detecting methylation marker in preparation of product for detecting cervical cancer and / or precancerous lesions of cervical cancer

Abstract

The invention discloses a methylation marker for detecting cervical cancer and / or cervical cancer precancerous lesions and application, and particularly relates to the technical field of molecular biomedicine. According to the application, methylation differences between patients with cervical cancer and / or precancerous lesions of cervical cancer and healthy people are researched through DNA of cervical exfoliated cells of Chinese population, candidate methylation markers with obvious differences are screened out, and by combining LASSO-Logistic modeling, AUC ranking and manual screening methods, the candidate methylation markers with obvious differences are screened out, so that the candidate methylation markers of cervical cancer and / or precancerous lesions of cervical cancer are obtained. 33 optimal methylation markers are screened for the first time to establish a Chinese population cervical cancer and / or cervical precancerous lesion methylation risk prediction method, and the method is suitable for predicting risk assessment of early-stage cervical cancer and / or cervical precancerous lesion occurrence of Chinese population and screening and diagnosis of cervical cancer and / or cervical precancerous lesion.

Description

Technical Field

[0001] This manual relates to the field of molecular biomedical technology, and in particular to the application of substances that detect methylation markers in the preparation of products for the detection of cervical cancer and / or precancerous cervical lesions. Background Technology

[0002] Cervical cancer poses a significant threat to women's health. Globally, cervical cancer ranks fourth in both incidence and mortality among malignant tumors. In my country, nearly 20% of new cervical cancer cases and deaths occur.

[0003] Only persistent infection with high-risk human papillomavirus (hrHPV) can lead to cervical lesions, progressing from mild cervical intraepithelial neoplasia (CIN), moderate CIN, and severe CIN, eventually developing into cervical cancer. Current screening guidelines recommend HPV DNA testing as the first-line method, or in combination with thin-layer cytologic test (CT). hrHPV testing is objective and highly reproducible, but it cannot distinguish between transient and transformative infections, potentially leading to increased colposcopy referrals and causing anxiety in women with positive results. Cytological screening methods such as TCT have high specificity, but limitations such as diagnostic subjectivity and low sensitivity can lead to missed diagnoses.

[0004] Recent studies have shown that early epigenetic alterations are important characteristics of tumor development and progression. DNA methylation detection is gradually becoming an emerging method for detecting cervical cancer and precancerous lesions. Various methylation genes, such as FAM19A4, Mir124-2, PAX1, ZNF582, SOX1, and EPB41L3, have been considered biomarkers for cervical cancer screening. However, their detection efficacy for cervical cancer and precancerous lesions in the Chinese population is generally limited. Therefore, exploring and developing more effective and objective biomarkers suitable for the Chinese population has significant clinical implications. Summary of the Invention

[0005] To address the lack of methylation biomarkers in the Chinese population, simplified genome methylation sequencing (RRBS) and targeted methylation sequencing (TBS) were used for initial screening of cervical exfoliated cell DNA from the Chinese population, followed by validation. High-throughput sequencing results were used to analyze differential methylation regions between cervical cancer, precancerous lesions, and healthy individuals, and a model was constructed to provide new ideas and detection methods for the early diagnosis and treatment of cervical cancer and precancerous lesions. The main contribution of this application is the discovery of these methylation biomarkers; any methylation level analysis method can be used to detect the methylation levels of the methylation biomarkers discovered in this application.

[0006] This application provides the use of substances that detect methylation markers in the preparation of products for detecting cervical cancer and / or precancerous lesions of the cervix, wherein the methylation markers include any one of the following genes or fragments thereof: FOXD3, ARHGEF4, WEE1P1, or NOL4.

[0007] This application also provides a product for detecting cervical cancer and / or precancerous lesions of the cervix, the product comprising a substance for detecting methylation markers, said methylation markers including any one of the following genes or fragments thereof: FOXD3, ARHGEF4, WEE1P1, or NOL4.

[0008] This application also provides a cervical cancer and / or cervical precancerous lesion detection device, comprising the following modules: a data acquisition module, used to provide methylation level data of a target biomarker in the sample to be tested, wherein the target biomarker is the methylation biomarker used in the above application; and a judgment module, used to assess the status of cervical cancer and / or cervical precancerous lesions based on the methylation level data of the target biomarker in the sample to be tested.

[0009] This application also provides a computer-readable storage medium storing computer instructions that, when executed by a processor, implement a method for detecting cervical cancer and / or precancerous cervical lesions. The method includes: acquiring methylation level data of a target biomarker in a sample to be tested, wherein the target biomarker is a methylation biomarker as described above; and assessing the status of cervical cancer and / or precancerous cervical lesions based on the methylation level data of the target biomarker in the sample to be tested.

[0010] This application also provides an electronic terminal, which includes: a processor, a memory, an input / output interface, and a communication port; the memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory, so that the terminal performs the method for detecting cervical cancer and / or precancerous lesions of the cervix described in the above-described computer-readable storage medium.

[0011] This application also provides a computer program product, including a computer program that, when executed by a processor, performs the following steps: acquiring methylation level data of a target biomarker in a test sample, and assessing cervical cancer and / or precancerous lesions based on the methylation level data of the target biomarker in the test sample.

[0012] The beneficial effects of this application include, but are not limited to: This application conducts precise screening based on cervical lesion samples of different grades in the Chinese population, and specifically seeks methylation biomarkers that are effective in detecting cervical cancer and precancerous lesions in the Chinese population. The screened methylation biomarkers are novel methylation biomarkers with clinical potential, and have good sensitivity and specificity, providing a rapid, effective and accurate new approach for early screening, auxiliary diagnosis and assessment of cervical cancer and precancerous lesions. Attached Figure Description

[0013] This application will be further described by way of exemplary embodiments, which will be described in detail with reference to the accompanying drawings. These embodiments are not limiting, wherein: Figure 1 This paper illustrates the development process of using methylation sequencing data to screen methylation biomarkers for detecting cervical cancer and precancerous cervical lesions.

[0014] Figure 2 A heatmap showing the difference in methylation levels of methylation markers between CIN1- and CIN3+ in simplified genome methylation sequencing in Example 1 of this application is shown.

[0015] Figure 3 The present invention illustrates the receiver operating characteristic (ROC) curves of 20 methylation biomarker compositions used in Example 2 of this application for screening cervical cancer and precancerous cervical lesions.

[0016] Figure 4 The present invention provides a receiver operating characteristic (ROC) curve for screening cervical cancer and precancerous cervical lesions using methylation markers with AUC Top15 in Example 2 of this application.

[0017] Figure 5 The present invention illustrates the receiver operating characteristic (ROC) curves of three methylation markers manually screened in Example 2 of this application for cervical cancer and precancerous lesions.

[0018] Figure 6 This is a block diagram of a cervical cancer and / or precancerous lesion detection device according to some embodiments of this application.

[0019] Figure 7 This is a flowchart illustrating a method for detecting cervical cancer and / or precancerous lesions of the cervix according to some embodiments of this application.

[0020] Figure 8 This is a schematic diagram of the architecture of an electronic terminal 800 according to some embodiments of this application. Detailed Implementation

[0021] To more clearly illustrate the technical solutions of the embodiments in this specification, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are merely some examples or embodiments of this specification. For those skilled in the art, these drawings can be applied to other similar scenarios without creative effort. Unless obvious from the context or otherwise specified, the same reference numerals in the drawings represent the same structures or operations.

[0022] As indicated in this specification and claims, unless the context clearly indicates otherwise, the words "a," "an," "an," and / or "the" do not specifically refer to the singular and may also include the plural. Generally speaking, the terms "comprising" and "including" only indicate the inclusion of expressly identified steps and elements, which do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

[0023] Flowcharts are used in this specification to illustrate the operations performed by the system according to embodiments of this specification. It should be understood that the preceding or following operations are not necessarily performed in exact order. Instead, the steps can be processed in reverse order or simultaneously. Furthermore, other operations can be added to these processes, or one or more steps can be removed from them.

[0024] This application provides the use of substances that detect methylation markers in the preparation of products for detecting cervical cancer and / or precancerous lesions of the cervix, wherein the methylation markers include any one of the following genes or fragments thereof: FOXD3, ARHGEF4, WEE1P1, or NOL4.

[0025] In some embodiments, based on the sequence of the human reference genome Hg19, the methylation marker may include any one of the following genes or fragments thereof: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604.

[0026] "Chr" and the following numbers (1-20) indicate the specific chromosome, and the number after the colon indicates the base position on the chromosome. For example, "Chr1: 220100662-220102096" represents the base sequence from base 220100662 to base 220102096 on human chromosome 1.

[0027] In some embodiments, the cervical precancerous lesion may be a severe cervical precancerous lesion.

[0028] In some embodiments, the sample for product testing may include any one or more of the following: blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells. Preferably, in some embodiments, the sample for product testing may be cervical exfoliated cells.

[0029] In some embodiments, the substance for detecting methylation markers may include a substance for detecting the methylation level of the methylation markers. Preferably, in some embodiments, the substance for detecting the methylation level of the methylation markers may include primer pairs and probes.

[0030] In some embodiments, more preferably, the primer pair and probe are specific primer pairs and probes corresponding to any one of the following genes or fragments: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604.

[0031] In this article, "corresponding" refers to the corresponding gene fragment after Bisulfite treatment. After Bisulfite treatment, the corresponding gene fragment will undergo different changes depending on whether it is methylated. Specifically, unmethylated cytosine (C) will be converted to uracil (U) during Bisulfite treatment, while methylated cytosine will remain unchanged.

[0032] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker FOXD3 gene or fragments thereof may be as shown in SEQ ID NO.40 and SEQ ID NO.41.

[0033] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker ARHGEF4 gene or fragments thereof may be as shown in SEQ ID NO.52 and SEQ ID NO.53.

[0034] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker WEE1P1 gene or fragments thereof may be as shown in SEQ ID NO.82 and SEQ ID NO.83.

[0035] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker NOL4 gene or fragments thereof may be as shown in SEQ ID NO. 91 and SEQ ID NO. 92.

[0036] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the FOXD3 gene or a fragment thereof may be as shown in SEQ ID NO.42.

[0037] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ARHGEF4 gene or a fragment thereof may be as shown in SEQ ID NO.54.

[0038] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the WEE1P1 gene or a fragment thereof may be as shown in SEQ ID NO.84.

[0039] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the NOL4 gene or a fragment thereof may be as shown in SEQ ID NO.93.

[0040] This application also provides a product for detecting cervical cancer and / or precancerous lesions of the cervix, the product comprising a substance for detecting methylation markers, said methylation markers including any one of the following genes or fragments thereof: FOXD3, ARHGEF4, WEE1P1, or NOL4.

[0041] In some embodiments, based on the sequence of the human reference genome Hg19, the methylation marker may include any one of the following genes or fragments thereof: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604.

[0042] In some embodiments, the cervical precancerous lesion may be a severe cervical precancerous lesion.

[0043] In some embodiments, the sample tested by the product may include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells. Preferably, in some embodiments, the sample tested by the product may be cervical exfoliated cells. In some embodiments, the product may be used to detect DNA in the sample.

[0044] In some embodiments, the product may be any of a kit, chip, membrane strip, protein array, composition, or detection system. In some embodiments, preferably, the product may include one or more of DNA polymerase, a mixture of deoxyribonucleotides (dNTPs), a buffer solution, primers, probes, sodium bisulfite, a positive control, or a negative control.

[0045] The term “kit” refers to a packaged collection of related components, such as one or more polynucleotides or compositions, and one or more related materials, such as delivery devices (e.g., syringes), solvents, solutions, buffers, instructions, or desiccants.

[0046] The term "membrane strip" refers to a diagnostic tool that utilizes the principle of specific biomolecular recognition. It involves immobilizing biomolecules such as antigens or antibodies on a membrane, allowing them to specifically bind to the analyte in a sample, and then using visualization or other signal detection methods to qualitatively or quantitatively analyze the target substance in the sample.

[0047] The term "chip" typically refers to a miniature device that integrates biosensors and microfluidics technology. It can perform various operations such as sample preparation, reaction, and detection in biological, chemical, and medical analysis processes at the microscopic level to achieve rapid and accurate detection of disease-related biomarkers.

[0048] Protein arrays, also known as protein microarrays, are high-throughput biotechnology tools that allow for the simultaneous analysis and study of large numbers of proteins. This technology enables rapid analysis of protein expression, protein-protein interactions, and protein-small molecule binding by arranging thousands of different proteins or protein-protein interaction probes in an orderly manner on a solid surface.

[0049] In some embodiments, the substance for detecting methylation markers may include a substance for detecting the methylation level of the methylation markers. Preferably, in some embodiments, the substance for detecting the methylation level of the methylation markers may include primer pairs and probes.

[0050] In some embodiments, more preferably, the primer pair and probe can be a specific primer pair and probe corresponding to any one of the following genes or fragments: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604.

[0051] As used in this application, the term "primer" refers to a naturally occurring oligonucleotide (e.g., a restriction fragment) or a synthetically produced oligonucleotide that can be used as a starting point for the synthesis of primer extension products, wherein the primer extension product is complementary to a nucleic acid strand (template or target sequence) under appropriate conditions (e.g., buffer, salt, temperature, and pH) and in the presence of nucleotides and reagents for nucleic acid polymerization (e.g., DNA-dependent or RNA-dependent polymerases). Typically, a primer set will consist of at least two primers, an "upstream primer" and a "downstream primer," which together define the amplicon (the sequence to be amplified using the primers).

[0052] The term "probe" refers to any molecule capable of selectively binding to a target biomolecule (e.g., a nucleic acid sequence that hybridizes with the probe). In some embodiments, the probe may be labeled, for example, with a fluorescent group and a quencher group. In some embodiments, the probe may be a Taqman probe with a fluorescent reporter group added to the 5' end and a fluorescent quencher group added to the 3' end.

[0053] Antibodies are protective proteins produced by the body in response to antigen stimulation. In some embodiments... In this context, antibodies can be used as detection reagents to detect gene expression.

[0054] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker FOXD3 gene or fragments thereof may be as shown in SEQ ID NO.40 and SEQ ID NO.41.

[0055] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker ARHGEF4 gene or fragments thereof may be as shown in SEQ ID NO.52 and SEQ ID NO.53.

[0056] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker WEE1P1 gene or fragments thereof may be as shown in SEQ ID NO.82 and SEQ ID NO.83.

[0057] In some embodiments, the primer pair nucleotide sequences for detecting the methylation marker NOL4 gene or fragments thereof may be as shown in SEQ ID NO. 91 and SEQ ID NO. 92.

[0058] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the FOXD3 gene or a fragment thereof may be as shown in SEQ ID NO.42.

[0059] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ARHGEF4 gene or a fragment thereof may be as shown in SEQ ID NO.54.

[0060] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the WEE1P1 gene or a fragment thereof may be as shown in SEQ ID NO.84.

[0061] In some embodiments, the nucleotide sequence of the probe for detecting the methylation level of the NOL4 gene or a fragment thereof may be as shown in SEQ ID NO.93.

[0062] This application also provides a diagnostic method for human cervical cancer and / or cervical precancerous lesions, wherein the method utilizes the above-mentioned product to detect whether the methylation level of FOXD3, ARHGEF4, WEE1P1 or NOL4 genes or fragments thereof from the test sample is elevated relative to that of healthy individuals.

[0063] In some embodiments, the method may include the following steps: (1) collecting the sample to be tested, extracting the sample genomic DNA, and performing Bisulfite treatment to obtain transformed DNA; (2) preparing a reaction system, the reaction system including the transformed DNA of the sample genomic DNA, DNA polymerase, a mixture of deoxyribonucleotides (dNTPs), a buffer solution, primers, and probes; (3) performing an amplification reaction; (4) analyzing the results to obtain the methylation level of the FOXD3, ARHGEF4, WEE1P1, or NOL4 genes or fragments of the sample to be tested; and (5) determining whether the person corresponding to the sample to be tested has cervical cancer and / or precancerous lesions of the cervix.

[0064] In some embodiments, the amplification reaction can be quantitative methylation-specific PCR (qMSP). The ΔCt value of the methylation marker is calculated based on the qMSP results. 目标基因 =Ct 目标基因 -Ct β-actin .

[0065] The ROC of each gene was obtained using SPSS statistic 21 software. The sensitivity (true positive rate) was plotted on the ordinate, and 1 - specificity (false positive rate) was plotted on the x-axis. The Youden index = sensitivity - (1 - specificity). The optimal critical point (the cutoff value of ΔCt) was determined based on the maximum value of the Youden index.

[0066] In some embodiments, when the ΔCt value of the FOXD3 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 15.33 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0067] In some embodiments, when the ΔCt value of the ARHGEF4 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 7.44 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0068] In some embodiments, when the ΔCt value of the WEE1P1 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 6.68 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0069] In some embodiments, when the ΔCt value of the NOL4 gene or its fragment in the test sample is lower than or significantly lower than the cutoff value of 7.15 for ΔCt, it can be determined that the person corresponding to the test sample has cervical cancer and / or cervical precancerous lesions.

[0070] This application also provides a device for detecting cervical cancer and / or precancerous cervical lesions, such as Figure 6 As shown, it includes the following modules: data acquisition module 610 and judgment module 620.

[0071] The data acquisition module 610 is used to provide methylation level data of the target marker of the sample to be tested, wherein the target marker is the methylation marker in the above application.

[0072] In some embodiments, the ΔCt value of the methylation biomarker can be calculated based on the methylation level data of the target biomarker in the test sample. Preferably, in some embodiments, the methylation level data can be the Ct value of the methylation biomarker detected by quantitative methylation-specific PCR. Preferably, in some embodiments, the ΔCt value can be the difference between the Ct value of the target biomarker and the Ct value of an internal reference gene. More preferably, in some embodiments, the internal reference gene can be β-actin.

[0073] The judgment module 620 is used to assess the status of cervical cancer and / or precancerous lesions based on the methylation level data of the target marker in the test sample. In some embodiments, the status of cervical cancer and / or precancerous lesions can be assessed based on the ΔCt value and a preset ΔCt cutoff value. In some embodiments, preferably, the test sample can be determined to be negative or positive based on the ΔCt value and a preset ΔCt cutoff value. In some embodiments, more preferably, the ΔCt cutoff value can be the ΔCt value that maximizes the Youden index.

[0074] In some embodiments, when the ΔCt value of the target marker in the test sample is less than or equal to the cutoff value of ΔCt, the test sample can be determined to be positive. In some embodiments, when the ΔCt value of the target marker in the test sample is greater than the cutoff value of ΔCt, the test sample can be determined to be negative.

[0075] In some embodiments, a positive result means that the individual corresponding to the test sample is a patient with severe cervical precancerous lesions or cervical cancer, and a negative result means that the individual corresponding to the test sample is a healthy person, a patient with mild cervical precancerous lesions, or a patient with moderate cervical precancerous lesions.

[0076] In some embodiments, the sample to be tested may include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells. In some embodiments, preferably, the sample to be tested may be cervical exfoliated cells.

[0077] This application also provides a computer-readable storage medium storing computer instructions that, when executed by a processor, implement a method for detecting cervical cancer and / or precancerous lesions of the cervix. A flowchart of the method is shown below. Figure 7 As shown.

[0078] In step S710, the methylation level data of the target marker of the sample to be tested is obtained, wherein the target marker is the methylation marker in the above application.

[0079] In some embodiments, the ΔCt value of the methylation biomarker can be calculated based on the methylation level data of the target biomarker in the test sample. Preferably, in some embodiments, the methylation level data can be the Ct value of the methylation biomarker detected by quantitative methylation-specific PCR. Preferably, in some embodiments, the ΔCt value can be the difference between the Ct value of the target biomarker and the Ct value of an internal reference gene. More preferably, in some embodiments, the internal reference gene can be β-actin.

[0080] In step S720, the status of cervical cancer and / or precancerous lesions is assessed based on the methylation level data of the target biomarker in the sample to be tested.

[0081] In some embodiments, the presence of cervical cancer and / or precancerous lesions can be assessed based on the ΔCt value and a preset ΔCt cutoff value. Preferably, in some embodiments, the test sample can be determined to be negative or positive based on the ΔCt value and a preset ΔCt cutoff value. More preferably, in some embodiments, the ΔCt cutoff value can be the ΔCt value that maximizes the Youden index.

[0082] In some embodiments, when the ΔCt value of the target marker in the test sample is less than or equal to the cutoff value of ΔCt, the test sample can be determined to be positive. In some embodiments, when the ΔCt value of the target marker in the test sample is greater than the cutoff value of ΔCt, the test sample can be determined to be negative.

[0083] In some embodiments, a positive result means that the individual corresponding to the test sample is a patient with severe cervical precancerous lesions or cervical cancer, and a negative result means that the individual corresponding to the test sample is a healthy person, a patient with mild cervical precancerous lesions, or a patient with moderate cervical precancerous lesions.

[0084] In some embodiments, the sample to be tested may include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells. In some embodiments, preferably, the sample to be tested may be cervical exfoliated cells.

[0085] This application also provides an electronic terminal 800, characterized in that the electronic terminal 800 includes: a processor 810, a memory 820, an input / output interface 830, and a communication port 840; the memory 820 is used to store a computer program, and the processor 810 is used to execute the computer program stored in the memory 820, so that the terminal executes the method for detecting cervical cancer and / or precancerous lesions of the cervix described in the above-mentioned computer-readable storage medium.

[0086] Processor 810 can execute computational instructions (program code) and perform the functions of the detection device described in this application. Computational instructions may include programs, objects, components, data structures, processes, modules, and functions (functions refer to the specific functions described in this application). For example, processor 810 can process instructions in a cervical cancer and / or precancerous lesion detection device for assessing cervical cancer and / or precancerous lesions. In some embodiments, processor 810 may include a microcontroller, microprocessor, reduced instruction set computer (RISC), application-specific integrated circuit (ASIC), application-specific instruction set processor (ASIP), central processing unit (CPU), graphics processing unit (GPU), physical processing unit (PPU), microcontroller unit, digital signal processor (DSP), field-programmable gate array (FPGA), advanced RISC machine (ARM), programmable logic device, and any circuit and processor capable of performing one or more functions, or any combination thereof. This is for illustrative purposes only. Figure 8 Only one processor 810 is described in this application, but it should be noted that this application may include multiple processors.

[0087] The memory 820 can store data / information obtained from any component of the cervical cancer and / or precancerous lesion detection device. In some embodiments, the memory 820 may include mass storage, removable storage, volatile read and write memory, and read-only memory (ROM), or any combination thereof. Exemplary mass storage may include disks, optical disks, and solid-state drives. Removable storage may include flash drives, floppy disks, optical disks, memory cards, USB flash drives, compact disks, and portable hard drives. Volatile read and write memory may include random access memory (RAM). RAM may include dynamic RAM (DRAM), double-rate synchronous dynamic RAM (DDRSDRAM), static RAM (SRAM), thyristor RAM (T-RAM), and zero-capacitance (Z-RAM), etc. ROM may include mask ROM (MROM), programmable ROM (PROM), erasable programmable ROM (PEROM), electrically erasable programmable ROM (EEPROM), optical disc ROM (CD-ROM), and digital universal disk ROM, etc.

[0088] The input / output interface 830 can be used to input or output signals, data, or information. In some embodiments, the input / output interface 830 can be used to enable interaction between a user (e.g., the person corresponding to the test sample, the user of a cervical cancer and / or precancerous lesion detection device, etc.) and the processor 710. In some embodiments, the user can input characteristic information of the person corresponding to the test sample through the input / output interface 830. In some embodiments, the input / output interface 830 may include an input device and an output device. Exemplary input devices may include a keyboard, mouse, touch screen, and microphone, or any combination thereof. Exemplary output devices may include a display device, speaker, printer, projector, or any combination thereof. Exemplary display devices may include a liquid crystal display (LCD), a light-emitting diode (LED) based display, a flat panel display, a curved display, a television device, a cathode ray tube (CRT), or any combination thereof.

[0089] Communication port 840 can be connected to a network for data communication. The connection can be wired, wireless, or a combination of both. Wired connections can include cables, fiber optic cables, or telephone lines, or any combination thereof. Wireless connections can include Bluetooth, WiFi, WiMax, WLAN, ZigBee, mobile networks (e.g., 3G, 4G, or 5G), or any combination thereof. In some embodiments, communication port 840 can be a standardized port, such as RS232 or RS485. In some embodiments, communication port 840 can be a specially designed port.

[0090] This application also provides a computer program product, including a computer program that, when executed by a processor, performs the following steps: acquiring methylation level data of a target biomarker in a test sample, and assessing cervical cancer and / or precancerous lesions based on the methylation level data of the target biomarker in the test sample.

[0091] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent companies. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0092] The individuals included in this example (Normal, CIN1, CIN2, CIN3, and cervical cancer patients) all met the diagnostic criteria. The diagnostic criteria were based on the fifth edition of the World Health Organization (WHO) classification of tumors of the female reproductive organs.

[0093] The cervical exfoliated cell specimens, reagents and materials used, and their sources in the examples are as follows: 1. Specimen The biological samples used in the examples were cervical exfoliated cells collected by the Institute of Clinical Pharmacology, Xiangya Hospital, Central South University, between January 2020 and December 2022, and all samples had known pathological information.

[0094] 2. Main Reagents and Materials The cervical exfoliated cell genomic DNA extraction kit was the HiPure Universal DNA Kit (Magen); the transformation kit was the EZ DNA Methylation Gold Kit (ZYMO); the primers and probes used were synthesized by Sangon Biotech (Shanghai) Co., Ltd.; the nuclease-free water, 10×Ex Buffer, Ex Taq HS enzyme, and dNTPs used were purchased from Takara Bio (Dalian) Co., Ltd.

[0095] The flowchart for screening methylation biomarkers for cervical cancer and precancerous cervical lesions using methylation sequencing data is as follows: Figure 1 As shown.

[0096] Example 1: Screening for candidate methylation biomarkers for cervical cancer and precancerous cervical lesions based on simplified genome methylation sequencing (RRBS). Genomic DNA samples were obtained from cervical exfoliated cells derived from patients with CIN1- (22 cases), CIN3 (7 cases), and cervical cancer (7 cases) for RRBS detection. Specifically, genomic DNA was extracted from cervical exfoliated cell samples using the HiPure Universal DNA Kit (Magen) extraction kit and evaluated by agarose gel electrophoresis and OD260 / 280 ratio.

[0097] 100-300 ng of quality-tested genomic DNA was collected and digested using MspI (methylation-insensitive restriction enzyme). End repair and adapter ligation were performed on the digested DNA fragments. After fragment selection via gel excision, the DNA was treated with bisulfite (BS) using the EZ DNA Methylation Gold Kit, followed by PCR amplification to obtain DNA libraries. After library quality control, different libraries were pooled according to effective concentration and target sequencing quantity requirements before sequencing. The quality of the sequencing data was assessed, and sequencing adapters and low-quality data were removed using trimming to obtain clean data for subsequent analysis. Bsmap software was used for alignment analysis of methylation data to a reference genome. Differentially methylated regions (DMR) analysis was performed using Metilene software (Ver 0.2-7).

[0098] The samples were grouped into CIN1- and CIN3+ groups. CIN2- was defined as negative individuals, CIN1, and CIN2 patients, and CIN3 and cervical cancer patients as CIN3+ patients. Hypermethylated DMRs were screened based on the following criteria: ① Annotated region; ② Average methylation difference between the CIN1- and CIN3+ groups was in the Top 500; ③ Average methylation value difference between the CIN1- and CIN3+ groups was in the Top 500; ④ Methylation level in the CIN1- group was <0.2. A total of 572 DMRs were screened for further validation. The results are shown in [link to results]. Figure 2 .

[0099] Example 2: Screening for methylation biomarkers in cervical cancer and precancerous cervical lesions based on targeted methylation sequencing (TBS). Genomic DNA samples were obtained from exfoliated cells derived from 36 negative, 35 CIN1, 46 CIN2, 33 CIN3, and 42 cervical cancer cases for TBS testing. Specifically, after sample testing, 1 μg of genomic DNA was taken and treated with bisulfite using the EZDNA Methylation Gold Kit (Zymo Research). Using 1 / 20 of the elution product as a template, PCR amplification was performed for 35 cycles using the KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA). For each sample, equal amounts of BSP products from multiple genes were pooled together. End repair, A-labeling, and adapter ligation were performed, followed by sequencing on an Illumina platform. Trimming was used to process the data, resulting in clean data. The clean data was aligned with the amplified target sequence using Bsmap software. After alignment, the methylation level of the CG sites was calculated using the Python program included with Bsmap.

[0100] according to p When the mean methylation level was < 0.05 and the difference in mean methylation level between CIN3+ and CIN2- groups was greater than 0.15, a total of 1103 hypermethylated sites were identified. Then, through ① the negative group (mean methylation level < 0.1) and ② the CIN3 vs negative group, p < 0.05; ③CIN3 vs CIN2 group, p < 0.10; A total of 125 DMCs were screened for further analysis. LASSO regression analysis was performed using R language, with patient pathological grade defined as the outcome variable (CIN2- defined as 0, CIN3+ defined as 1), and the 125 DMCs as the independent variables of the model. After LASSO regression analysis, 20 DMCs were screened out. SPSS statistic21 software was used to model the 20 differentially methylated markers (Table 1), with the following formula: Table 1. 20 methylation markers for LASSO-Logistic modeling Receiver operating curves (ROCs) for 20 combinations of methylation biomarkers were obtained using SPSS Statistic 21 software. Sensitivity (true positive rate) was plotted on the ordinate, and 1 - specificity (false positive rate) on the x-axis. The Youden index was calculated as sensitivity - (1 - specificity). The optimal cutoff point (the threshold for methylation levels) was determined based on the maximum value of the Youden index. Figure 3 The area under the curve (AUC) of the composition for predicting the risk of cervical cancer and precancerous lesions is shown, with a predictive sensitivity of 86.67%, a specificity of 94.87%, and an AUC of 0.979.

[0101] SPSS statistic 21 software was used to obtain the ROC and AUC of 1103 candidate methylation markers to distinguish CIN2- and CIN3+ sites. The AUC of the 1103 hypermethylated sites was sorted and the top 15 sites with high AUC were selected (Table 2). Figure 4 The ROCs of the Top 15 methylation markers for predicting the risk of cervical cancer and precancerous cervical lesions are shown.

[0102] Table 2. Methylation markers of the top 15 AUC values ​​and AUC values ​​for screening CIN3+. Thirty-six negative, 35 CIN1, 46 CIN2, 33 CIN3, and 42 cervical cancer samples were collected, totaling 117 CIN2- and 75 CIN3+ samples. The sites of 1103 candidate methylation markers corresponding to the 117 CIN2- and 75 CIN3+ samples were manually analyzed. Methylation markers that could detect the CIN3+ samples missed by the preceding 35 methylation markers were screened, resulting in three supplementary methylation markers, as shown in Table 3. Figure 5 The ROCs of supplementary methylation markers for predicting the risk of cervical cancer and precancerous cervical lesions are shown.

[0103] Table 3 Supplementary methylation markers and AUC for screening CIN3+ Example 3: Quantitative methylation-specific PCR (qMSP) to validate screened methylation markers for cervical cancer and precancerous cervical lesions. Genomic DNA samples were obtained from exfoliated cells derived from 19 negative, 13 CIN1, 37 CIN2, 33 CIN3, and 44 cervical cancer cases for qMSP detection. This further validated the performance of the screened methylation markers, successfully establishing a detection system for 33 methylation markers. Specifically, after sample testing, 1.5 μL of genomic DNA was taken and treated with Bisulfite using the EZ DNA Methylation Gold Kit (Zymo Research). The transformed product was then used for PCR. The total PCR reaction solution was 20 μL, composed of: nuclease-free water, 1×PCR buffer, dNTPs (0.25 mM), Ex Taq HS enzyme (1 U / reaction), β-actin-QF (0.4 μM), β-actin-QR (0.4 μM), β-actin-P (0.4 μM), target gene-QF (0.45 μM), target gene-QR (0.45 μM), and target gene-P (0.45 μM). The primers and probes were prepared in μM and 1.5 μL respectively. Primer and probe sequences are shown in Tables 4, 5, 6, 7, and 8. PCR was performed according to the following reaction program: 95℃ for 5 min, then 95℃ for 15 sec, followed by 60℃ for 30 sec, for a total of 50 cycles. The ΔCt value of the methylation marker was calculated based on the qMSP results. 目标基因 =Ct 目标基因 -Ct β-actin .

[0104] The ROC of each gene was obtained using SPSS Statistic 21 software, with sensitivity (true positive rate) as the ordinate and 1-specificity (false positive rate) as the abscissa. The Youden index was calculated as sensitivity - (1-specificity), and the optimal cutoff point (ΔCt cutoff value) was determined based on the maximum value of the Youden index. Tables 9 and 10 show the performance of qMSP in detecting methylation markers for cervical cancer and precancerous lesions.

[0105] Table 4 Primer Sequences Table 5 Primer sequences Table 6 Primer sequences Table 7 Probe Sequences Table 8 Probe Sequences Table 9. Sensitivity and specificity of qMSP method for detecting CIN3+ using methylation markers. Table 10. Sensitivity and specificity of qMSP method for detecting CIN3+ using methylation markers. The above results demonstrate that candidate methylation biomarkers for cervical cancer and / or precancerous lesions in Chinese patients and healthy individuals were screened using DNA from exfoliated cervical cells. Combining LASSO-Logistic modeling, AUC ranking, and manual screening, 33 methylation biomarkers for screening cervical cancer and / or precancerous lesions in the Chinese population were identified for the first time. Based on these biomarkers, a methylation risk prediction model for cervical cancer and / or precancerous lesions in the Chinese population was established. This model is suitable for risk assessment of early cervical cancer and / or precancerous lesions in the Chinese population, as well as for screening and diagnosis of cervical cancer and / or precancerous lesions, providing a rapid, effective, and accurate new approach for early screening, auxiliary diagnosis, and assessment of cervical cancer and / or precancerous lesions.

[0106] The basic concepts have been described above. Obviously, for those skilled in the art, the detailed disclosure above is merely illustrative and does not constitute a limitation of this specification. Although not explicitly stated herein, those skilled in the art may make various modifications, improvements, and corrections to this specification. Such modifications, improvements, and corrections are suggested in this specification and therefore remain within the spirit and scope of the exemplary embodiments described herein.

[0107] Furthermore, this specification uses specific terms to describe embodiments thereof. For example, "an embodiment," "one embodiment," and / or "some embodiments" refer to a particular feature, structure, or characteristic associated with at least one embodiment of this specification. Therefore, it should be emphasized and noted that references to "an embodiment," "one embodiment," or "an alternative embodiment" in different locations throughout this specification do not necessarily refer to the same embodiment. Moreover, certain features, structures, or characteristics in one or more embodiments of this specification can be appropriately combined.

[0108] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are modified in some examples with the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±20%. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed depending on the characteristics required by individual embodiments. In some embodiments, numerical parameters should take into account specified significant digits and employ a general method of digit reservation. Although the numerical ranges and parameters used to confirm their breadth of range in some embodiments of this specification are approximate values, in specific embodiments, such values ​​are set as precisely as feasible.

[0109] Finally, it should be understood that the embodiments described in this specification are merely illustrative of the principles of the embodiments described herein. Other variations may also fall within the scope of this specification. Therefore, alternative configurations of the embodiments described herein are intended to be illustrative rather than limiting, and should be considered consistent with the teachings of this specification. Accordingly, the embodiments described herein are not limited to those explicitly introduced and described herein.

Claims

1. The use of a substance for detecting methylation markers in the preparation of products for detecting cervical cancer and / or precancerous lesions of the cervix, wherein the methylation markers include any one of the following genes or fragments thereof: FOXD3, ARHGEF4, WEE1P1, or NOL4.

2. Use according to claim 1, wherein Based on the sequence of the human reference genome Hg19, the methylation markers include any one of the following genes or fragments: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604; And / or, the cervical precancerous lesion is a severe cervical precancerous lesion; And / or, the samples for product testing include any one or more of blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells, preferably, the samples for product testing are cervical exfoliated cells.

3. The use according to claim 1, wherein the compound is ###0002### The substance for detecting methylation markers includes a substance for detecting the methylation level of methylation markers. Preferably, the substance for detecting the methylation level of methylation markers includes primer pairs and probes. More preferably, the primer pairs and probes are specific primer pairs and probes corresponding to any one of the following genes or fragments: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604.

4. The use according to claim 1, wherein The primer pair nucleotide sequences for detecting the methylation marker FOXD3 gene or fragments thereof are shown in SEQ ID NO.40 and SEQ ID NO.41; And / or, the primer pair nucleotide sequences for detecting the methylation marker ARHGEF4 gene or fragments thereof are as shown in SEQ ID NO. 52 and SEQ ID NO. 53; And / or, the primer pair nucleotide sequences for detecting the methylation marker WEE1P1 gene or fragments thereof are as shown in SEQ ID NO. 82 and SEQ ID NO. 83; And / or, the primer pair nucleotide sequences for detecting the methylation marker NOL4 gene or fragments thereof are shown in SEQ ID NO. 91 and SEQ ID NO.

92.

5. The application as described in claim 1, characterized in that, The nucleotide sequence of the probe for detecting the methylation level of the FOXD3 gene or its fragments, a methylation marker, is shown in SEQ ID NO.42; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ARHGEF4 gene or fragments thereof is shown in SEQ ID NO.54; And / or, the nucleotide sequence of the probe for detecting the methylation level of the WEE1P1 gene or fragment thereof, a methylation marker, is shown in SEQ ID NO.84; And / or, the nucleotide sequence of the probe for detecting the methylation level of the NOL4 gene or fragment thereof is shown in SEQ ID NO.

93.

6. A product for detecting cervical cancer and / or precancerous lesions of the cervix, the product comprising a substance for detecting methylation markers, said methylation markers comprising any one of the following genes or fragments thereof: FOXD3, ARHGEF4, WEE1P1, or NOL4.

7. The product of claim 6, wherein, Based on the sequence of the human reference genome Hg19, the methylation markers include any one of the following genes or fragments: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604; And / or, the cervical precancerous lesion is a severe cervical precancerous lesion.

8. The product of claim 6, wherein, The samples for product testing include any one or more of the following: blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells. Preferably, the samples for product testing are cervical exfoliated cells.

9. The product of claim 6, wherein, The product is any one of a reagent kit, chip, membrane strip, protein array, composition or detection system. Preferably, the product includes one or more of the following: DNA polymerase, deoxynucleotide (dNTP) mixture, buffer solution, primer, probe, sodium bisulfite, positive control or negative control.

10. The product of claim 6, wherein, The substance for detecting methylation markers includes a substance for detecting the methylation level of methylation markers. Preferably, the substance for detecting the methylation level of methylation markers includes primer pairs and probes. More preferably, the primer pairs and probes are specific primer pairs and probes corresponding to any one of the following genes or fragments: Chr1: 63784781-63786348, Chr2: 131721013-131722519, Chr4: 107146-108577, Chr18: 31804106-31805604.

11. The product of claim 6, wherein, The primer pair nucleotide sequences for detecting the methylation marker FOXD3 gene or fragments thereof are shown in SEQ ID NO.40 and SEQ ID NO.41; And / or, the primer pair nucleotide sequences for detecting the methylation marker ARHGEF4 gene or fragments thereof are shown in SEQ ID NO. 52 and SEQ ID NO. 53; And / or, the primer pair nucleotide sequences for detecting the methylation marker WEE1P1 gene or fragments thereof are as shown in SEQ ID NO. 82 and SEQ ID NO. 83; And / or, the primer pair nucleotide sequences for detecting the methylation marker NOL4 gene or fragments thereof are shown in SEQ ID NO. 91 and SEQ ID NO.

92.

12. The product of claim 6, wherein, The nucleotide sequence of the probe for detecting the methylation level of the FOXD3 gene or its fragments, a methylation marker, is shown in SEQ ID NO.42; And / or, the nucleotide sequence of the probe for detecting the methylation level of the methylation marker ARHGEF4 gene or fragments thereof is shown in SEQ ID NO.54; And / or, the nucleotide sequence of the probe for detecting the methylation level of the WEE1P1 gene or fragment thereof, a methylation marker, is shown in SEQ ID NO.84; And / or, the nucleotide sequence of the probe for detecting the methylation level of the NOL4 gene or fragment thereof is shown in SEQ ID NO.

93.

13. A device for detecting cervical cancer and / or precancerous lesions of the cervix, comprising the following modules: a data acquisition module, used to provide methylation level data of a target biomarker in a sample to be tested, wherein the target biomarker is a methylation biomarker as described in any one of claims 1 to 5; The judgment module is used to assess cervical cancer and / or precancerous lesions based on the methylation level data of the target biomarker in the test sample.

14. The detection device of claim 13, wherein, Based on the methylation level data of the target biomarker in the sample to be tested, the ΔCt value of the methylation biomarker is calculated. Preferably, the methylation level data is the Ct value of the methylation biomarker detected by quantitative methylation-specific PCR, and the ΔCt value is the difference between the Ct value of the target biomarker and the Ct value of the internal reference gene. More preferably, the internal reference gene is β-actin.

15. The detection device of claim 14, wherein, The status of cervical cancer and / or precancerous lesions is assessed based on the ΔCt value and a preset ΔCt cutoff value. Preferably, the test sample is determined to be negative or positive based on the ΔCt value and the preset ΔCt cutoff value. More preferably, the ΔCt cutoff value is the ΔCt value that maximizes the Youden index.

16. The detection device of claim 13, wherein, The sample to be tested includes any one or more of the following: blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells. Preferably, the sample to be tested is cervical exfoliated cells.

17. The detection device of claim 15, wherein, When the ΔCt value of the target marker in the test sample is less than or equal to the cutoff value of ΔCt, the test sample is determined to be positive. And / or, when the ΔCt value of the target marker of the test sample is greater than the cutoff value of ΔCt, the test sample is determined to be negative; And / or, the positive means that the individual corresponding to the test sample is a patient with severe cervical precancerous lesions or cervical cancer, and the negative means that the individual corresponding to the test sample is a healthy person, a patient with mild cervical precancerous lesions, or a patient with moderate cervical precancerous lesions.

18. A computer-readable storage medium, characterized in that, The storage medium stores computer instructions, which, when executed by a processor, implement a method for detecting cervical cancer and / or precancerous cervical lesions, the method comprising: Acquire methylation level data of the target biomarker in the sample to be tested, wherein the target biomarker is the methylation biomarker in any one of the applications of claims 1 to 5; Based on the methylation level data of the target biomarkers in the test samples, assess the status of cervical cancer and / or precancerous lesions of the cervix.

19. The computer-readable storage medium as claimed in claim 18, characterized in that, Based on the methylation level data of the target biomarker in the sample to be tested, the ΔCt value of the methylation biomarker is calculated. Preferably, the methylation level data is the Ct value of the methylation biomarker detected by quantitative methylation-specific PCR, and the ΔCt value is the difference between the Ct value of the target biomarker and the Ct value of the internal reference gene. More preferably, the internal reference gene is β-actin.

20. The computer-readable storage medium of claim 19, wherein, The status of cervical cancer and / or precancerous lesions is assessed based on the ΔCt value and a preset ΔCt cutoff value. Preferably, the test sample is determined to be negative or positive based on the ΔCt value and the preset ΔCt cutoff value. More preferably, the ΔCt cutoff value is the ΔCt value that maximizes the Youden index.

21. The computer-readable storage medium of claim 18, wherein, The sample to be tested includes any one or more of the following: blood, serum, plasma, lymph, urine, cervical scraping cells or tissue, biopsy tissue, surgical tissue, or cervical exfoliated cells. Preferably, the sample to be tested is cervical exfoliated cells.

22. The computer-readable storage medium of claim 20, wherein, When the ΔCt value of the target marker in the test sample is less than or equal to the cutoff value of ΔCt, the test sample is determined to be positive. And / or, when the ΔCt value of the target marker of the test sample is greater than the cutoff value of ΔCt, the test sample is determined to be negative; And / or, the positive means that the individual corresponding to the test sample is a patient with severe cervical precancerous lesions or cervical cancer, and the negative means that the individual corresponding to the test sample is a healthy person, a patient with mild cervical precancerous lesions, or a patient with moderate cervical precancerous lesions.

23. An electronic terminal, characterized in that The electronic terminal includes: a processor, a memory, an input / output interface, and a communication port; the memory is used to store a computer program, and the processor is used to execute the computer program stored in the memory to cause the terminal to perform the method for detecting cervical cancer and / or precancerous lesions of the cervix as described in any one of claims 18 to 22 in a computer-readable storage medium.

24. A computer program product comprising a computer program, wherein when the computer program is executed by a processor, a method for performing the following steps: acquiring methylation level data of a target biomarker in a test sample, and assessing cervical cancer and / or precancerous lesions based on the methylation level data of the target biomarker in the test sample.

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