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

Abstract

The present invention provides biomarkers for diagnosing lymph node metastasis of cervical cancer and their applications, belonging to the field of bio-detection technology. Through bioinformatics analysis methods such as multivariate statistical analysis, differential screening, random forest, and ROC analysis, the present invention obtains three biomarkers, namely, quiescent thiol oxidase-1, paraoxonase 1, and keratin 6A, for diagnosing lymph node metastasis of cervical cancer. Based on the biomarkers of the present invention, kits for diagnosing lymph node metastasis of cervical cancer are constructed, etc., for accurately judging whether lymph node metastasis has occurred in cervical cancer patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to biomarkers for diagnosing lymph node metastasis of cervical cancer and their applications. Background Art

[0002] In recent years, cancer has become one of the important challenges in the field of global public health, and a large number of new cancer cases are diagnosed every day. Among them, cervical cancer is one of the most common gynecological malignancies, and its pathogenesis is mainly closely related to the infection of high-risk human papillomavirus (HPV).

[0003] Among cervical cancer patients, lymph node metastasis is one of the main pathological processes and has an important impact on the prognosis of patients. 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 lymph node metastasis of cervical cancer have attracted much attention. For patients with positive lymph node metastasis, if appropriate treatment is not given in time, rapid recurrence may occur. At the same time, for patients who need to evaluate the lymph node metastasis situation 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 are crucial.

[0004] Currently, the methods for evaluating lymph node metastasis of cervical cancer 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 the prognosis of patients.

[0005] A biomarker refers to a molecule, cell or tissue that exists in a living organism and has certain characteristics and functions, and can reflect the internal physiological state, pathological process or response to treatment of the living organism. In the prediction and diagnosis of lymph node metastasis of cervical cancer, it is of great significance to find applicable biomarkers. However, it is not easy to find reliable biomarkers. First, due to the extremely complex pathological process of cervical cancer, involving the regulation of multiple biological mechanisms, the selection of biomarkers needs to take this complexity into account. Second, the biomarker must have sufficient specificity and sensitivity to ensure that it can be reliably detected at the early stage of lymph node metastasis. In addition, the detection method of the biomarker needs to have high accuracy and reproducibility to ensure its reliability and effectiveness in clinical applications.

[0006] Therefore, finding biomarkers applicable to cervical cancer lymph node metastasis is a challenging task that requires interdisciplinary collaboration and extensive experimental validation. Researchers need to utilize technical means in multiple fields such as molecular biology, cell biology, and biochemistry to conduct extensive screening and identification of candidate biomarkers. Then, these biomarkers need to be verified and evaluated in clinical samples to determine their exact role and clinical application prospects in cervical cancer lymph node metastasis.

[0007] Although there are certain difficulties in finding suitable biomarkers, once successfully found and verified, it will bring substantial progress to the early diagnosis and individualized treatment of cervical cancer lymph node metastasis. Therefore, despite the challenges, finding biomarkers is still essential for improving the treatment effect and prognosis of cervical cancer patients. Summary of the Invention

[0008] To solve the above technical problems, the present invention provides biomarkers for diagnosing cervical cancer lymph node metastasis and their applications.

[0009] For this purpose, the present invention adopts the following technical solutions:

[0010] 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 biomarker present in human plasma; the cervical cancer lymph node metastasis biomarker is a combination of one or more of resting thiol oxidase-1, paraoxonase 1, and keratin 6A.

[0011] Preferably, the cervical cancer lymph node metastasis biomarker is a combination of 3 biomarkers: resting thiol oxidase-1, paraoxonase 1, and keratin 6A.

[0012] In the second aspect, the present invention provides an application of a reagent for detecting the cervical cancer lymph node metastasis biomarker as described in the first aspect in the preparation of a diagnostic kit or detection device for cervical cancer lymph node metastasis.

[0013] In the 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 biomarker as described in the first aspect.

[0014] Preferably, the kit is an ELISA kit.

[0015] In the fourth aspect, the present invention provides an application of the cervical cancer lymph node metastasis biomarker as described in the first aspect in the diagnosis of cervical cancer lymph node metastasis for non-disease diagnosis or treatment purposes.

[0016] Fifth aspect, the present invention provides a method for diagnosing whether a subject has lymph node metastasis of cervical cancer, which includes detecting the level of any one of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A in the plasma of the subject, and optionally detecting the levels of all of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A.

[0017] 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 includes using any one or any two or all 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.

[0018] Seventh aspect, the present invention provides a method for screening compounds that can prevent or treat lymph node metastasis of cervical cancer, which includes using any one or any two or all three of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A as biomarkers to evaluate the effect of the compound.

[0019] The present invention has the following beneficial effects compared with the prior art:

[0020] The present invention provides novel molecular markers that can be used to discriminate lymph node metastasis of cervical cancer, which can be used for early detection, diagnosis, and prediction of lymph node metastasis of cervical cancer, and thus achieve more precise drug use.

[0021] The biomarkers provided by the present invention for diagnosing lymph node metastasis of cervical cancer are any combination of one, two, or all three of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A. By comparing their levels in the plasma with those in the plasma of patients with lymph node metastasis of cervical cancer, lymph node metastasis of cervical cancer can be diagnosed with high precision. Description of the Drawings

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 OPLS-DA analysis of the proteomic results in the examples.

[0024] Figure 2 OPLS-DA analysis of the metabolomic results in the examples.

[0025] Figure 3 Volcano plot of differential proteins in the examples.

[0026] Figure 4Volcano plot of differential metabolites in the examples.

[0027] Figure 5 ROC analysis results of biomarkers for lymph node metastasis in cervical cancer in the examples.

[0028] Figure 6 ROC of the combined model of three biomarkers for lymph node metastasis in cervical cancer in the examples.

[0029] Figure 7 ROC curve analysis of three biomarkers among the biomarkers for lymph node metastasis in cervical cancer.

[0030] Figure 8 Displays the ROC analysis results of the combined model of three biomarkers for lymph node metastasis in cervical cancer. Detailed implementation manners

[0031] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific implementation manners of the present invention will be made with reference to the accompanying drawings. Many specific details are set forth in the following description in order to fully understand 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 departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below. The technical features in various embodiments of the present invention can be combined correspondingly without conflict.

[0032] Lymph node metastasis in cervical cancer is one of the common metastasis ways of cervical cancer. The present invention provides corresponding biomarkers for this phenomenon, which are selected from any 1, 2 or 3 of quiescent thiol oxidase-1, paraoxonase 1 and keratin 6A.

[0033] It should be noted that quiescent thiol oxidase-1, paraoxonase 1 and keratin 6A in the present invention are all known proteins in the human body. They can be queried through public protein databases. For example, the protein IDs in uniprot are shown in Table 1 below.

[0034] The accession number of quiescent thiol oxidase-1 (QSOX1) in the protein database uniprot is O00391. It is an enzyme that catalyzes oxidation-reduction reactions and plays an important role in both the internal and external environments of cells. It can catalyze the oxidation reaction of free thiols (i.e., groups containing sulfhydryl groups) to form disulfide bonds. This process helps in the folding and formation of intracellular proteins and regulates the formation and stability of the extracellular matrix in the external environment of cells. QSOX1 plays a key role in the intracellular redox balance and is also closely related to the occurrence and development of some diseases, especially cancer.

[0035] The accession number of paraoxonase-1 (PON1) in the protein database Uniprot is P27169. It is an antioxidant enzyme and plays a key role in maintaining the in vivo oxidation-antioxidation balance. It has been found that PON1 is associated with systemic oxidative stress and cardiovascular events, but there are few reports on its association with lymph node metastasis of cervical cancer or its use alone as a biomarker for lymph node metastasis of cervical cancer.

[0036] The accession number of keratin 6A (KRT 6A) in the protein database Uniprot is P0253. It is a member of the keratin family and belongs to cytoskeletal proteins. It is mainly present 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, protect cells from damage by the external environment, and also participate in regulating the proliferation and differentiation processes of keratinocytes. In some diseases such as skin diseases and cancers, the expression level of Keratin 6A may change and is related to the occurrence and development of the diseases.

[0037] Table 1 IDs and Chinese and English names of proteins

[0038] Lymphatic metastasis markers of cervical cancer Protein ID Sulfhydryl oxidase 1 (Quiescent sulfhydryl oxidase-1) O00391 Paraoxonase-1 P27169 Keratin 6A P02538

[0039] It should be noted that among the above-mentioned biomarkers for lymph node metastasis of cervical cancer, any one of them alone can be used as the basis for diagnosing lymph node metastasis of cervical cancer. However, due to the possibility of misjudgment with a single biomarker, to improve the accuracy and reliability of the diagnosis, a more preferred option is to combine these three biomarkers as the biomarker for lymph node metastasis of cervical cancer.

[0040] By combining the three biomarkers of quiescent thiol oxidase-1, paraoxonase-1, and keratin 6A, the sensitivity and specificity for lymph node metastasis of cervical cancer can be improved. This combination strategy helps to reduce the possibility of misjudgment and improve the accurate diagnosis rate for lymph node metastasis of cervical cancer, providing a more reliable basis for the treatment and management of patients.

[0041] Therefore, although a single biomarker also has certain diagnostic value, the combination of the three biomarkers is still preferentially considered as the basis for diagnosing lymph node metastasis of cervical cancer to ensure the accuracy and reliability of the diagnostic results.

[0042] In another embodiment of the present invention, there is provided the use of a reagent for detecting the above-mentioned biomarkers for lymph node metastasis of cervical cancer (a single biomarker or a combination of multiple biomarkers) in the preparation of a kit or detection device for diagnosing lymph node metastasis of cervical cancer.

[0043] Since the above-mentioned cervical cancer lymph node metastasis markers may be single markers or combinations of multiple markers, the corresponding diagnostic kits or detection devices can also take various forms. Exemplarily, it can be a diagnostic kit or detection device for a single marker, that is, a kit or device specifically for detecting any one of resting thiol oxidase-1, paraoxonase 1, or keratin 6A. Further exemplarily, it can also be a combination of diagnostic kits or detection devices for each of the multiple markers, that is, a kit or device for simultaneously detecting resting thiol oxidase-1, paraoxonase 1, and keratin 6A.

[0044] This diverse design enables the diagnostic kit or detection device to have a wider scope of application and greater flexibility, and an appropriate detection scheme 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.

[0045] The reagents contained in the specific kit in the present invention can be set according to the detection means for each marker. This means that for the three markers of resting thiol oxidase-1, paraoxonase 1, and keratin 6A, specific methods and reagents suitable for their detection can be selected.

[0046] 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 biology techniques such as polymerase chain reaction (PCR), in situ hybridization, etc. can also be used to detect the gene expression levels of these markers. In addition, mass spectrometry analysis and other means can also be adopted.

[0047] Based on the rich experience and mature methods of the prior art, the most suitable method can be selected to detect the above-mentioned markers (single marker or 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 procedures of the prior art.

[0048] ELISA is a commonly used method for detecting biomarkers. It uses specific antibodies to bind to the biomarkers to be detected, and then generates a measurable signal through the reaction of enzymes. The ELISA method has high sensitivity and specificity and can be applied to the detection of single markers or multiple markers.

[0049] Immunofluorescence analysis uses fluorescently labeled antibodies to bind to the biomarkers to be detected, and the intensity of the fluorescent signal is detected by a fluorescence microscope or a fluorometer to quantitatively analyze the amount of the biomarker present.

[0050] Mass spectrometry can perform accurate quantitative analysis of 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.

[0051] The PCR technique can be used to detect the transcribed RNA of biomarkers. By amplifying the RNA or its fragments to be detected through PCR, and then performing quantitative analysis through methods such as gel electrophoresis or real-time fluorescence quantitative PCR.

[0052] Protein chip technology can detect multiple biomarkers simultaneously. By immobilizing a variety of antibodies on the chip surface, and then binding to the proteins in the sample to be detected (such as plasma), and finally detecting the biomarkers through the signals of fluorescence or radioactive labeling.

[0053] Exemplarily, considering the detection efficiency and convenience, the kit can be an ELISA kit.

[0054] In another embodiment of the present invention, there is provided an application of the above-mentioned biomarker (a single biomarker or a combination of multiple biomarkers) 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 for purposes such as scientific research, non-medical commercial detection or testing, etc.

[0055] For example, biomarkers of cervical cancer lymph node metastasis can be used to study the disease occurrence mechanism, pathophysiological process, and potential therapeutic targets. By analyzing the changes of these biomarkers in the disease process, the development and metastasis mechanism of the disease can be deeply understood, providing a deeper understanding for the prevention and treatment of the disease.

[0056] Biomarkers also play an important role in the process of drug research and development. They can be used as indicators for evaluating drug efficacy, helping 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.

[0057] 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 diseases can be predicted, and corresponding health management measures can be taken, such as adjusting lifestyle, eating habits, or drug treatment, to maintain health and delay the progression of diseases.

[0058] Biomarkers can be used to evaluate the impact of lifestyle on health. By monitoring the changes of these biomarkers, the degree of impact of different lifestyles on health can be evaluated, and individuals can be guided to take appropriate lifestyle intervention measures, such as losing weight, quitting smoking, increasing exercise, etc., so as to improve health status and reduce the risk of diseases.

[0059] The present invention also provides a method for diagnosing whether a subject has lymph node metastasis of cervical cancer. The key to this method lies in detecting the level of any one of resting sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A in the plasma of the subject, or optionally detecting any combination of these three markers.

[0060] First, the implementation of this method involves the collection and processing of samples. Extracting a plasma sample from the venous blood of the subject requires following strict standard operating procedures to ensure the quality and integrity of the sample.

[0061] Secondly, for the obtained plasma sample, appropriate techniques and reagents can be selected 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 biology techniques such as PCR, in situ hybridization, etc., or mass spectrometry means, etc. Through these methods, the content level of a certain marker in the plasma of the subject can be accurately quantified.

[0062] At the same time, this 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 situations. For example, the contents of these three markers can be detected simultaneously, or one or more combinations can be selected based on previous research or clinical experience.

[0063] After obtaining the detection results of the markers, they need to be compared and analyzed with the relevant data in patients without lymph node metastasis. Based on these comparison results, it can be judged whether the subject has the risk of lymph node metastasis of cervical cancer. If the level of a certain marker or marker combination is significantly lower than the normal range, it can be inferred that the subject has a higher possibility of lymph node metastasis of cervical cancer.

[0064] 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, in combination with clinical practice experience and data analysis, interpret and diagnose the results. In addition, for the detection results of any one marker, careful evaluation and interpretation should be carried out to avoid the uncertainty of the diagnosis result due to the possible misjudgment of a single marker.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] Finally, based on the obtained test results, the therapeutic effect of the drug can be evaluated and determined. If the level of the selected biomarker has changed significantly and there are obvious differences compared with that before treatment, it can be inferred that the drug may have the potential to prevent or treat lymph node metastasis of cervical cancer. On the contrary, if the change in the biomarker level is not obvious or fails to achieve the expected effect, it may be necessary to re-evaluate the treatment plan or try other treatment strategies.

[0072] The present invention provides a method for screening compounds capable of preventing or treating lymph node metastasis of cervical cancer, which includes using any one or any two or three of resting thiol oxidase-1, paraoxonase 1, and keratin 6A as biomarkers to evaluate the effect of the compound.

[0073] First, the implementation of this method requires the establishment of a suitable experimental system and model. A cell line or animal model of lymph node metastasis of cervical cancer can be used to simulate the physiological process of lymph node metastasis of cervical cancer and evaluate the effect of the compound on it.

[0074] Secondly, select suitable biomarkers for evaluating the effect of the compound. These three biomarkers, resting thiol oxidase-1, paraoxonase 1, and keratin 6A, can reflect the biological process and pathological state of lymph node metastasis of cervical cancer. Therefore, they can be used as biomarkers for screening the effect of the compound to evaluate the intervention effect of the compound on lymph node metastasis of cervical cancer.

[0075] Next, conduct a screening experiment of the compound. Add the compound to be screened into cells or animals, and observe its effect on resting thiol oxidase-1, paraoxonase 1, and keratin 6A. Appropriate techniques and reagents can be used to detect the levels of these biomarkers, such as immunological techniques and molecular biological techniques, etc. Based on the test results, the potential therapeutic effect of the compound on lymph node metastasis of cervical cancer can be preliminarily evaluated.

[0076] After obtaining the preliminary screening results, further verification and confirmation experiments can be carried out. By using a more refined and strict experimental design and operation process, the effect of the compound on the biomarker level can be verified, and its therapeutic effect on lymph node metastasis of cervical cancer can be further evaluated.

[0077] Finally, according to the experimental results, further research and development are carried out on the compounds with potential efficacy. Their pharmacological properties, toxic and side effects, pharmacokinetics and other drug properties can be further evaluated, and preclinical studies and clinical trials are conducted.

[0078] Next, the present invention further demonstrates the selection principle, process, and effect of the above-mentioned biomarkers through a specific embodiment, so as to facilitate those skilled in the art to understand the essence of the present invention.

[0079] 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 of analytical purity or above. Among them, the chromatographic column used has the model: Acclaim Pep Map100 C18 column, and the manufacturer is Thermo; the liquid chromatograph has the model: EASY-nLC 1200, and the manufacturer is Thermo.

[0080] Example 1: Selection of Biomarkers

[0081] 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. Venous blood from the elbow was collected in an EDTA anticoagulant tube, gently inverted up and down to mix evenly, centrifuged at 1600g for 10 min at 4°C, the supernatant was collected after centrifugation, transferred to a 1.5 ml centrifuge tube, centrifuged at 13000g for 2 min at 4°C, the supernatant was collected and aliquoted into multiple centrifuge tubes. After the collected plasma was labeled, it was immediately transferred to -80°C for storage in the dark to avoid repeated freezing and thawing.

[0082] The reagents used in this example: formic acid, methanol, ammonium formate, ammonium bicarbonate, acetonitrile, dithiothreitol (DTT), iodoacetamide (IAM).

[0083] I. For protein biomarkers

[0084] The high-abundance proteins in the plasma samples were removed, the protein concentration of the samples was measured, and then 100 μg of protein in the blood samples was purified, and then the purified samples were subjected to a reduction alkylation reaction. The specific operation steps are as follows: removal of high-abundance proteins, protein purification, enrichment quality control, proteolysis. The resulting peptide fragments were then desalted on ZipTip Pipette Tips (Merck, Germany), dried under vacuum centrifugation, and stored at -80°C until detection.

[0085] Proteomic analysis was performed using an Easy-nLC 1200 ultra-high performance liquid chromatography (Thermo Fisher, USA) with a nano-liter flow rate, coupled with an Orbitrap Exploris 480 high-resolution mass spectrometer (Thermo Fisher, USA). The chromatographic column was an Acclaim Pep Map 100 C18 column (inner diameter 75 μm, column packing particle size 2 μm, column length approximately 25 cm). Separation was carried out at a flow rate of 300 nL / min using the following effective gradient: 0 - 1.0 minute, 3% - 35% solution B (98% acetonitrile, 0.1% formic acid); 1 - 56 minutes, 35% - 95% solution B; 56 - 59 minutes, 95% solution B; 59 - 65 minutes, 95% - 3% solution B. The column temperature was maintained at 40 °C, and the sample manager temperature was set at 4 °C. The end of the nano-liquid phase separation was directly connected to the mass spectrometer and detected according to the following parameters. The peptides separated by liquid chromatography were ionized by a nano ESI source and then entered the tandem mass spectrometer Orbitrap Exploris 480 (Thermo Fisher, USA) for detection in the DDA (Data Dependent Acquisition) mode.

[0086] II. For metabolite markers

[0087] First, metabolite extraction: After slowly thawing the plasma samples at 4 °C, 100 μL was taken and placed in a 1.5 ml EP tube. Then, 300 μL of extraction solution (methanol:acetonitrile = 2:1, v:v, pre-cooled at -20 °C), 10 μL of internal standard 1, and 10 μL of internal standard 2 were added. The mixture was vortexed for 1 min, left standing at -20 °C for 30 min, and then centrifuged at 4 °C, 16000 g for 30 min. After centrifugation, 300 μL of the supernatant was taken, dried in a freeze-drying vacuum concentrator, and then re-dissolved by adding 150 μL of reconstitution solution (methanol:water = 1:1, v:v). The mixture was vortexed, centrifuged at 14000 g, 4 °C for 15 min, and the supernatant was taken and placed in an injection vial. 15 μL of the supernatant from each sample was taken and mixed to form a QC quality control sample for evaluating the repeatability and stability of the LC-MS analysis process.

[0088] Subsequently, a Vanquish ultra-high performance liquid chromatography UPLC (Thermo Fisher, USA) coupled with an Orbitrap Exploris 480 high-resolution mass spectrometer (Thermo Fisher, USA) was used for the separation and detection of metabolites.

[0089] III. Bioinformatics analysis to find markers

[0090] Bioinformatics analysis methods such as differential screening, random forest, and ROC analysis are mainly used to obtain biomarkers. The criteria for screening differences are mainly fold change (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 ranking, and the comprehensive results are used to select the final biomarkers. According to the above bioinformatics analysis, 3 biomarkers are selected as cervical cancer lymph node metastasis biomarkers, which are one or more combinations of quiescent thiol oxidase-1, paraoxonase 1, and keratin 6A, and all three biomarkers belong to proteins.

[0091] Figure 1 and Figure 2 respectively show the OPLS-DA results of the proteome and metabolome in this example. In the figure, Non-Met represents the non-metastatic group of cervical cancer, and Met-Lymph represents the lymph node metastatic group of cervical cancer. The OPLS-DA analysis diagram reflects the overall distribution of each group of samples.

[0092] Figure 3 This is a volcano plot of differential proteins between the non-metastatic group and the lymph node metastatic group of cervical cancer, which shows the results of differential proteins in the lymph node metastatic group of cervical cancer using a volcano plot. Among them, a total of 58 proteins were screened, and there were significant differences in the recurrence-free metastasis of patients with lymph node metastasis of cervical cancer. Among these proteins, 31 were down-regulated and 27 were up-regulated. Figure 4 This is a volcano plot of differential metabolites between the non-metastatic group and the lymph node metastatic group of cervical cancer, which shows the results of differential metabolites in the lymph node metastatic group of cervical cancer using a volcano plot. Among them, a total of 288 metabolites were screened, and there were significant differences in the lymph node metastatic patients and recurrence-free metastasis of cervical cancer. Among these metabolites, 183 were down-regulated and 105 were up-regulated.

[0093] Figure 5 This is the ROC curve (receiver operating characteristic curve) analysis of the three biomarkers among the cervical cancer lymph node metastasis biomarkers obtained by bioinformatics analysis in this example, that is, the receiver operating characteristic curve analysis. By plotting the ROC and calculating the area under the curve (AUC) to evaluate the sensitivity and specificity of the three biomarkers in diagnosing cervical cancer lymph node metastasis. The judgment criteria are as follows: when AUC < 0.5, it indicates that the diagnosis is meaningless; when AUC = 0.5 - 0.7, it indicates that the diagnostic accuracy is low; when AUC = 0.7 - 0.9, it indicates that the diagnostic accuracy is medium; when AUC > 0.9, it indicates that the diagnostic accuracy is high. That is, in the ROC curve, the AUC value is the area covered by the ROC curve, and the larger its value, the higher the accuracy of the model prediction.

[0094] From Figure 5It can be seen that among the three markers, keratin 6A, quiescent sulfhydryl oxidase-1 (QSOX1), and paraoxonase 1 (PON1), each has a relatively high diagnostic accuracy for cervical cancer lymph node metastasis when used alone. Among them, the AUC value of keratin 6A (KRT6A) is 0.909, the AUC value of quiescent 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, quiescent sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A, can be used to diagnose the presence of cervical cancer lymph node metastasis.

[0095] IV. Using logistic regression to build a marker combination model

[0096] For the cervical cancer lymph node metastasis group, the three candidate biomarkers for cervical cancer lymph node metastasis obtained previously were combined to construct a logistic regression model, and ROC curve analysis was performed for evaluation. Figure 6 The ROC analysis results of the three-marker combination model for cervical cancer lymph node metastasis are shown. The results indicate that the AUC value of the biomarker combination is 0.973. Compared with the aforementioned single markers, the combination of the three markers, quiescent sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A, has better predictive accuracy.

[0097] Example 2: Further confirmation of biomarkers

[0098] 1. Plasma samples from another 8 patients with cervical cancer lymph node metastasis and 10 patients without cervical cancer lymph node metastasis were selected, and the contents of the three biomarkers, keratin 6A, quiescent sulfhydryl oxidase-1 (QSOX1), and paraoxonase 1 (PON1), were measured with reference to Example 1, and compared with the contents of these three biomarkers in the plasma of patients without cervical cancer lymph node metastasis. The criterion for difference was fold change ≥ 1.2 or ≤ 0.83, and P value < 0.05.

[0099] The results showed that for any one of the three biomarkers, 8 out of the 8 selected patients with cervical cancer lymph node metastasis had significant differences from the control group, that is, 8 positive patients.

[0100] Analysis was performed according to the combined model constructed in Example 1, and the index value of each biomarker was set as the ratio between the level of the biomarker in the plasma of the subject and the level in the plasma of patients without cervical cancer lymph node metastasis. The risk ranges for each biomarker are as follows: the risk range indicating cervical cancer lymph node metastasis corresponding to quiescent sulfhydryl oxidase-1 is that the index value is less than 0.59; the risk range indicating cervical cancer lymph node metastasis corresponding to paraoxonase 1 is that the index value is less than 0.56; the risk range indicating cervical cancer lymph node metastasis corresponding to keratin 6A is that the index value is less than 0.24. It was found that all 8 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 better accuracy compared to a single biomarker.

[0101] 2. ROC curve analysis was performed on the three biomarkers respectively, and the AUC value of keratin 6A (KRT6A) was 0.625, the AUC value of quiescent sulfhydryl oxidase-1 (QSOX1) was 0.975, and the AUC value of paraoxonase 1 (PON1) was 0.912. This shows that any one of the biomarkers quiescent sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A can be used to diagnose the presence of cervical cancer lymph node metastasis. Figure 7 ROC curve analysis of three biomarkers among the biomarkers for cervical cancer lymph node metastasis obtained by bioinformatics analysis in Example 2.

[0102] 3. In another selected 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 ROC curve analysis was used for evaluation. Figure 8 The ROC analysis results of the combined model of 3 biomarkers for cervical cancer lymph node metastasis are shown. The results show that the AUC value of the biomarker combination is 0.988. Compared with the aforementioned single biomarker, the combination of the three biomarkers quiescent sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A has better predictive accuracy.

[0103] Example 3: ELISA kit for cervical cancer lymph node metastasis

[0104] This example provides an ELISA kit for diagnosing cervical cancer lymph node metastasis. The design of this kit aims to detect the content levels of the three biomarkers quiescent sulfhydryl oxidase-1, paraoxonase 1, and keratin 6A in the plasma of the subject, so as to achieve accurate diagnosis of cervical cancer lymph node metastasis.

[0105] Composition of the kit:

[0106] Quiescent sulfhydryl oxidase-1 (Prx-1) ELISA assay:

[0107] Reagents and materials including antibodies, standards, substrates, washing buffers, etc. for detecting quiescent thiol oxidase-1.

[0108] For detecting the content level of quiescent thiol oxidase-1 in the plasma of a subject and performing quantitative analysis according to the standard curve.

[0109] Paraoxonase 1 (Tyr-1) ELISA assay:

[0110] Reagents and materials including antibodies, standards, substrates, washing buffers, etc. for detecting paraoxonase 1.

[0111] For detecting the content level of paraoxonase 1 in the plasma of a subject and performing quantitative analysis according to the standard curve.

[0112] Keratin 6A (CK6A) ELISA assay:

[0113] Reagents and materials including antibodies, standards, substrates, washing buffers, etc. for detecting keratin 6A. For detecting the content level of keratin 6A in the plasma of a subject and performing quantitative analysis according to the standard curve.

[0114] The detection process can be as follows:

[0115] Take out the plasma sample of the subject and perform specimen processing and pretreatment steps according to the kit instructions.

[0116] Add the pretreated plasma sample into the respective ELISA plate wells to specifically react with the antibodies of quiescent thiol oxidase-1, paraoxonase 1, and keratin 6A.

[0117] Use the washing buffer to wash the plate wells to remove unbound substances.

[0118] Add the substrate and perform the reaction under appropriate conditions to produce a measurable color.

[0119] Use an enzyme-linked immunosorbent assay reader to measure the absorbance of the reaction product and calculate the contents of quiescent thiol oxidase-1, paraoxonase 1, and keratin 6A in the sample according to the standard curve.

[0120] According to the content levels of each biomarker and in combination with the preset diagnostic criteria, determine whether the subject has lymph node metastasis of cervical cancer.

[0121] The above-described embodiments are only a preferred solution of the present invention, but they are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can still make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, all technical solutions obtained by adopting equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

[0122] The serial numbers of the embodiments of the present invention above are only for description and do not represent the advantages or disadvantages of the embodiments.

[0123] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A plasma biomarker for diagnosing lymph node metastasis of cervical cancer, characterized in that, The biomarker is a combination of three biomarkers: quiescent thiol oxidase-1, paraoxonase 1, and keratin 6A.

2. Use of a reagent for detecting plasma biomarkers in the preparation of a diagnostic kit or detection device for cervical cancer lymph node metastasis, wherein the biomarker is a combination of quiescent thiol oxidase-1 and other biomarkers, and the other biomarker is any one or two of paraoxonase 1 and keratin 6A.

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

4. A kit for diagnosing lymph node metastasis of cervical cancer, characterized in that, It includes a reagent for detecting a biomarker, wherein the biomarker is the biomarker according to claim 1, and optionally, the detection is a quantitative detection of the level of the biomarker in the plasma of a subject.

5. The kit according to claim 4, wherein the reagent is an antibody against the biomarker or a primer for PCR.

6. The kit for diagnosing lymphatic metastasis of cervical cancer according to claim 4, wherein The kit is an ELISA kit.

7. Use of a plasma biomarker as a biomarker for cervical cancer lymph node metastasis in the preparation of a preparation for diagnosing cervical cancer lymph node metastasis, wherein the biomarker is a combination of quiescent thiol oxidase-1 and other biomarkers, and the other biomarker is any one or two of paraoxonase 1 and keratin 6A.

8. A method for screening a compound capable of preventing or treating cervical cancer lymph node metastasis, which comprises using a plasma biomarker as a biomarker to evaluate the effect of the compound, wherein the biomarker is a combination of quiescent thiol oxidase-1 and other biomarkers, and the other biomarker is any one or two of paraoxonase 1 and keratin 6A.

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