Biomarker for lymphatic metastasis of cervical cancer and diagnostic application of biomarker

Abstract

The invention provides a biomarker for diagnosing lymphatic metastasis of cervical cancer and application of the biomarker, and belongs to the field of medical diagnosis. According to the invention, three biomarkers of resting sulfydryl oxidase 1, paraoxonase 1 and keratin 6A for diagnosing lymphatic metastasis of cervical cancer are obtained through multivariate statistical analysis, difference screening, random forest, ROC analysis and other biosignal analysis methods. Based on the biomarker provided by the invention, a kit and the like for diagnosing lymphatic metastasis of cervical cancer are constructed, and the kit and the like are used for accurately judging whether lymphatic metastasis occurs in cervical cancer patients or not.

Description

[0001] This application is a divisional application filed for an application with an application date of July 11, 2024, application number 2024109296052, and invention name “Biomarkers for diagnosing cervical cancer lymph node metastasis and their applications”. Technical Field

[0002] The present invention belongs to the technical field of medical diagnosis, and in particular relates to biomarkers for diagnosing cervical cancer lymph node metastasis and applications thereof. Background Art

[0003] In recent years, cancer has become a major challenge in global public health, with a large number of new cancer cases diagnosed daily. Cervical cancer is one of the most common gynecological malignancies, and its pathogenesis is closely related to infection with high-risk human papillomavirus (HPV). According to the World Health Organization (WHO), 604,000 new cases of cervical cancer were reported worldwide in 2020, of which approximately 342,000 patients died. In China, cervical cancer ranks sixth among female malignant tumor deaths and ranks first in the incidence of female reproductive system malignancies.

[0004] In patients with cervical cancer, lymph node metastasis is one of the main pathological processes and has a significant impact on the patient's prognosis. The 5-year overall survival rate of patients without lymph node metastasis exceeds 90%, while that of patients with lymph node metastasis is only about 50%. Therefore, the accurate diagnosis and treatment of cervical cancer lymph node metastasis has attracted much attention. For patients with positive lymph node metastasis, if appropriate treatment is not given in time, they may relapse rapidly. At the same time, for patients who need to be evaluated for lymph node metastasis before surgery, if unnecessary extensive lymph node dissection surgery is performed, the risk of unnecessary complications may increase. Therefore, for early-stage patients or patients with non-surgical indications, the detection and accurate diagnosis of lymph node metastasis is crucial.

[0005] Currently, the main methods for evaluating cervical cancer lymph node metastasis include transabdominal / laparoscopic pathological biopsy and imaging diagnosis, such as computed tomography (CT) and magnetic resonance imaging (MRI). However, some patients may have low sensitivity to imaging examinations, which may lead to underestimation of lymph node metastasis, thus affecting treatment efficacy and patient prognosis.

[0006] Biomarkers are molecules, cells, or tissues with specific characteristics and functions that exist within an organism and can reflect the organism's physiological state, pathological processes, or response to treatment. Finding suitable biomarkers is of great significance in the prediction and diagnosis of cervical cancer lymph node metastasis. However, finding reliable biomarkers is not easy. First, because the pathological process of cervical cancer is extremely complex and involves the regulation of multiple biological mechanisms, the selection of biomarkers needs to take this complexity into account. Second, biomarkers must have sufficient specificity and sensitivity to ensure that they can be reliably detected in the early stages of lymph node metastasis. In addition, the detection method of biomarkers needs to be highly accurate and reproducible to ensure their reliability and effectiveness in clinical applications.

[0007] Therefore, identifying biomarkers for cervical cancer lymph node metastasis is a challenging task that requires interdisciplinary collaboration and extensive experimental validation. Researchers need to utilize techniques from multiple fields, including molecular biology, cell biology, and biochemistry, to extensively screen and identify candidate markers. These markers then need to be validated and evaluated in clinical samples to determine their precise role in cervical cancer lymph node metastasis and their potential clinical application.

[0008] Although finding suitable biomarkers is difficult, once successfully identified and validated, it will bring substantial progress to the early diagnosis and personalized treatment of cervical cancer lymph node metastasis. Therefore, despite the challenges, the search for biomarkers remains essential to improve the treatment and prognosis of cervical cancer patients. Summary of the Invention

[0009] In order to solve the above technical problems, the present invention provides biomarkers for diagnosing cervical cancer lymph node metastasis and applications thereof.

[0010] To this end, the present invention adopts the following technical solutions:

[0011] In the first aspect, the present invention provides a biomarker for diagnosing cervical cancer lymph node metastasis, which is a cervical cancer lymph node metastasis marker present in human plasma; the cervical cancer lymph node metastasis marker is a combination of one or more markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A.

[0012] Preferably, the cervical cancer lymph node metastasis marker is a combination of three markers: resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A.

[0013] In a second aspect, the present invention provides a use of a reagent for detecting a cervical cancer lymph node metastasis marker as described in the first aspect in preparing a diagnostic kit or detection device for cervical cancer lymph node metastasis.

[0014] In a third aspect, the present invention provides a kit for diagnosing cervical cancer lymph node metastasis, including a kit for detecting the cervical cancer lymph node metastasis marker described in the first aspect.

[0015] Preferably, the kit is an ELISA kit.

[0016] In a fourth aspect, the present invention provides a use of the cervical cancer lymph node metastasis marker as described in the first aspect in the diagnosis of cervical cancer lymph node metastasis for purposes other than disease diagnosis or treatment.

[0017] In a fifth aspect, the present invention provides a method for diagnosing whether a subject has cervical cancer lymph node metastasis, which comprises detecting the level of any one of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A in the subject's plasma, and optionally detecting the levels of all three of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A.

[0018] In a sixth aspect, the present invention provides a method for pre-evaluating whether a drug can prevent or treat cervical cancer lymph node metastasis, which comprises using any one, any two, or any three of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A as biomarkers to evaluate the effect of the drug, so as to achieve the purpose of precision medicine.

[0019] In the seventh aspect, the present invention provides a method for screening compounds that can prevent or treat cervical cancer lymph node metastasis, which comprises using any one, any two, or any three of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A as biomarkers to evaluate the effect of the compound.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a new molecular marker that can be used to identify cervical cancer lymph node metastasis, which can be used for early detection, diagnosis and prediction of cervical cancer lymph node metastasis, thereby achieving more accurate medication.

[0022] The biomarkers for diagnosing cervical cancer lymph node metastasis provided by the present invention are a combination of any one, two or three of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A. By comparing their levels in plasma with the levels in the plasma of patients with cervical cancer lymph node metastasis, cervical cancer lymph node metastasis can be diagnosed with high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1 OPLS-DA analysis of the proteomic results in the examples.

[0025] Figure 2 OPLS-DA analysis of the metabolomics results in the examples.

[0026] Figure 3 This is a volcano plot of differential proteins in the examples.

[0027] Figure 4 This is a volcano plot of differential metabolites in the examples.

[0028] Figure 5 4 is the ROC analysis result of cervical cancer lymph node metastasis biomarkers in the examples.

[0029] Figure 6 This is the ROC of the combined model of three markers for cervical cancer lymph node metastasis in the example.

[0030] Figure 7 This is the ROC curve analysis of three markers of cervical cancer lymph node metastasis.

[0031] Figure 8 The ROC analysis results of the three marker combination model for cervical cancer lymph node metastasis are shown. DETAILED DESCRIPTION

[0032] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below. The technical features in the various embodiments of the present invention can be combined accordingly without conflicting with each other.

[0033] Lymphatic metastasis of cervical cancer is one of the common metastatic modes of cervical cancer. The present invention provides corresponding biomarkers for this phenomenon, which are selected from any one, two or three of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A.

[0034] It should be noted that the resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A in the present invention are all known proteins in the human body. They can be searched through public protein databases, for example, the protein IDs in Uniprot are shown in Table 1 below.

[0035] Quiescent sulfhydryl oxidase-1 (QSOX1), accession number O00391 in the protein database Uniprot, is an enzyme that catalyzes redox reactions and plays an important role both inside and outside the cell. It catalyzes the oxidation of free sulfhydryl groups (i.e., groups containing sulfhydryl groups) to form disulfide bonds. This process contributes to the folding and formation of proteins within the cell and regulates the formation and stability of the matrix in the extracellular environment. QSOX1 plays a key role in intracellular redox homeostasis and is closely associated with the occurrence and progression of several diseases, particularly cancer.

[0036] Paraoxonase-1 (PON1), accession number P27169 in the protein database Uniprot, is an antioxidant enzyme that plays a key role in maintaining oxidation-antioxidation balance in the body. PON1 has been found to be associated with systemic oxidative stress and cardiovascular events, but there are few reports on its association with cervical cancer lymph node metastasis or its use as a biomarker for cervical cancer lymph node metastasis.

[0037] Keratin 6A (KRT 6A), accession number P0253 in the protein database Uniprot, is a member of the keratin family and a cytoskeletal protein. It is primarily found in keratinized tissues such as skin, hair, and nails, where it maintains cellular morphology and mechanical support. Keratin 6A is highly expressed in the epidermis. Its primary function is to provide structural support for cells, protecting them from environmental damage. It also regulates keratinocyte proliferation and differentiation. In certain diseases, such as skin diseases and cancer, Keratin 6A expression levels may vary, correlating with the onset and progression of the disease.

[0038] Table 1 Protein IDs and Chinese and English names

[0039]

[0040]

[0041] It should be noted that any one of the above-mentioned cervical cancer lymph node metastasis markers can be used alone as a basis for diagnosing cervical cancer lymph node metastasis. However, due to the possibility of misdiagnosis using a single marker, in order to improve the accuracy and reliability of diagnosis, a more preferred approach is to use a combination of these three markers as a marker for cervical cancer lymph node metastasis.

[0042] Combining three markers—resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A—can improve the sensitivity and specificity of cervical cancer lymph node metastasis. This combination strategy helps reduce the likelihood of misdiagnosis and improves the accurate diagnosis rate of cervical cancer lymph node metastasis, providing a more reliable basis for patient treatment and management.

[0043] Therefore, although a single marker also has certain diagnostic value, it is still preferred to use a combination of three markers as the basis for the diagnosis of cervical cancer lymph node metastasis to ensure the accuracy and reliability of the diagnostic results.

[0044] In another embodiment of the present invention, there is provided a use of a reagent for detecting the above-mentioned cervical cancer lymph node metastasis marker (a single marker or a combination of multiple markers) in the preparation of a kit or detection device for diagnosing cervical cancer lymph node metastasis.

[0045] Because the aforementioned cervical cancer lymph node metastasis markers may be single markers or combinations of multiple markers, the corresponding diagnostic kits or detection devices may also take various forms. For example, they may be diagnostic kits or detection devices for a single marker, i.e., kits or detection devices specifically for detecting any one of resting sulfhydryl oxidase-1, paraoxonase 1, or keratin 6A. Furthermore, for example, they may be combinations of diagnostic kits or detection devices that include multiple markers, i.e., kits or detection devices that simultaneously detect resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A.

[0046] This diverse design allows for greater applicability and flexibility in diagnostic kits or test devices, allowing for the selection of appropriate testing protocols based on specific clinical needs and diagnostic requirements. However, whether a diagnostic kit is based on a single marker or a combination of multiple markers, it is expected to provide relatively reliable and accurate diagnostic results.

[0047] The reagents included in the specific kit of the present invention can be set according to the detection method of each marker. This means that for the three markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A, specific methods and reagents suitable for their detection can be selected.

[0048] Since these three markers are known substances, extensive and reliable detection methods already exist in the prior art. For example, immunological methods such as enzyme-linked immunosorbent assay (ELISA) and immunofluorescence analysis can be used to detect the levels of these markers. Molecular biology techniques such as polymerase chain reaction (PCR) and in situ hybridization can also be used to detect the gene expression levels of these markers. In addition, mass spectrometry and other methods can also be used.

[0049] Based on the rich experience and mature methods of the prior art, the most suitable method for detecting the above-mentioned markers (single marker or combination of multiple markers) can be selected by referring to existing detection methods and reagents. Then, based on the selected detection method, the corresponding diagnostic kit or detection device can be designed according to the standards and procedures of the prior art.

[0050] ELISA is a commonly used biomarker detection method that utilizes specific antibodies to bind to the biomarker being detected, followed by an enzymatic reaction to produce a measurable signal. The ELISA method is highly sensitive and specific and can be applied to the detection of a single biomarker or multiple biomarkers.

[0051] Immunofluorescence analysis uses fluorescently labeled antibodies to bind to the biomarker to be detected and detects the intensity of the fluorescent signal using a fluorescence microscope or fluorescence photometer to quantitatively analyze the presence of the biomarker.

[0052] Mass spectrometry can accurately quantify biomarkers and includes methods such as proton nuclear magnetic resonance (NMR) and mass spectrometry (MS), which can provide very precise molecular structure and relative content information.

[0053] PCR technology can be used to detect biomarker RNA transcripts. The RNA or its fragments to be detected are amplified by PCR and then quantitatively analyzed by methods such as gel electrophoresis or real-time fluorescence quantitative PCR.

[0054] Protein chip technology can detect multiple biomarkers simultaneously by fixing multiple antibodies on the chip surface, which then bind to proteins in the sample to be tested (such as plasma) and finally detect the biomarkers through fluorescent or radioactive labeled signals.

[0055] Exemplarily, considering detection efficiency and convenience, the kit may be an ELISA kit.

[0056] In another embodiment of the present invention, the use of the aforementioned biomarker (a single marker or a combination of multiple markers) in the diagnosis of cervical cancer lymph node metastasis for non-disease diagnosis or treatment purposes is provided. The non-disease diagnosis or treatment purpose of cervical cancer lymph node metastasis diagnosis can be used for scientific research, non-medical commercial detection or testing, etc.

[0057] For example, biomarkers of cervical cancer lymph node metastasis can be used to study disease mechanisms, pathophysiological processes, and potential therapeutic targets. By analyzing changes in these markers during the disease process, we can gain a deeper understanding of the disease's development and metastasis mechanisms, providing a deeper understanding of disease prevention and treatment.

[0058] Biomarkers also play a crucial role in drug development. They can serve as indicators for evaluating drug efficacy, helping researchers assess the safety and effectiveness of new drugs. By monitoring the effects of drugs on biomarkers, potential drug toxicities and adverse reactions can be identified early, guiding dosage adjustments and optimization.

[0059] Biomarkers can also be used in individual health management and preventive medicine. By regularly monitoring specific biomarkers, changes in individual health status can be detected, disease risk can be predicted, and appropriate health management measures can be taken, such as adjusting lifestyle, dietary habits, or taking medication, to maintain health and slow disease progression.

[0060] Biomarkers can be used to assess the impact of lifestyle on health. By monitoring changes in these biomarkers, we can evaluate the extent to which different lifestyles affect health and guide individuals to adopt appropriate lifestyle interventions, such as weight loss, smoking cessation, and increased physical activity, thereby improving health and reducing disease risk.

[0061] The present invention also provides a method for diagnosing whether a subject has cervical cancer lymph node metastasis. The key to this method is to detect the level of any one of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A in the subject's plasma, or optionally any combination of these three markers.

[0062] First, the implementation of this method involves sample collection and processing. Plasma samples are extracted from the subject's venous blood, a process that requires strict standard operating procedures to ensure sample quality and integrity.

[0063] Secondly, for the plasma samples already obtained, appropriate techniques and reagents can be used to detect the levels of any of the following: resting sulfhydryl oxidase-1, paraoxonase-1, and keratin 6A. These detection methods can include immunological techniques such as ELISA and immunofluorescence analysis, molecular biology techniques such as PCR and in situ hybridization, or mass spectrometry. These methods can accurately quantify the level of a specific marker in the subject's plasma.

[0064] This method also provides another option, namely, optionally detecting any combination of the three markers: resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A. The choice of this combination can be determined based on actual needs and clinical circumstances. For example, the levels of these three markers can be detected simultaneously, or one or more combinations can be selected based on previous research or clinical experience.

[0065] After obtaining marker test results, they need to be compared and analyzed with relevant data from patients without lymph node metastasis. Based on these comparisons, the subject's risk of cervical cancer lymph node metastasis can be determined. If the level of a particular marker or marker combination is significantly lower than the normal range, it can be inferred that the subject is at high risk of cervical cancer lymph node metastasis.

[0066] The reliability and accuracy of this method depend on the sensitivity and specificity of the selected detection method, as well as the determination of the reference range or threshold. Therefore, when implementing this method, it is necessary to strictly control the experimental conditions and combine clinical practice experience and data analysis to interpret the results and make a diagnosis. In addition, the test results of any one marker should be carefully evaluated and interpreted to avoid uncertainty in the diagnostic results due to the possibility of misdiagnosis due to the potential for misdiagnosis of a single marker.

[0067] Preferably, an index value is set for each marker, which is the ratio of the marker's level in the subject's plasma to the level in the plasma of patients without cervical cancer lymph node metastasis. The risk range for each marker is exemplified as follows: the risk range for resting sulfhydryl oxidase-1, indicating cervical cancer lymph node metastasis, is an index value less than 0.59; the risk range for paraoxonase 1, indicating cervical cancer lymph node metastasis, is an index value less than 0.56; and the risk range for keratin 6A, indicating cervical cancer lymph node metastasis, is an index value less than 0.24. The results showed that all 10 selected patients with cervical cancer lymph node metastasis fell within the risk range, indicating that the combination of the three biomarkers of the present invention has superior accuracy compared to a single biomarker.

[0068] The present invention provides a method for pre-evaluating whether a drug can prevent or treat cervical cancer lymph node metastasis, which comprises using any one, any two, or any three of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A as biomarkers to evaluate the effect of the drug, so as to achieve the purpose of precision medicine.

[0069] Implementation of this method begins with experimental design and sample collection. During the design phase of a study or clinical trial, the drug to be evaluated, the treatment regimen, and the evaluation criteria must be clearly defined. Furthermore, subject selection criteria must be determined, and appropriate biological samples, such as plasma, tissue, or cell samples, must be collected for subsequent experimental analysis.

[0070] For a selected drug or treatment regimen, any one, any two, or all three of resting sulfhydryl oxidase-1, paraoxonase-1, and keratin 6A can be used as biomarkers to assess the therapeutic effect. The selection of these markers can be determined based on previous research or clinical experience, or can be inferred based on the drug's mechanism of action and expected effect.

[0071] After obtaining a biological sample, appropriate techniques and reagents can be used to detect the levels of the selected markers. These detection methods can include immunological techniques such as ELISA and immunofluorescence analysis, molecular biology techniques such as PCR and in situ hybridization, or mass spectrometry. These methods can accurately quantify the levels of the markers in the subject's biological sample.

[0072] Next, the drug or treatment regimen is administered to the subject, and treatment is continued according to the prescribed regimen. During treatment, the subject's biological samples are regularly monitored, and the levels of selected markers are repeatedly measured. This allows for timely monitoring of the drug's therapeutic effects and whether the intended treatment goals have been achieved.

[0073] Finally, based on the test results obtained, the therapeutic effect of the drug can be evaluated and determined. If the levels of the selected markers change significantly, with a clear difference compared to pre-treatment levels, it can be inferred that the drug may have the potential to prevent or treat cervical cancer lymph node metastasis. Conversely, if the marker levels do not change significantly or do not achieve the expected effect, it may be necessary to re-evaluate the treatment plan or try other treatment strategies.

[0074] The present invention provides a method for screening compounds capable of preventing or treating cervical cancer lymph node metastasis, which comprises using any one, any two or three of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A as biomarkers to evaluate the effect of the compound.

[0075] First, the implementation of this method requires the establishment of a suitable experimental system and model. Cell lines or animal models of cervical cancer lymphatic metastasis can be used to simulate the physiological process of cervical cancer lymphatic metastasis and evaluate the effects of compounds on it.

[0076] Secondly, appropriate biomarkers should be selected to evaluate the compound's efficacy. Resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A are biomarkers that reflect the biological processes and pathological status of cervical cancer lymph node metastasis. Therefore, they can be used as biomarkers to screen compounds and assess their efficacy in preventing cervical cancer lymph node metastasis.

[0077] Next, compound screening experiments are conducted. The compounds to be screened are added to cells or animals to observe their effects on resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A. Appropriate techniques and reagents, such as immunology and molecular biology, can be used to measure the levels of these markers. Based on these test results, a preliminary assessment of the compound's potential therapeutic effect on cervical cancer lymph node metastasis can be made.

[0078] After obtaining preliminary screening results, further verification and confirmation experiments can be conducted. By using more sophisticated and rigorous experimental designs and operational procedures, the effects of the compound on marker levels can be verified and its therapeutic effect on cervical cancer lymph node metastasis can be further evaluated.

[0079] Finally, based on the experimental results, compounds with potential therapeutic effects are further researched and developed. Their pharmacological properties, toxicity, side effects, pharmacokinetics, and other drug attributes can be further evaluated, and preclinical studies and clinical trials can be conducted.

[0080] The present invention further illustrates the selection principle, process and effect of the above-mentioned biomarkers through a specific example below, so that those skilled in the art can understand the essence of the present invention.

[0081] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. All reagents used in the present invention are of analytical grade or higher. The chromatographic column used is Acclaim Pep Map100C18 column, manufactured by Thermo; and the liquid chromatograph is EASY-nLC 1200, manufactured by Thermo.

[0082] Example 1: Selection of biomarkers

[0083] Plasma samples were collected from 40 participants, including 25 patients with cervical cancer without lymph node metastasis and 15 patients with cervical cancer with lymph node metastasis. Blood was collected from the elbow vein in EDTA-anticoagulant tubes, gently inverted to mix, and centrifuged at 1600 g for 10 minutes at 4°C. The supernatant was collected and transferred to a 1.5 ml centrifuge tube. Centrifuged at 13,000 g for 2 minutes at 4°C, the supernatant was collected and aliquoted into multiple centrifuge tubes. After labeling, the collected plasma was immediately transferred to -80°C and stored in the dark to avoid repeated freezing and thawing.

[0084] Reagents used in this example include formic acid, methanol, ammonium formate, ammonium bicarbonate, acetonitrile, dithiothreitol (DTT), and iodoacetamide (IAM).

[0085] 1. Targeting protein markers

[0086] Plasma samples were depleted of high-abundance proteins. After protein concentration was determined, 100 μg of blood protein was purified and then subjected to reductive alkylation. The specific steps were as follows: high-abundance protein removal, protein purification, enrichment and quality control, and proteolysis. The resulting peptides were then desalted on ZipTip Pipette Tips (Merck, Germany), dried by vacuum centrifugation, and stored at −80°C until analysis.

[0087] Proteomic analysis was performed using an EASY-nLC 1200 nanoliter ultra-high-performance liquid chromatography (UHPLC) system (Thermo Fisher, USA) coupled to an Orbitrap Exploris 480 high-resolution mass spectrometer (Thermo Fisher, USA). The chromatographic column was an Acclaim Pep Map 100C18 column (75 μm inner diameter, 2 μm particle size, approximately 25 cm column length). Separation was performed at a flow rate of 300 nL / min using the following efficient gradient: 0–1.0 min, 3%–35% Solution B (98% acetonitrile, 0.1% formic acid); 1–56 min, 35%–95% Solution B; 56–59 min, 95% Solution B; 59–65 min, 95%–3% Solution B. The column temperature was maintained at 40°C, and the sample manager temperature was set to 4°C. The end of the nanoliter liquid phase separation was directly connected to a mass spectrometer and detected according to the following parameters. The peptides separated by the liquid phase were ionized by a nano ESI source and then entered the tandem mass spectrometer Orbitrap Exploris 480 (Thermo Fisher, USA) for DDA (Data Dependent Acquisition) mode detection.

[0088] 2. Targeting metabolite markers

[0089] First, metabolite extraction was performed: After slowly thawing the plasma sample at 4°C, 100 μL was transferred to a 1.5 mL EP tube. 300 μL of extraction solution (methanol:acetonitrile = 2:1, v:v, pre-chilled at -20°C), 10 μL of internal standard 1, and 10 μL of internal standard 2 were added. The sample was vortexed for 1 minute, allowed to stand at -20°C for 30 minutes, and then centrifuged at 16,000 g for 30 minutes at 4°C. After centrifugation, 300 μL of the supernatant was collected and placed in a freeze-vacuum concentrator to dryness. The supernatant was then reconstituted with 150 μL of reconstitution solution (methanol:water = 1:1, v:v), vortexed, and centrifuged at 14,000 g at 4°C for 15 minutes. The supernatant was then transferred to a sample injection vial. 15 μL of the supernatant from each sample was mixed to form a QC sample for evaluating the reproducibility and stability of the LC-MS analysis.

[0090] The metabolites were then separated and detected using Vanquish ultra-performance liquid chromatography (UPLC) (Thermo Fisher, USA) coupled with a high-resolution mass spectrometer Orbitrap Exploris 480 (Thermo Fisher, USA).

[0091] 3. Bioinformatics Analysis to Find Biomarkers

[0092] Biomarkers are mainly obtained through bioinformatics analysis methods such as differential screening, random forest, and ROC analysis. The main criteria for screening differences are fold change ≥ 1.2 or ≤ 0.83, and P value < 0.05. ROC curve analysis is then performed on the differential proteins and metabolites, and then combined with random forest sorting, the results are integrated to select the final biomarkers. Based on the above bioinformatics analysis, three markers were screened as markers of cervical cancer lymph node metastasis. They are one or more combinations of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A, and all three markers are proteins.

[0093] Figure 1 and Figure 2 The OPLS-DA results for the proteome and metabolome of this example are shown separately. The Non-Met group represents the cervical cancer non-metastasis group, and the Met-Lymph group represents the cervical cancer lymph node metastasis group. The OPLS-DA analysis chart reflects the overall distribution of samples in each group.

[0094] Figure 3 This is a volcano plot of differentially expressed proteins between the cervical cancer non-metastasis group and the cervical cancer lymph node metastasis group. A total of 58 proteins were screened, showing significant differences between patients with cervical cancer lymph node metastasis and those without recurrence or metastasis. Of these, 31 were downregulated and 27 were upregulated. Figure 4 This is a volcano plot showing the differential metabolites between the cervical cancer non-metastasis group and the cervical cancer lymph node metastasis group. A total of 288 metabolites were screened to show significant differences between patients with cervical cancer lymph node metastasis and those without recurrence or metastasis. Of these, 183 were downregulated and 105 were upregulated.

[0095] Figure 5 This is the ROC curve (receiver operating characteristic curve) analysis of the three markers in the cervical cancer lymph node metastasis marker obtained by bioinformatics analysis in this embodiment, that is, the receiver operating characteristic curve analysis. The sensitivity and specificity of the three markers in diagnosing cervical cancer lymph node metastasis are evaluated by plotting ROC and calculating the area under the curve (AUC). The judgment criteria are as follows: when AUC < 0.5, it means that the diagnosis is meaningless; when AUC = 0.5-0.7, it means that the diagnostic accuracy is low; when AUC = 0.7-0.9, it means that the diagnostic accuracy is medium; when AUC > 0.9, it means that the diagnostic accuracy is high. That is, in the ROC curve, the AUC value is the area covered by the ROC curve. The larger the value, the higher the accuracy of the model prediction.

[0096] from Figure 5It can be seen that the three markers screened in this example, keratin 6A, resting sulfhydryl oxidase-1 (QSOX1), and paraoxonase 1 (PON1), all have high diagnostic accuracy for cervical cancer lymph node metastasis. The AUC value of keratin 6A (KRT6A) is 0.909, the AUC value of resting sulfhydryl oxidase-1 (QSOX1) is 0.907, and the AUC value of paraoxonase 1 (PON1) is 0.907. This indicates that any one of the markers, resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A, can be used to diagnose the presence of cervical cancer lymph node metastasis.

[0097] 4. Marker combination modeling using logistic regression

[0098] For the cervical cancer lymph node metastasis group, the three candidate biomarkers of cervical cancer lymph node metastasis obtained previously were combined to construct a logistic regression model, and ROC curve analysis and evaluation were performed. Figure 6 The results of a receiver operating characteristic (ROC) analysis of a three-marker combination model for cervical cancer lymph node metastasis were presented. The results showed that the area under the curve (AUC) for the biomarker combination was 0.973. Compared with the single markers described above, the combination of resting sulfhydryl oxidase-1, paraoxonase-1, and keratin 6A demonstrated superior predictive accuracy.

[0099] Example 2: Further confirmation of biomarkers

[0100] 1. Plasma samples from 8 additional patients with cervical cancer with lymph node metastasis and 10 patients without lymph node metastasis were collected and the levels of three biomarkers, keratin 6A, resting sulfhydryl oxidase-1 (QSOX1), and paraoxonase 1 (PON1), were measured according to Example 1. The levels of these three biomarkers were compared with the plasma levels of the patients with cervical cancer without lymph node metastasis. The difference was determined by a fold change ≥ 1.2 or ≤ 0.83, with a P value < 0.05.

[0101] The results showed that for any of the three biomarkers, 8 out of the 8 selected patients with cervical cancer lymph node metastasis had significant differences compared with the control group, that is, 8 were positive patients.

[0102] According to the combined model analysis constructed in Example 1, and setting the index value of each marker as the ratio between the level of the marker in the human plasma of the subject and the level in the plasma of patients without cervical cancer lymph node metastasis, the risk range of each marker is as follows: the risk range corresponding to resting sulfhydryl oxidase-1 indicating cervical cancer lymph node metastasis is an index value less than 0.59; the risk range corresponding to paraoxonase 1 indicating cervical cancer lymph node metastasis is an index value less than 0.56; the risk range corresponding to keratin 6A indicating cervical cancer lymph node metastasis is an index value less than 0.24. The results showed that all 8 selected patients with cervical cancer lymph node metastasis fell into the risk range, indicating that the combination of the three biomarkers of the present invention has better accuracy than a single biomarker.

[0103] 2. Receiver operating characteristic (ROC) curve analysis of the three biomarkers revealed an AUC value of 0.625 for keratin 6A (KRT6A), 0.975 for quiescent sulfhydryl oxidase-1 (QSOX1), and 0.912 for paraoxonase 1 (PON1). This suggests that any one of these markers, QSOX1, paraoxonase 1, and keratin 6A, can be used to diagnose lymph node metastasis in cervical cancer. Figure 7 This is the ROC curve analysis of the three cervical cancer lymph node metastasis markers obtained by the bioinformatics analysis in Example 2.

[0104] 3. In another cohort of 8 patients with lymph node metastasis and 10 patients without metastasis, the three biomarkers were combined to construct a logistic regression model, and the ROC curve was used for analysis and evaluation. Figure 8 The results of a receiver operating characteristic (ROC) analysis of a three-marker combination model for cervical cancer lymph node metastasis were presented. The results showed that the area under the curve (AUC) for the biomarker combination was 0.988. Compared with the aforementioned single markers, the combination of resting sulfhydryl oxidase-1, paraoxonase-1, and keratin 6A demonstrated superior predictive accuracy.

[0105] Example 3: ELISA kit for cervical cancer lymph node metastasis

[0106] This embodiment provides an ELISA kit for diagnosing cervical cancer lymph node metastasis. The kit is designed to detect the levels of three markers, namely resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A, in the plasma of subjects, thereby achieving accurate diagnosis of cervical cancer lymph node metastasis.

[0107] Kit Composition:

[0108] Resting sulfhydryl oxidase-1 (Prx-1) ELISA analysis:

[0109] Includes reagents and materials such as antibodies, standards, substrates, and wash buffers for detecting resting sulfhydryl oxidase-1.

[0110] Used to detect the level of resting sulfhydryl oxidase-1 in the subject's plasma and perform quantitative analysis based on the standard curve.

[0111] Paraoxonase 1 (Tyr-1) ELISA analysis:

[0112] Includes reagents and materials such as antibodies, standards, substrates, wash buffer, etc. for detecting paraoxonase 1.

[0113] Used to detect the level of paraoxonase 1 in the subject's plasma and perform quantitative analysis based on the standard curve.

[0114] Keratin 6A (CK6A) ELISA analysis:

[0115] The kit includes antibodies, standards, substrates, washing buffers and other reagents and materials for detecting keratin 6A. It is used to detect the level of keratin 6A in the plasma of subjects and perform quantitative analysis based on the standard curve.

[0116] The detection process can be as follows:

[0117] Take the subject's plasma sample and perform specimen processing and pretreatment steps according to the kit instructions.

[0118] The pretreated plasma samples were added to the respective ELISA plate wells and reacted specifically with antibodies against resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A.

[0119] Wash the plate with wash buffer to remove unbound material.

[0120] A substrate is added and the reaction is allowed to proceed under appropriate conditions to produce a measurable color.

[0121] The absorbance of the reaction product was measured using a microplate reader, and the contents of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A in the sample were calculated based on the standard curve.

[0122] Based on the content levels of each marker and the preset diagnostic criteria, it is determined whether the subject has cervical cancer lymph node metastasis.

[0123] The embodiment described above is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Persons skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent substitution or equivalent transformation falls within the scope of protection of the present invention.

[0124] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0125] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A biomarker for diagnosing cervical cancer lymph node metastasis, characterized in that: It is a cervical cancer lymph node metastasis marker present in human plasma; the cervical cancer lymph node metastasis marker is resting sulfhydryl oxidase-1, or a combination thereof with any one of keratin 6A and paraoxonase 1 markers; or the cervical cancer lymph node metastasis marker is paraoxonase 1, or a combination thereof with any one of keratin 6A and resting sulfhydryl oxidase-1 markers; or the cervical cancer lymph node metastasis marker is keratin 6A, or a combination thereof with any one of keratin 6A and resting sulfhydryl oxidase-1 markers.

2. Use of a reagent for detecting the cervical cancer lymph node metastasis marker according to claim 1 in the preparation of a cervical cancer lymph node metastasis diagnostic kit or detection device.

3. The use according to claim 2, wherein the reagent is an antibody against the marker, a primer for PCR, a reagent for mass spectrometry analysis, or a reagent for chromatography analysis.

4. A kit for diagnosing cervical cancer lymph node metastasis, characterized in that: The invention comprises a reagent for detecting the cervical cancer lymph node metastasis marker according to claim 1. The kit according to claim 4 , wherein the detection is a quantitative detection of the biomarker level in the subject's plasma. 6 . The kit according to claim 4 , wherein the reagent is an antibody against the marker, a primer for PCR, a reagent for mass spectrometry analysis, or a reagent for chromatography analysis.

7. The kit for diagnosing cervical cancer lymph node metastasis according to claim 4 or 5, characterized in that: The kit is an ELISA kit.

8. Use of the cervical cancer lymph node metastasis marker according to claim 1 in the diagnosis of cervical cancer lymph node metastasis for purposes other than disease diagnosis or treatment.

9. Use of the marker according to claim 1 in preparing a preparation for diagnosing lymph node metastasis of cervical cancer.

10. A method for evaluating whether a drug can prevent or treat cervical cancer lymph node metastasis, comprising using the marker of claim 1 as a biomarker to evaluate the effect of the drug; or a method for screening a compound that can prevent or treat cervical cancer lymph node metastasis, comprising using the marker of claim 1 as a biomarker to evaluate the effect of the compound.

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