Prognostic molecular markers specific for squamous and adenocarcinoma of the cervix and uses thereof

Abstract

The application provides a molecular marker for evaluating the prognosis of cervical squamous cell carcinoma and adenocarcinoma and application thereof, and belongs to the technical field of biological detection. Through bioinformatics analysis methods such as multivariate statistical analysis, difference screening, single factor COX regression analysis and multi-factor COX regression analysis, the application obtains an adenine kinase 4 used for evaluating the prognosis marker of cervical squamous cell carcinoma and a brain sponge-shaped deformity 2 protein used for evaluating the prognosis biomarker of cervical adenocarcinoma. Based on the biomarkers of the application, a kit for evaluating cervical squamous cell carcinoma or adenocarcinoma is constructed, which is used for accurately evaluating the prognosis of cervical squamous cell carcinoma or adenocarcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to specific prognostic biomarkers for cervical squamous cell carcinoma and adenocarcinoma and their application in prognostic assessment and personalized treatment. Background Technology

[0002] Cervical cancer is a malignant tumor that typically originates from the epithelial cells of the cervical mucosa and is closely associated with human papillomavirus (HPV) infection. According to the World Health Organization (WHO), cervical cancer is the fourth leading cause of cancer death among women worldwide, causing more than 2.6 million deaths annually, mostly in low- and middle-income countries, making it a global public health issue. High-risk HPV infection is considered a major contributing factor to cervical cancer.

[0003] The main pathological types of cervical cancer include squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, and neuroendocrine carcinoma. The most common subtype of cervical cancer is squamous cell carcinoma, accounting for approximately 75% of cervical cancer cases. It is usually associated with high-risk HPV infection. Clinically, squamous cell carcinoma typically occurs at the external os of the cervix and the squamous-epithelial junction. It grows slowly and is relatively easy to detect early. It is classified into three grades based on histological differentiation: Grade I is well-differentiated squamous cell carcinoma, Grade II is moderately differentiated squamous cell carcinoma (non-keratinizing large cell type), and Grade III is poorly differentiated squamous cell carcinoma (small cell type), mostly undifferentiated small cells. Adenocarcinoma accounts for 15%–20% of cervical cancers. Unlike squamous cell carcinoma, adenocarcinoma often occurs at the internal os of the cervix and in glandular tissue. Its pathogenesis involves multiple factors, including HPV infection, genetic variations, and hormonal influences. Adenocarcinoma grows relatively quickly and is often discovered at a later stage, posing a challenge to treatment. Adenosquamous carcinoma accounts for 3%–5% of cervical cancers. It is formed by the simultaneous differentiation of reserve cells into glandular cells and squamous cells. The cancerous tissue contains both adenocarcinoma and squamous cell carcinoma components. This type exhibits pathological diversity, making treatment and prognosis more complex. Neuroendocrine carcinoma: This is a relatively rare subtype of cervical cancer with a poor prognosis. Studies have reported that different pathological types of cervical cancer may be driven by different molecular mechanisms.

[0004] Currently, squamous cell carcinoma antigen (SCC-Ag) and carbohydrate antigen 125 (CA125) are mainly used as serum biomarkers for cervical cancer in clinical practice. However, these biomarkers are not specific to cervical cancer and have insufficient sensitivity and specificity, making it impossible to effectively differentiate the prognosis of squamous cell carcinoma and adenocarcinoma. Adenocarcinoma, due to its rapid progression and low sensitivity to radiotherapy and chemotherapy, generally has a poor prognosis, but research on the different biological characteristics of squamous cell carcinoma and adenocarcinoma is still insufficient.

[0005] With the rapid development of proteomics and bioinformatics technology, differential analysis based on protein expression profile has become an important method for finding new biomarkers. By screening and verifying specific molecular markers that can reflect the prognosis difference between squamous carcinoma and adenocarcinoma, scientific basis can be provided for precise diagnosis and individualized treatment, clinical needs can be met, and patient survival rate and quality of life can be improved. SUMMARY

[0006] To solve the above technical problems, the present application provides a prognostic biomarker for cervical squamous carcinoma and adenocarcinoma and its application.

[0007] To this end, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a biomarker combination for prognosing cervical squamous carcinoma and cervical adenocarcinoma, which is adenosine kinase 4 and brain sponge malformation 2 protein, or the present application provides a biomarker for prognosing cervical squamous carcinoma or cervical adenocarcinoma, which is a prognostic marker for cervical squamous carcinoma or adenocarcinoma present in human tissues, the prognostic marker for cervical squamous carcinoma is adenosine kinase 4, and the prognostic marker for cervical adenocarcinoma is brain sponge malformation 2 protein.

[0009] In a second aspect, the present application provides use of a reagent for detecting the cervical squamous carcinoma and adenocarcinoma marker of the first aspect in the preparation of a cervical squamous carcinoma or adenocarcinoma prognostic product (such as a kit or a detection device), or the present application provides use of a reagent for detecting the cervical squamous carcinoma or adenocarcinoma marker of the first aspect in the preparation of a cervical squamous carcinoma or adenocarcinoma prognostic product (such as a kit or a detection device).

[0010] In a third aspect, the present application provides a kit for prognosing cervical squamous carcinoma and a kit for prognosing cervical adenocarcinoma, comprising a kit for detecting the cervical squamous carcinoma or adenocarcinoma prognostic marker of the first aspect.

[0011] As a preferred, the kit is an ELISA kit.

[0012] In a fourth aspect, the present application provides use of the cervical adenocarcinoma or squamous carcinoma prognostic marker of the first aspect in non-prognostic evaluation, pathological diagnosis or treatment of cervical squamous carcinoma or adenocarcinoma, or the present application provides use of the cervical adenocarcinoma or squamous carcinoma prognostic marker of the first aspect in prognostic evaluation, pathological diagnosis or treatment of cervical squamous carcinoma or adenocarcinoma.

[0013] In a fifth aspect, the present application provides a method for evaluating the prognosis of cervical squamous carcinoma and adenocarcinoma, which comprises detecting the level of adenylate kinase 4 in the cervical squamous carcinoma tissue of a subject and the level of brain spongiform malformation 2 protein in the cervical adenocarcinoma tissue, or using adenylate kinase 4 alone or in combination with other biomarkers as a prognostic marker for cervical squamous carcinoma in the preparation of a preparation for evaluating the prognosis of cervical squamous carcinoma, and using brain spongiform malformation 2 protein alone or in combination with other biomarkers as a prognostic marker for cervical adenocarcinoma in the preparation of a preparation for evaluating the prognosis of cervical adenocarcinoma.

[0014] In a sixth aspect, the present application provides a method for evaluating whether a drug can prevent or treat cervical squamous carcinoma and adenocarcinoma, which comprises using adenylate kinase 4 as a biomarker for cervical squamous carcinoma to evaluate the effect of the drug and using brain spongiform malformation 2 protein as a biomarker for cervical adenocarcinoma to evaluate the effect of the drug, so as to achieve the purpose of precision medicine.

[0015] In a seventh aspect, the present application provides a method for screening a drug active molecule or a drug capable of preventing or treating cervical squamous carcinoma or adenocarcinoma, which comprises using adenylate kinase 4 as a biomarker for cervical squamous carcinoma to evaluate the effect of the drug active molecule or the drug and using brain spongiform malformation 2 protein as a biomarker for cervical adenocarcinoma to evaluate the effect of the drug active molecule or the drug.

[0016] Compared with the prior art, the present application has the following beneficial effects:

[0017] The present application provides a new prognostic marker for evaluating cervical squamous carcinoma or adenocarcinoma, which can be used for diagnosing pathological types and evaluating the prognosis of cervical cancer, and thus more precise medication can be achieved.

[0018] The biomarker for evaluating the prognosis of cervical squamous carcinoma provided by the present application is adenylate kinase 4, and the biomarker for evaluating the prognosis of cervical adenocarcinoma is brain spongiform malformation 2 protein, and according to the level in the cervical cancer tissue, the prognosis of the cancer can be evaluated. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1 OPLS-DA analysis of proteomic results in the embodiments.

[0021] Figure 2 Comparison of AK4 (adenylate kinase 4) protein abundance of four subtypes in the examples.

[0022] Figure 3 Protein abundance comparison of the four subtypes of CCM2 (Cerebral cavernous malformation 2 protein) in the example.

[0023] Figure 4 Survival curve of squamous cell carcinoma patients stratified by AK4 in the example.

[0024] Figure 5 Survival curve of adenocarcinoma patients stratified by CCM2 in the example. DETAILED DESCRIPTION

[0025] In order to make the above objectives, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below. In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application. However, the present application 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 concept of the present application, so the present application is not limited to the specific embodiments disclosed below. The technical features in each embodiment of the present application can be combined accordingly without conflict.

[0026] The two most common subtypes of cervical cancer are squamous cell carcinoma and adenocarcinoma. The present application provides a corresponding prognostic biomarker in view of this phenomenon, which is selected from Adenylate kinase 4 in squamous cell carcinoma and Cerebral cavernous malformation 2 protein in adenocarcinoma.

[0027] It should be noted that both Adenylate kinase 4 and Cerebral cavernous malformation 2 protein are known proteins in the human body. They can be queried through public protein databases, for example, the protein ID in uniprot is shown in Table 1 below.

[0028] Adenylate kinase 4 (AK4) has the accession number P27144 in the protein database uniprot, belongs to the adenylate kinase family, and plays a key role in cellular energy metabolism and maintaining the adenine nucleotide balance of various subcellular regions. Specifically, adenylate kinase promotes the reversible transfer of a phosphate group from ATP to AMP, thereby forming two molecules of ADP. Studies have shown that AK4 interacts with ADP / ATP transporters, indirectly regulating mitochondrial membrane permeability, indicating its potential role in regulating mitochondrial function. There is also evidence that the increase in AK4 expression is closely related to tumor metastasis and drug resistance, mainly through mitochondrial activity and oxidative stress. However, there are few reports that it is related to the prognosis of cervical squamous cell carcinoma or can be used as a prognostic biomarker for cervical squamous cell carcinoma alone.

[0029] Cerebral cavernous malformations 2 protein (CCM2) has the accession number Q9BSQ5 in the protein database uniprot, which is a scaffold protein and plays a role in the stress-activated p38 mitogen-activated protein kinase (MAPK) signaling cascade. The protein interacts with SMAD specific E3 ubiquitin protein ligase 1 (also known as SMURF1) through a phosphotyrosine binding domain, promoting RhoA degradation. The protein is required for normal cytoskeletal structure, intercellular interactions, and lumen formation of endothelial cells. Mutations in this gene can cause cerebral cavernous malformations. However, there are few reports that it is related to the prognosis of cervical adenocarcinoma or can be used as a prognostic biomarker of cervical adenocarcinoma alone.

[0030] Table 1 ID and Chinese and English names of proteins

[0031]

[0032] It should be noted that the above-mentioned prognostic markers of cervical squamous carcinoma and adenocarcinoma can also be used as the basis for diagnosing cervical squamous carcinoma and adenocarcinoma. Adenylate kinase 4 can be used for diagnosing cervical squamous carcinoma, and cerebral cavernous malformations 2 protein can be used for diagnosing cervical adenocarcinoma. As a prognostic biomarker, it can also become a potential therapeutic drug target.

[0033] The reagents contained in the specific products (such as detection devices or kits) in the present application can be set according to the detection means of each marker. This means that for the two different prognostic markers of adenylate kinase 4 and cerebral cavernous malformations 2 protein, specific methods and reagents suitable for their detection can be selected.

[0034] Since the two markers are known substances, there are 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 level of these markers. In addition, mass spectrometry and other means can also be used.

[0035] Based on the rich experience and mature methods of the prior art, the existing detection means and reagents can be referred to, and the most suitable method can be selected to detect the above-mentioned markers. Then, according to the selected detection method, the corresponding diagnostic kit or detection device can be designed according to the standard and process of the prior art.

[0036] ELISA is a commonly used biomarker detection method, which uses specific antibodies to bind to the biomarker to be detected, and then produces a measurable signal through enzyme reaction. ELISA method has high sensitivity and specificity, and can be applied to the detection of single marker or multiple markers.

[0037] Immunofluorescence analysis is to use fluorescently labeled antibodies to bind to the biomarker to be detected, and then detect the intensity of the fluorescent signal through fluorescence microscope or fluorescence spectrophotometer, so as to quantitatively analyze the amount of biomarker.

[0038] Mass spectrometry analysis can accurately quantify biomarkers, including proton nuclear magnetic resonance (NMR) and mass spectrometry (MS) methods, which can provide very accurate molecular structure and relative content information.

[0039] PCR technology can be used to detect the transcriptional 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 fluorescent quantitative PCR method.

[0040] Protein chip technology can detect multiple biomarkers at the same time. Multiple antibodies are fixed on the surface of the chip, then combined with the proteins in the sample (such as tissue) to be detected, and finally the biomarkers are detected by fluorescence or radioactively labeled signal.

[0041] Exemplarily, considering the detection efficiency and convenience, the kit can be an ELISA kit. Further, the kit can be a product for detecting the mRNA expression level or protein expression level of AK4 and / or CCM2. Further, the kit for detecting the mRNA expression level or protein expression level of AK4 and / or CCM2 comprises a nucleic acid capable of binding to AK4 and / or CCM2 or a substance capable of binding to AK4 and / or CCM2 protein.

[0042] More specifically, the product for detecting the mRNA expression level of AK4 and / or CCM2 can comprise a molecule capable of binding to AK4 and / or CCM2 nucleic acid. The product can realize the function through known nucleic acid analysis methods, such as polymerase chain reaction (PCR), Southern hybridization, Northern hybridization, dot hybridization, fluorescence in situ hybridization (FISH), DNA microarray, high-throughput sequencing and chip detection, etc., especially PCR technology, including real-time fluorescent quantitative PCR and isothermal amplification technology (such as rolling circle amplification, loop-mediated isothermal amplification, strand displacement amplification). Using these products can qualitatively, quantitatively or semi-quantitatively analyze mRNA.

[0043] The nucleic acids contained in the products can be obtained by chemical synthesis or extracted from biological materials and amplified by primers. The mRNA can be reverse transcribed into cDNA and then amplified by specific primers to determine the corresponding mRNA expression level. In addition, the nucleic acids can also include primers specific for amplifying AK4 and / or CCM2 or probes that recognize the target genes. The probe is usually a polynucleotide sequence with a detection label and complementary to the target gene. These products come in various forms, including reagents, kits, test papers, and gene chips, and the primers or probes can also be immobilized on a carrier through solid-phase chip technology. High-throughput sequencing platforms can also detect AK4 and / or CCM2 by combining specific primers or probes.

[0044] The products for detecting the expression level of AK4 and / or CCM2 protein can contain substances that can bind to the target protein, such as antibodies or their fragments. The products can use classical methods for protein detection, such as ELISA, radioimmunoassay, immunohistochemistry, Western blotting, or proteomics techniques (such as antibody chips, mass spectrometry). The antibodies or their fragments can be monoclonal or polyclonal antibodies, or functional fragments thereof, including F(ab')2, Fab', Fab, single-chain Fv (scFv), disulfide-bonded Fv (dsFv), etc. Antibody fragments can also be prepared by nucleic acids encoding their amino acid sequences and expressed as vectors or cells.

[0045] The products can be made into reagents, kits, test papers, or gene chips, or as instrument platforms containing measurement modules and analysis modules, for determining the expression level of the target protein and its difference from the reference sample.

[0046] In another embodiment of the present application, the use of the above-mentioned biomarkers in the prognosis of cervical squamous cell carcinoma or adenocarcinoma for non-prognosis evaluation, pathological diagnosis, or therapeutic purposes is provided. The prognosis of cervical squamous cell carcinoma or adenocarcinoma for non-prognosis evaluation, pathological diagnosis, or therapeutic purposes can be for scientific research, non-medical commercial testing, or other purposes.

[0047] For example, the prognostic biomarkers of cervical squamous cell carcinoma or adenocarcinoma can be used to study the mechanisms of disease occurrence, pathophysiological processes, and potential therapeutic targets. By analyzing the changes of these markers in the disease process, the development and metastasis mechanisms of the disease can be better understood, providing a deeper understanding of the prevention and treatment of the disease.

[0048] Biomarkers also play an important role in drug development. They can be used as indicators for efficacy evaluation to help researchers assess 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 drug dosage adjustments and optimization can be guided.

[0049] Biomarkers can also be used in individual health management and preventive medicine. By regularly monitoring specific biomarkers, changes in an individual's health status can be detected in a timely manner, predicting the risk of disease, and taking appropriate health management measures, such as adjusting lifestyle, dietary habits or drug treatment, to maintain health and delay disease progression.

[0050] Biomarkers can be used to assess the impact of lifestyle on health. By monitoring changes in these markers, the extent to which different lifestyles affect health can be assessed, and individuals can be guided to take appropriate lifestyle interventions, such as weight loss, smoking cessation, increased exercise, etc., to improve health status and reduce disease risk.

[0051] The present application also provides a method for evaluating the prognosis of cervical squamous cell carcinoma and adenocarcinoma in a subject. The key to this method is to detect the level of adenylate kinase 4 in the tissue of a subject with cervical squamous cell carcinoma and the level of brain sponge malformation 2 protein in the tissue of a subject with cervical adenocarcinoma.

[0052] First of all, the implementation of this method involves sample collection and processing. The tissue sample is extracted from the cervical cancer tissue of the subject, and this process needs to follow strict standard operating procedures to ensure the quality and integrity of the sample.

[0053] Secondly, for the obtained tissue sample, appropriate techniques and reagents can be selected to detect the level of adenylate kinase 4 in cervical squamous cell carcinoma and the level of brain sponge malformation 2 protein in cervical adenocarcinoma. 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 methods, etc. Through these methods, the content level of a certain marker in the subject's tissue can be accurately quantified.

[0054] After obtaining the detection results of the markers, they need to be compared and analyzed with the relevant data of patients with poor prognosis. According to these comparison results, the prognosis prediction of the subject can be judged. If the level of a certain marker or marker combination is significantly higher than the normal range, it can be inferred that the subject has a higher probability of poor prognosis.

[0055] 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, in the implementation of this method, strict control of experimental conditions is required, combined with clinical practice experience and data analysis, to interpret the results and make diagnoses.

[0056] The present application provides a method for evaluating whether a drug can prevent or treat cervical squamous carcinoma or adenocarcinoma, which comprises using adenosine kinase 4 as a biomarker for cervical squamous carcinoma to evaluate the effect of the drug and using brain sponge malformation 2 protein as a biomarker for cervical adenocarcinoma to evaluate the effect of the drug, so as to achieve the purpose of precision medicine.

[0057] The implementation of the present method requires starting from experimental design and sample collection. In the design stage of a study or clinical trial, the drug to be evaluated, the treatment regimen, and the evaluation indicators need to be clearly defined. At the same time, the selection criteria for the subjects need to be determined, and the corresponding biological samples such as plasma, tissue or cell samples need to be collected for subsequent experimental analysis.

[0058] After obtaining the biological samples, the levels of the selected markers can be detected using appropriate techniques and reagents. 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 analysis. Through these methods, the content level of the markers in the biological samples of the subjects can be accurately quantified.

[0059] Next, the drug or treatment regimen needs to be applied to the subjects and treated according to the predetermined regimen. During the treatment process, the biological samples of the subjects need to be monitored regularly, and the levels of the selected markers need to be repeatedly detected. In this way, the therapeutic effect of the drug can be timely understood, and whether the expected treatment goal has been achieved can be determined.

[0060] Finally, based on the obtained detection results, the therapeutic effect of the drug can be evaluated and determined. If the levels of the selected markers have changed significantly and there is a significant difference compared with before treatment, it can be inferred that the drug may have the potential to prevent or treat cervical squamous carcinoma or adenocarcinoma. On the contrary, if the levels of the markers do not change significantly or do not achieve the expected effect, the treatment regimen may need to be re-evaluated or other treatment strategies may need to be tried.

[0061] The present application provides a method for screening drug active molecules or drugs capable of preventing or treating cervical squamous carcinoma or adenocarcinoma, which comprises using adenosine kinase 4 as a biomarker for cervical squamous carcinoma to evaluate the effect of the drug active molecules or drugs and using brain sponge malformation 2 protein as a biomarker for cervical adenocarcinoma to evaluate the effect of the drug active molecules or drugs, so as to achieve the purpose of precision medicine.

[0062] Firstly, the implementation of the present method requires the establishment of a suitable experimental system and model. The cell lines or animal models of cervical squamous carcinoma (adenocarcinoma) can be used to simulate the physiological processes of cervical squamous carcinoma (adenocarcinoma) and evaluate the effects of drug active molecules or drugs on them.

[0063] Secondly, suitable biomarkers are selected for evaluating the effects of drug active molecules or drugs. Adenylate kinase 4 can reflect the biological processes and pathological states of cervical squamous cell carcinoma as a biomarker of cervical squamous cell carcinoma, and brain sponge malformation 2 protein can reflect the biological processes and pathological states of cervical adenocarcinoma as a biomarker of cervical adenocarcinoma. Therefore, they can be used as biomarkers or individual biomarkers for screening the effects of drug active molecules or drugs to evaluate the intervention effects of drug active molecules or drugs on cervical squamous cell carcinoma (adenocarcinoma).

[0064] Next, screening experiments of drug active molecules or drugs are carried out. The drug active molecules or drugs to be screened are added to cells or animals, and the effects on adenylate kinase 4 in cervical squamous cell carcinoma and brain sponge malformation 2 protein in cervical adenocarcinoma are observed. Appropriate techniques and reagents can be used to detect the levels of these markers, such as immunological techniques and molecular biological techniques. According to the detection results, the potential therapeutic effects of drug active molecules or drugs on cervical squamous cell carcinoma or adenocarcinoma can be preliminarily evaluated.

[0065] After obtaining the preliminary screening results, further verification and confirmation experiments can be carried out. By using more refined and strict experimental design and operation process, the effects of drug active molecules or drugs on the levels of markers can be verified, and the therapeutic effects on cervical squamous cell carcinoma or adenocarcinoma can be further evaluated.

[0066] Finally, according to the experimental results, further research and development are carried out on drug active molecules or drugs with potential therapeutic effects. Their pharmacological properties, toxic side effects, pharmacokinetics and other drug properties can be further evaluated, and preclinical studies and clinical trials can be carried out.

[0067] The selection principle, process and effect of the above biomarkers are further demonstrated by a specific embodiment of the present application below, so as to facilitate the understanding of the essence of the present application by those skilled in the art.

[0068] If not specifically indicated, the technical means used in the examples is the conventional means familiar to those skilled in the art, and the reagents used in the present application are all analytical pure or above specifications, wherein the chromatographic column used is of the model Acclaim Pep Map100C18 column and the manufacturer is Thermo; the liquid chromatograph is of the model EASY-nLC 1200 and the manufacturer is Thermo.

[0069] Example 1: Selection of biomarkers

[0070] Sample inclusion criteria: 1. Patients diagnosed as cervical cancer by pathology (including histology and cytology). 2. Patients have collected cervical tissue samples before treatment. 3. Age > 18 years old.

[0071] Sample exclusion criteria: concurrent other primary malignancy.

[0072] Based on the above inclusion and exclusion criteria, 404 participants from Zhejiang Cancer Hospital from 2015 to 2020 were obtained. The 404 participants included 298 cases of cervical cancer squamous carcinoma, 74 cases of cervical cancer adenocarcinoma, 22 cases of cervical cancer adenosquamous carcinoma, and 10 cases of cervical cancer neuroendocrine carcinoma. The demographic and baseline characteristics of all patients are shown in Table 2. The collected fresh cervical cancer tissues of the subjects were directly placed into cryogenic tubes and stored in a -80°C refrigerator, and dry ice was transported.

[0073] Table 2 Demographic and baseline characteristics of cervical cancer patients

[0074]

[0075]

[0076] Reagents used in the present application: urea, thiourea, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid inner salt (CHAPS), tributylphosphine, formic acid, methanol, ammonium formate, ammonium bicarbonate, acetonitrile, dithiothreitol (DTT), iodoacetamide (IAM).

[0077] I. Sample processing and machine

[0078] Sample processing: The sample was homogenized in a lysis buffer (7M urea, 2M thiourea, 5% CHAPS, 2mM tributylphosphine) with protease inhibitors. After standing for 20 minutes, centrifugation was performed at 14000g for 10 minutes to remove tissue debris. The supernatant was collected and the protein concentration was determined by Bradford protein method. After that, protein extraction and digestion were performed. 100 μg of protein was purified by methanol / chloroform precipitation method, and the obtained protein precipitate was dried. The dried precipitate was resuspended in 50 mM ammonium bicarbonate, reduced in 0.25 M dithiothreitol at 56°C for 30 min, and then alkylated in 0.3 M iodoacetamide at 37°C in the dark for 30 min. After the protein sample was diluted with 100 mM ammonium bicarbonate, trypsin digestion was performed (enzyme substrate ratio 1:50, 37°C, 16 hours). The trypsin digestion was stopped by adding 20 μL of 0.1% formic acid aqueous solution. Then the peptides were desalted using ZipTip Pipette Tips (Merch Millipore, MA) according to the standard procedure.

[0079] The polypeptides of each sample were resuspended in mobile phase A (0.1% FA) and mixed with 0.8 μL of 2.5x iRT standard (Biognosys, Zurich, Switzerland).

[0080] Peptide separation was performed using an EASY-nLC 1200 (Thermo Fisher Scientific, San Jose, USA) equipped with an Acclaim Pep Map 100 C18 column (250 mm x 75 pm, 2 pm, Thermo Scientific Inc.). The mobile phase used for separation included solvent A (0.1% formic acid in water) and solvent B (80% acetonitrile in water, 0.1% formic acid in water). For each acquisition, the peptide separation was performed with a 65 min gradient (0-1 min, 3-8% buffer B; 1-52 min, 8%~35% buffer B; 52-56 min, 35%~95% buffer B; 56~65 min, 95% buffer B) at a flow rate of 300 pi / min. Peptides eluted from the analytical column were ionized into an Orbitrap Exploris 480 (Thermo Fisher Scientific, San Jose, USA) at a potential of +2.2 kV. The target column was loaded with a total of 500 ng of peptides per injection (measured by NanoDrop before loading) at a pressure of 550 bar. The ion transfer tube temperature was maintained at 320 °C. For MS1 full scans, the Orbitrap mass analyzer acquired ions at a high resolution of 120,000 over an m / z range of 350~1500. The maximum ion injection time was 50 ms. MS2 acquisition was performed in the highest speed mode with a duty cycle time of 3 s. Precursor ions were selected and fragmented using high-energy collisional dissociation (HCD) with a normalized collision energy of 32%. Each acquisition cycle included one survey scan at a resolution of 30000 (normalized automatic gain control target (AGC) of 2000%, injection time (IT) of 54 ms), and 30 DIA cycles with an isolation window of 5.6.

[0081] MS / MS spectra were searched using Spectronaut v.18 in the SwissProt human database at the UniProt website (www.UniProt.org) (Biognosys, Zurich, Switzerland). The data filtering was set to “Qvalue”. Default settings were used unless otherwise stated. FDR was set to 1% at both protein and peptide precursor levels.

[0082] II. Biomarker search

[0083] Figure 1 OPLS-DA results of the proteome in this example are shown in the figure, where SCC represents squamous carcinoma, AC represents adenocarcinoma, ASC represents adenosquamous carcinoma, and NEC represents neuroendocrine carcinoma. The OPLS-DA analysis plot reflects the overall distribution of samples in each group.

[0084] The biological prognostic markers were obtained by bioinformatics analysis methods such as differential screening, single factor COX regression analysis, and multi-factor COX regression analysis. The screening criteria for differences are mainly P value < 0.05, and the standard of COX regression analysis is P value < 0.05.

[0085] Screening of squamous carcinoma biomarkers:

[0086] First, the differential proteins of squamous carcinoma were screened by comparing with the other three subtypes respectively. Then, single factor COX regression analysis was performed on each subtype to screen out the proteins with significant prognostic significance only for squamous carcinoma. Subsequently, the intersection of the above two groups of proteins was taken to obtain the proteins that were both differentially expressed and had prognostic significance. Finally, multi-factor COX regression analysis was performed on the above potential prognostic biomarkers together with age and stage to obtain the final prognostic biomarker adenylate kinase 4 (AK4). The hazard ratio (HR) of the biomarker is shown in Table 3.

[0087] Figure 2 Figure 4 is a comparison of the protein abundance of adenylate kinase 4 (AK4) in the four subtypes. Compared with the other three subtypes, adenylate kinase 4 (AK4) was significantly up-regulated in squamous carcinoma. In the figure, SCC represents the cervical squamous carcinoma group, AC represents the cervical adenocarcinoma group, ASC represents the cervical adenoid squamous carcinoma group, and NEC represents the cervical neuroendocrine carcinoma group.

[0088] Figure 4 Figure 5 is the survival curve of the two groups of patients in the cervical squamous carcinoma group using the surv_cutpoint function to find the best cut point. The P value is < 0.0001, indicating that the group with high expression of adenylate kinase 4 has a poorer prognosis.

[0089] Table 3 Hazard ratio (HR) of biomarkers

[0090] Gene name Protein ID P value HR(CI) Squamous carcinoma AK4 P27144 0.002964 1.541(1.159-2.049) Adenocarcinoma CCM2 Q9BSQ5 0.0146 4.6(1.352-15.65)

[0091] Screening of adenocarcinoma biomarkers:

[0092] First, the differential proteins of adenocarcinoma were screened by comparing with the other three subtypes respectively. Then, single factor COX regression analysis was performed on each subtype to screen out the proteins with significant prognostic significance only for adenocarcinoma. Subsequently, the intersection of the above two groups of proteins was taken to obtain the proteins that were both differentially expressed and had prognostic significance. Finally, multi-factor COX regression analysis was performed on the above potential prognostic biomarkers together with age and stage to obtain the final prognostic biomarker cerebral cavernous malformation 2 protein (CCM2). The protein abundance box plot of cerebral cavernous malformation 2 protein in the four subtypes is shown in Figure 6, and the stratified survival curve in adenocarcinoma is shown in Figure 7. The hazard ratio (HR) of the biomarker is shown in Table 3. Figure 3 Figure 5 ​​

[0093] Figure 3 The protein abundance of cerebral cavernous malformation 2 (CCM2) is compared for the four subtypes. Adenocarcinoma is significantly down-regulated compared with the other three subtypes, and SCC represents the cervical squamous carcinoma group, AC represents the cervical adenocarcinoma group, ASC represents the cervical adenosquamous carcinoma group, and NEC represents the cervical neuroendocrine carcinoma group in the figure.

[0094] Figure 5 The cerebral cavernous malformation 2 (CCM2) protein is used to find the best cut point in cervical adenocarcinoma patients using the surv_cutpoint function, and the patients are divided into two groups according to the protein expression. The survival curve of the two groups is shown in the figure. The P value is 0.00015, indicating that the group with high expression of cerebral cavernous malformation 2 protein has a poorer prognosis.

[0095] Example 2: Further confirmation of biomarkers

[0096] Randomly selected 100 cervical cancer tissue samples from patients who visited Zhejiang Cancer Hospital from 2020 to 2023. The 100 patients include cervical cancer squamous carcinoma patients and cervical cancer adenocarcinoma patients. The collected fresh cervical cancer tissues of the subjects are directly placed into cryopreservation tubes, and the cryopreservation tubes are stored in a -80°C refrigerator and transported with dry ice.

[0097] The abundance of adenylate kinase 4 (AK4) in cervical squamous carcinoma patients and the abundance of cerebral cavernous malformation 2 protein (CCM2) in cervical adenocarcinoma patients are detected respectively, and the best cut point is found using the surv_cutpoint function. The patients are divided into two groups according to the protein expression.

[0098] Survival curve analysis is performed on the two markers, and the survival time of the two groups of squamous carcinoma patients with high and low expression of adenylate kinase 4 protein is subjected to Log-rank test. The result P is less than <0.001, and the group with high expression has a poorer prognosis. The survival curves of the two groups of adenocarcinoma patients with high and low expression of cerebral cavernous malformation 2 protein are subjected to Log-rank test, and the result P is <0.001, and the group with high expression has a poorer prognosis.

[0099] The above results show that adenylate kinase 4 can be used to evaluate the prognosis of cervical squamous carcinoma, and the group with high expression of adenylate kinase 4 has a poorer prognosis; cerebral cavernous malformation 2 protein can be used to evaluate the prognosis of cervical adenocarcinoma, and the group with high expression of cerebral cavernous malformation 2 protein has a poorer prognosis.

[0100] Example 3: ELISA kit for cervical squamous carcinoma or adenocarcinoma

[0101] The embodiment provides an ELISA kit for prognosis or diagnosis of cervical squamous cell carcinoma or adenocarcinoma, which is designed to detect the content level of adenylate kinase 4 in cervical squamous cell carcinoma tissue or the content level of cerebral cavernous malformation 2 protein in cervical adenocarcinoma tissue of a subject, so as to realize accurate diagnosis of cervical squamous cell carcinoma or adenocarcinoma.

[0102] Kit composition:

[0103] Adenylate kinase 4 (AK4) ELISA analysis:

[0104] The kit comprises reagents and materials for detecting adenylate kinase 4, standards, substrates, washing buffers and the like.

[0105] The content level of adenylate kinase 4 in the cervical cancer tissue of the subject is detected, and quantitative analysis is performed according to a standard curve.

[0106] Cerebral cavernous malformation 2 protein (CCM2) ELISA analysis:

[0107] The kit comprises reagents and materials for detecting cerebral cavernous malformation 2 protein, standards, substrates, washing buffers and the like.

[0108] The content level of cerebral cavernous malformation 2 protein in the cervical cancer tissue of the subject is detected, and quantitative analysis is performed according to a standard curve.

[0109] The detection process can be as follows:

[0110] The cervical cancer tissue sample of the subject is taken out, and sample processing and pretreatment steps are performed according to the kit instructions.

[0111] The pretreated tissue sample is added to the respective ELISA plate holes, and specific reactions occur with the antibodies of adenylate kinase 4 and cerebral cavernous malformation 2 protein.

[0112] The hole plate is washed with a washing buffer to remove unbound substances.

[0113] The substrate is added, and the reaction is performed under appropriate conditions to produce a measurable color.

[0114] The absorbance of the reaction product is measured using an enzyme label instrument, and the content of adenylate kinase 4 (AK4) and cerebral cavernous malformation 2 protein (CCM2) in the sample is calculated according to a standard curve.

[0115] According to the content level of each marker, the prognosis of the cervical cancer of the subject is evaluated in combination with a preset diagnosis standard.

[0116] The above-described embodiments are only the preferred ones of the present application, and are not intended to limit the present application. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present application. Therefore, any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present application.

Claims

1. Use of cerebral cavernous malformation 2 protein as a prognostic marker for cervical adenocarcinoma in the preparation of a formulation for assessing the prognosis of cervical adenocarcinoma.

2. Use of a reagent for detecting cerebral cavernous malformation 2 protein in the preparation of a kit or a detection device for assessing the prognosis of cervical adenocarcinoma.

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

4. The use according to claim 2, wherein the detection is a quantitative detection of the level of cerebral cavernous malformation 2 protein in the cervical cancer tissue of a subject.

5. The use according to claim 2, characterized in that, The kit is an ELISA kit.

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