CN118759189A - Biomarkers for diagnosing lymph node metastasis of cervical cancer and their applications - Google Patents

Abstract

The invention provides a biomarker for diagnosing cervical cancer lymphatic metastasis and application thereof, belonging to the technical field of biological detection. The invention obtains 3 biomarkers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A for diagnosing cervical cancer lymphatic metastasis through multivariate statistical analysis, differential screening, random forest, ROC analysis and other biological analysis methods. Based on the biomarker, a kit for diagnosing cervical cancer lymphatic metastasis and the like are constructed and used for accurately judging whether cervical cancer patients have lymphatic metastasis or not.

Description

Biomarkers for diagnosing lymph node metastasis of cervical cancer and their applications

Technical Field

The present invention belongs to the technical field of biological detection, and in particular relates to a biomarker for diagnosing lymphatic metastasis of cervical cancer and its application.

Background Art

In recent years, cancer has become one of the major challenges in the field of global public health, with a large number of new cancer cases being diagnosed every day. Among them, cervical cancer is one of the most common gynecological malignancies, and its pathogenesis is closely related to the infection of high-risk human papillomavirus (HPV).

In patients with cervical cancer, lymph node metastasis is one of the main pathological processes and has an important impact on the patient's prognosis. The 5-year overall survival rate of patients without lymph node metastasis is over 90%, while that of patients with lymph node metastasis is only about 50%. Therefore, the accurate diagnosis and treatment of lymph node metastasis in cervical cancer 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 be increased. Therefore, for early-stage patients or patients with non-surgical indications, the detection and accurate diagnosis of lymph node metastasis is crucial.

Currently, the methods for evaluating cervical cancer lymph node metastasis mainly include transabdominal/laparoscopic pathological sampling 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 the treatment effect and patient prognosis.

Biomarkers refer to molecules, cells or tissues with certain characteristics and functions that exist in organisms and can reflect the physiological state, pathological process or response to treatment in organisms. In the prediction and diagnosis of cervical cancer lymph node metastasis, it is of great significance to find suitable biomarkers. However, it is not easy to find reliable biomarkers. 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. Secondly, 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 have a high degree of accuracy and repeatability to ensure its reliability and effectiveness in clinical applications.

Therefore, finding biomarkers for cervical cancer lymph node metastasis is a challenging task that requires interdisciplinary collaboration and extensive experimental validation. Researchers need to use technical means from multiple fields such as 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 exact role in cervical cancer lymph node metastasis and their prospects for clinical application.

Although it is difficult to find a suitable biomarker, once successfully found and verified, it will bring substantial progress to the early diagnosis and personalized treatment of cervical cancer lymph node metastasis. Therefore, despite the challenges, it is still necessary to find biomarkers to improve the treatment effect and prognosis of cervical cancer patients.

Summary of the invention

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

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

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.

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

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 the preparation of a cervical cancer lymph node metastasis diagnosis kit or a detection device.

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.

Preferably, the kit is an ELISA kit.

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.

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.

In a sixth aspect, the present invention provides a method for pre-evaluating whether a drug can prevent or treat lymph node metastasis of cervical cancer, 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.

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 three of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A as biomarkers to evaluate the effect of the compound.

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

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

The biomarkers for diagnosing cervical cancer lymph node metastasis provided by the present invention are a combination of any 1, 2 or 3 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

In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. 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.

FIG1 is an OPLS-DA analysis of the proteomic results in the examples.

FIG. 2 is an OPLS-DA analysis of the metabolomics results in the examples.

FIG. 3 is a volcano diagram of differential proteins in the examples.

FIG. 4 is a volcano plot of differential metabolites in the examples.

FIG. 5 is the ROC analysis result of the cervical cancer lymph node metastasis biomarker in the embodiment.

FIG. 6 is a ROC diagram of a combined model of three markers for cervical cancer lymph node metastasis in an embodiment.

FIG. 7 is a ROC curve analysis of three markers of cervical cancer lymph node metastasis.

FIG8 shows the ROC analysis results of the three marker combination model for cervical cancer lymph node metastasis.

DETAILED DESCRIPTION

In order to make the above-mentioned purpose, features and advantages of the present invention more obvious and easy to understand, the specific implementation mode of the present invention is described in detail below in conjunction with 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 different from 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 each embodiment of the present invention can be combined accordingly without conflicting with each other.

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

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

Quiescent sulfhydryl oxidase-1 (QSOX1), with accession number O00391 in the protein database uniprot, is an enzyme that catalyzes redox reactions and plays an important role in the cell's internal and external environment. It can catalyze the oxidation of free sulfhydryl groups (i.e., groups containing sulfhydryl groups) to form disulfide bonds. This process helps fold and form proteins within cells and regulates the formation and stability of the matrix in the cell's external environment. QSOX1 plays a key role in intracellular redox balance and is also closely related to the occurrence and development of some diseases, especially cancer.

Paraoxonase-1 (PON1), whose accession number in the protein database uniprot is P27169, is an antioxidant enzyme that plays a key role in maintaining the oxidation-antioxidation balance in the body. It has been found that PON1 is associated with systemic oxidative stress and cardiovascular events, but there are few reports that it is associated with lymphatic metastasis of cervical cancer or can be used alone as a biomarker for lymphatic metastasis of cervical cancer.

Keratin 6A (KRT 6A) has the accession number P0253 in the protein database uniprot. It is a member of the keratin family and a cytoskeletal protein. It is mainly found in keratinized tissues such as skin, hair, and nails, and plays a role in maintaining cell morphology and mechanical support. Keratin 6A is highly expressed in the epidermis. Its main function is to provide structural support for cells and protect cells from damage by the external environment. It also participates in regulating the proliferation and differentiation of keratinocytes. In certain diseases such as skin diseases and cancers, the expression level of Keratin 6A may change, which is related to the occurrence and development of the disease.

Table 1 Protein IDs and Chinese and English names

Cervical cancer lymph node metastasis markers Protein ID Sulfhydryl oxidase 1 (Sulfhydryl oxidase 1) O00391 Paraoxonase-1 P27169 Keratin 6A P02538

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, since a single marker may have a certain possibility of misdiagnosis, in order to improve the accuracy and reliability of diagnosis, a more preferred solution is to combine these three markers as markers for cervical cancer lymph node metastasis.

By combining the three markers of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A, the sensitivity and specificity of cervical cancer lymph node metastasis can be improved. This combination strategy helps to reduce the possibility of misdiagnosis and improve the accurate diagnosis rate of cervical cancer lymph node metastasis, providing a more reliable basis for the treatment and management of patients.

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.

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 a detection device for diagnosing cervical cancer lymph node metastasis.

Since the above-mentioned cervical cancer lymph node metastasis marker may be a single marker or a combination of multiple markers, the corresponding diagnostic kit or detection device may also be in various forms. Exemplarily, it may be a diagnostic kit or detection device for a single marker, that is, a kit or detection device specifically used to detect any one of resting sulfhydryl oxidase-1, paraoxonase 1 or keratin 6A. Further exemplarily, it may also be a combination of diagnostic kits or detection devices containing multiple markers, that is, a kit or detection device for simultaneously detecting resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A.

This diverse design allows diagnostic kits or detection devices to have a wider range of applications and flexibility, and appropriate detection schemes can be selected according to specific clinical needs and diagnostic requirements. However, whether it is a diagnostic kit for a single marker or a combination of multiple markers, it is expected to provide relatively reliable and accurate diagnostic results.

The reagents contained 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.

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

Based on the rich experience and mature methods of the prior art, the most suitable method can be selected to detect the above markers (single marker or a combination of multiple markers) with reference to the existing detection means and reagents. Then, according to the selected detection method, the corresponding diagnostic kit or detection device can be designed according to the standards and processes of the prior art.

ELISA is a commonly used biomarker detection method that uses specific antibodies to bind to the biomarker to be detected, and then produces a measurable signal through an enzyme reaction. The ELISA method has high sensitivity and specificity and can be applied to the detection of a single marker or multiple markers.

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

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

PCR technology can be used to detect the transcribed RNA of biomarkers. The RNA or its fragments to be detected are amplified by PCR, and then quantitatively analyzed by gel electrophoresis or real-time fluorescence quantitative PCR.

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

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

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

For example, biomarkers of cervical cancer lymph node metastasis can be used to study the disease mechanism, pathophysiological process and potential therapeutic targets. By analyzing the changes in these markers during the course of the disease, we can gain a deeper understanding of the development and metastasis mechanism of the disease and provide a deeper understanding for the prevention and treatment of the disease.

Biomarkers also play an important role in the drug development process. They can be used as indicators for drug efficacy evaluation to help researchers evaluate the safety and effectiveness of new drugs. By monitoring the effects of drugs on biomarkers, potential drug toxicity and adverse reactions can be detected early, and the adjustment and optimization of drug dosage can be guided.

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 in a timely manner, the risk of disease can be predicted, and appropriate health management measures can be taken, such as adjusting lifestyle, dietary habits or drug treatment, to maintain health and delay disease progression.

Biomarkers can be used to assess the impact of lifestyle on health. By monitoring changes in these markers, the impact of different lifestyles on health can be assessed, and individuals can be guided to take appropriate lifestyle intervention measures, such as losing weight, quitting smoking, increasing exercise, etc., to improve health and reduce disease risks.

The present invention also provides a method for diagnosing whether a subject has cervical cancer lymph node metastasis. The key to the 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 detect any combination of these three markers.

First, the implementation of this method involves the collection and processing of samples. Plasma samples are extracted from the subject's venous blood, and this process needs to follow strict standard operating procedures to ensure the quality and integrity of the samples.

Secondly, for the plasma samples that have been obtained, appropriate techniques and reagents can be used to detect the level of any one of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A. These detection methods can be immunological techniques, such as ELISA, immunofluorescence analysis, etc., or molecular biological techniques, such as PCR, in situ hybridization, etc., or mass spectrometry, etc. Through these methods, the content level of a certain marker in the subject's plasma can be accurately quantified.

At the same time, the method also provides another option, that is, optionally, detecting any combination of the three markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A. The selection of this combination can be determined according to actual needs and clinical conditions, for example, the contents of these three markers can be detected simultaneously, or one or more combinations thereof can be selected based on previous research or clinical experience.

After obtaining the test results of the markers, it is necessary to compare and analyze them with the relevant data of patients without lymph node metastasis. Based on these comparison results, it can be determined whether the subject is at risk of cervical cancer lymph node metastasis. If the level of a certain marker or a combination of markers is significantly lower than the normal range, it can be inferred that the subject is more likely to have cervical cancer lymph node metastasis.

The reliability and accuracy of this method depends 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 and diagnose the results. In addition, the test results of any marker should be carefully evaluated and interpreted to avoid uncertainty in the diagnostic results due to the possibility of misdiagnosis of a single marker.

Preferably, the index value of each marker is set, which is the ratio between the level of the marker in the human plasma of the subject and the level in the plasma of the patient without cervical cancer lymph node metastasis. The risk range of each marker is exemplified 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. It was found that all 10 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.

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.

The implementation of this method needs to start with experimental design and sample collection. In the design stage of a study or clinical trial, it is necessary to clarify the drugs to be evaluated, the treatment regimen, and the evaluation indicators. At the same time, it is necessary to determine the selection criteria for the subjects and collect the corresponding biological samples, such as plasma, tissue or cell samples, for subsequent experimental analysis.

For the selected drug or treatment regimen, any one, any two, or all three of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A can be selected as biomarkers to evaluate its therapeutic effect. The selection of these markers can be determined based on previous studies or clinical experience, or can be inferred based on the mechanism of action and expected effect of the drug.

After obtaining the biological sample, appropriate techniques and reagents can be used to detect the level of the selected marker. These detection methods can be immunological techniques, such as ELISA, immunofluorescence analysis, etc., or molecular biology techniques, such as PCR, in situ hybridization, etc., or mass spectrometry. Through these methods, the content level of the marker in the subject's biological sample can be accurately quantified.

Next, the drug or treatment regimen needs to be applied to the subject and the treatment is carried out according to the predetermined regimen. During the treatment process, the subject's biological samples need to be monitored regularly and the levels of the selected markers need to be repeatedly tested. This allows timely understanding of the therapeutic effect of the drug and whether the desired treatment goal has been achieved.

Finally, based on the test results obtained, the therapeutic effect of the drug can be evaluated and determined. If the level of the selected marker has changed significantly and is significantly different from before treatment, it can be inferred that the drug may have the potential to prevent or treat cervical cancer lymph node metastasis. On the contrary, if the level of the marker does not change significantly or does not achieve the expected effect, it may be necessary to re-evaluate the treatment plan or try other treatment strategies.

The present invention provides a method for screening compounds capable of preventing or treating lymphatic metastasis of cervical cancer, 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.

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.

Secondly, select appropriate biomarkers to evaluate the effect of the compound. The three markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A as biomarkers can reflect the biological process and pathological state of cervical cancer lymphatic metastasis. Therefore, they can be used as biomarkers to screen the effect of compounds to evaluate the intervention effect of compounds on cervical cancer lymphatic metastasis.

Next, a compound screening experiment is performed. 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 can be used to detect the levels of these markers, such as immunological techniques and molecular biological techniques. Based on the test results, the potential therapeutic effect of the compound on cervical cancer lymph node metastasis can be preliminarily evaluated.

After obtaining the preliminary screening results, further verification and confirmation experiments can be carried out. By using more sophisticated and rigorous experimental designs and operating procedures, the effects of compounds on marker levels can be verified and their therapeutic effects on cervical cancer lymph node metastasis can be further evaluated.

Finally, based on the experimental results, further research and development of compounds with potential therapeutic effects can be carried out. Their pharmacological properties, toxicity, pharmacokinetics and other drug properties can be further evaluated, and preclinical studies and clinical trials can be carried out.

The present invention further demonstrates 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.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The reagents used in the present invention are all analytical grade or above. The chromatographic column used is model: Acclaim Pep Map100C18 column, manufacturer: Thermo; the liquid chromatograph is model: EASY-nLC 1200, manufacturer: Thermo.

Example 1: Selection of biomarkers

Plasma samples were obtained from 40 participants. These 40 participants included 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 anticoagulation tubes, gently inverted to mix, centrifuged at 1600g for 10 min at 4°C, and the supernatant was collected after centrifugation. The supernatant was transferred to a 1.5 ml centrifuge tube and centrifuged at 13000g for 2 min at 4°C. The supernatant was collected and divided into multiple centrifuge tubes. After the collected plasma was marked, it was immediately transferred to -80°C and stored in the dark to avoid repeated freezing and thawing.

The reagents used in this example are: formic acid, methanol, ammonium formate, ammonium bicarbonate, acetonitrile, dithiothreitol (DTT), and iodoacetamide (IAM).

1. Targeting protein markers

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

Proteomic analysis was performed using a nano-level ultra-high performance liquid chromatography EASY-nLC 1200 (Thermo Fisher, USA) coupled to a high-resolution mass spectrometer Orbitrap Exploris 480 (Thermo Fisher, USA). The chromatographic column was an AcclaimPep Map 100C18 column (75 μm inner diameter, 2 μm column particle size, approximately 25 cm column length). Separation was performed at a flow rate of 300 nL/min using the following effective 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 into a tandem mass spectrometer Orbitrap Exploris 480 (Thermo Fisher, USA) for DDA (Data Dependent Acquisition) mode detection.

2. Targeting metabolite markers

First, metabolite extraction: After slowly thawing the plasma sample at 4°C, take 100μL and place it in a 1.5ml EP tube, add 300μL of extraction solution (methanol:acetonitrile = 2:1, v:v, -20°C pre-cooling), 10μL of internal standard 1 and 10μL of internal standard 2, vortex mix for 1min, let stand at -20°C for 30min, and centrifuge at 4°C, 16000g for 30min. After centrifugation, take 300μL of supernatant, place it in a freeze vacuum concentrator to dry, add 150μL of reconstitution solution (methanol: water = 1:1, v:v) for reconstitution, vortex shake, 14000g, 4°C centrifuge for 15min, take the supernatant and place it in the injection bottle. Take 15μL of the supernatant of each sample and mix it into a QC quality control sample to evaluate the repeatability and stability of the LC-MS analysis process.

Then, Vanquish ultra-performance liquid chromatography (UPLC) (Thermo Fisher, USA) was combined with a high-resolution mass spectrometer (Orbitrap Exploris 480, Thermo Fisher, USA) to separate and detect metabolites.

3. Bioinformatics analysis to find markers

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

Figures 1 and 2 show the OPLS-DA results of the proteome and metabolome in this example, respectively. In the figure, Non-Met represents the cervical cancer non-metastasis group, and Met-Lymph represents the cervical cancer lymph node metastasis group. The OPLS-DA analysis diagram reflects the overall distribution of samples in each group.

Figure 3 is a volcano map of differential proteins between the cervical cancer non-metastasis group and the cervical cancer lymph node metastasis group, and the volcano map is used to show the results of differential proteins in the cervical cancer lymph node metastasis group. Among them, a total of 58 proteins were screened, and there were significant differences in patients with cervical cancer lymph node metastasis compared with those without recurrence and metastasis. Among these proteins, 31 were downregulated and 27 were upregulated. Figure 4 is a volcano map of differential metabolism between the cervical cancer non-metastasis group and the cervical cancer lymph node metastasis group, and the volcano map is used to show the results of differential metabolites in the cervical cancer lymph node metastasis group. Among them, a total of 288 metabolites were screened, which were significantly different between patients with cervical cancer lymph node metastasis and those without recurrence and metastasis. Among these metabolites, 183 were downregulated and 105 were upregulated.

Figure 5 is an ROC curve (receiver operating characteristic curve) analysis of the three markers of cervical cancer lymph node metastasis markers 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 drawing 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.

As can be seen from Figure 5, the three markers of keratin 6A, resting sulfhydryl oxidase-1 (QSOX1) and paraoxonase 1 (PON1) screened in this example all have high diagnostic accuracy for cervical cancer lymph node metastasis, among which 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. It shows that any one of the markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A can be used to diagnose whether there is cervical cancer lymph node metastasis.

4. Marker combination modeling using logistic regression

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 shows the ROC analysis results of the three marker combination model of cervical cancer lymph node metastasis, and the results show that the AUC value of the biomarker combination is 0.973. Compared with the aforementioned single markers, the combination of the three markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A has better predictive accuracy.

Example 2: Further confirmation of biomarkers

1. Plasma samples of 8 other patients with cervical cancer lymph node metastasis and 10 patients with cervical cancer without lymph node metastasis were selected and the contents of three biomarkers, keratin 6A, resting sulfhydryl oxidase-1 (QSOX1) and paraoxonase 1 (PON1), were determined according to Example 1, and compared with the contents of the three biomarkers in the plasma of patients with cervical cancer without lymph node metastasis. The difference standard was fold change ≥ 1.2 or ≤ 0.83, and P value < 0.05.

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 positive patients.

According to the combined model analysis constructed in Example 1, and the index value of each marker is set as the ratio between the level value of the marker in the human plasma of the subject and the level value in the plasma of the patient 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. It was found that all the selected 8 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.

2. The ROC curve analysis of the three biomarkers was performed respectively, and the AUC values of keratin 6A (KRT6A) were 0.625, the AUC value of resting sulfhydryl oxidase-1 (QSOX1) was 0.975, and the AUC value of paraoxonase 1 (PON1) was 0.912. It shows that any one of the markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A can be used to diagnose whether there is cervical cancer lymph node metastasis. Figure 7 is the ROC curve analysis of the three markers of cervical cancer lymph node metastasis markers obtained by bioinformatics analysis in Example 2.

3. In the 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 shows the ROC analysis results of the three marker combination model for cervical cancer lymph node metastasis, and the results show that the AUC value of the biomarker combination is 0.988. Compared with the above single markers, the combination of the three markers of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A has better predictive accuracy.

Example 3: ELISA kit for cervical cancer lymph node metastasis

This embodiment provides an ELISA kit for diagnosing lymph node metastasis of cervical cancer. 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, so as to achieve accurate diagnosis of lymph node metastasis of cervical cancer.

Kit composition:

Resting sulfhydryl oxidase-1 (Prx-1) ELISA analysis:

Includes antibodies, standards, substrates, wash buffers and other reagents and materials for detecting resting sulfhydryl oxidase-1.

Used to detect the level of resting sulfhydryl oxidase-1 in the subjects' plasma and perform quantitative analysis based on the standard curve.

Paraoxonase 1 (Tyr-1) ELISA analysis:

Includes reagents and materials such as antibodies, standards, substrates, washing buffer, etc. for detecting paraoxonase 1.

Used to detect the level of paraoxonase 1 in the plasma of subjects and perform quantitative analysis based on the standard curve.

Keratin 6A (CK6A) ELISA analysis:

It includes antibodies, standards, substrates, washing buffer and other reagents and materials for detecting keratin 6A. It is used to detect the content level of keratin 6A in the plasma of the subject and perform quantitative analysis according to the standard curve.

The detection process can be as follows:

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

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.

Wash the plate with wash buffer to remove unbound material.

Substrate is added and the reaction is allowed to proceed under appropriate conditions to produce a measurable color.

The absorbance of the reaction product was measured using an enzyme-labeled instrument, and the contents of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A in the sample were calculated based on the standard curve.

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.

The above-described embodiment is only a preferred solution of the present invention, but it is not intended to limit the present invention. A person skilled in the relevant technical field may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

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

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 principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

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 any one of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A or a combination of any two or three of the markers. 2. The biomarker for diagnosing cervical cancer lymph node metastasis according to claim 1, characterized in that the cervical cancer lymph node metastasis marker is a combination of three markers: resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A. 3. Use of a reagent for detecting the cervical cancer lymph node metastasis marker as claimed in claim 1 or 2 in the preparation of a cervical cancer lymph node metastasis diagnosis kit or detection device. 4. The use according to claim 3, wherein the reagent is an antibody to the marker, a primer for PCR, a reagent for mass spectrometry analysis, or a reagent for chromatography analysis. 5. A kit for diagnosing cervical cancer lymph node metastasis, characterized in that it comprises a reagent for detecting the cervical cancer lymph node metastasis marker as described in claim 1 or 2, and optionally, the detection is a quantitative detection of the biomarker level in the subject's plasma. 6 . The kit according to claim 5 , wherein the reagent is an antibody to 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 5, characterized in that the kit is an ELISA kit. 8. Use of the cervical cancer lymph node metastasis marker as claimed in claim 1 or 2 in the diagnosis of cervical cancer lymph node metastasis for purposes other than disease diagnosis or treatment. 9. Use of any one of resting sulfhydryl oxidase-1, paraoxonase 1 and keratin 6A or a combination of any two or three of the markers as a marker of cervical cancer lymph node metastasis in the preparation of a preparation for diagnosing cervical cancer lymph node metastasis. 10. A method for evaluating whether a drug can prevent or treat lymphatic metastasis of cervical cancer, 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 drug; or a method for screening a compound that can prevent or treat lymphatic metastasis of cervical cancer, 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.