Use of vamp8 in preparation of products for diagnosis and treatment of cervical diseases related to hpv16 virus infection

Abstract

The application discloses application of VAMP8 in preparation of products for diagnosing and treating HPV16 virus infection related cervical diseases. Through research on expression and functions of VAMP8 in HPV16 infection and cervical disease progression, the application provides a new potential marker and treatment target for prevention and treatment of HPV16 related cervical diseases, and has important clinical application value. Compared with the prior art, the application can more accurately treat HPV16 infection and induced cervical diseases, reduce side effects, and improve treatment effect.

Description

[0001] This application is a divisional application of application number "202311067977.0", application date "August 23, 2023", and invention title "Application of VAMP8 in the preparation of products for the diagnosis and treatment of HPV16 virus infection-related cervical diseases". Technical Field

[0002] This invention relates to the field of biotechnology, specifically to the application of VAMP8 in the preparation of products for the diagnosis and treatment of cervical diseases associated with HPV16 virus infection. Background Technology

[0003] In 2018, the International Agency for Research on Cancer (IARC) published a list of major cancer-related infectious pathogens worldwide, including high-risk human papillomavirus (HPV), Helicobacter pylori (HP), hepatitis B virus (HBV), and hepatitis C virus (HCV). These pathogens collectively contribute to over 90% of infection-related cancers globally. HPV, in particular, is predominantly found in sub-Saharan Africa and Asia. This virus is closely linked to benign lesions of the genitals and skin, as well as malignant tumors such as cervical cancer, head and neck cancer, and anal cancer. Cervical cancer is the fourth most common cancer among women worldwide and is the leading cause of cancer death among women in developing countries. According to the World Health Organization, approximately 570,000 new cases of cervical cancer are diagnosed globally each year, resulting in 311,000 deaths. In China, there are approximately 140,000 new cases of cervical cancer and about 37,000 deaths annually.

[0004] Human papillomavirus type 16 (HPV16) is a major carcinogenic factor for cervical cancer. Current diagnostic and treatment technologies, including HPV testing, pathological biopsy, early screening, and treatments such as surgery, radiotherapy, and chemotherapy, have significant limitations. For recurrent or advanced cervical cancer, current methods are of limited effectiveness, and the treatment process can impose a significant physical burden and severely impact patients' quality of life and fertility. Although there are vaccines to protect against HPV infection and early screening methods such as HPV DNA testing, liquid-based thin-layer cytology, and Pap smears, they cannot protect against all types of HPV and cannot completely prevent the disease, especially in women already infected with HPV. These screening methods have limitations in sensitivity and specificity, making it difficult to accurately identify high-risk patients or accurately assess disease progression in the early stages, easily leading to overdiagnosis or missed diagnosis, increasing patient stress and treatment costs.

[0005] The current state of clinical treatment for cervical cancer remains worrying. On the one hand, traditional treatments, such as surgery, radiotherapy, and chemotherapy, cannot completely control or eliminate the occurrence and metastasis of cervical cancer. On the other hand, the development of targeted drugs for cervical cancer is still in its early stages, and targeted drugs are scarce on the market. Existing target research mainly focuses on epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), mTOR, oncogenes E6 / E7, and immune checkpoints PD-1 / PD-L1 and CTLA-4. In addition, some researchers have discovered abnormal expression of multiple genes in cervical cancer tissues. These genes may drive tumor occurrence and development. In-depth research on these abnormally expressed genes is expected to find effective targets for anti-tumor therapy. Finding a new drug with durable efficacy, diverse targets, and relatively low cost has become an important research direction. Research on cervical lesions should further explore other possible therapeutic targets and optimize current treatment strategies to provide more effective and affordable treatment options.

[0006] Current technologies primarily focus on the detection and treatment of the virus itself, neglecting changes in host cells after viral infection, such as autophagy. These intracellular changes and autophagy have a significant impact on viral infection and disease development, but have not yet been thoroughly studied. Vesicle-associated membrane protein 8 (VAMP8), also known as endobrevin, is a member of the SNARE (soluble NSF attachment protein receptor) family. This protein family is involved in vesicle transport, facilitating the fusion of transport vesicles with their target membranes. SNARE proteins, including VAMP8, are characterized by the SNARE motif, a 60-70 amino acid-long region essential for protein-protein interactions. VAMP8 is located on chromosome 2, is approximately 10 kilobases long, and consists of 8 exons. The protein encoded by this gene is a type IV membrane protein, known to be primarily involved in the fusion of transport vesicles into their target compartments.

[0007] Functionally, VAMP8 participates in several important intracellular processes, including autophagy, endocytosis, exocytosis, and membrane repair. More specifically, VAMP8 participates in the final stage of exocytosis by promoting the fusion of secretory vesicles with the plasma membrane, a process crucial for the secretion of many proteins, neurotransmitters, and hormones. Furthermore, VAMP8 is involved in autophagosome-lysosome fusion, a key step in the autophagy pathway. In recent years, increasing research has focused on the potential role of VAMP8 in the pathogenesis of various diseases. However, there are currently no mature technologies or theories to elucidate its specific role in HPV16 infection and related cervical diseases, or how to combat HPV16 infection and control the progression of cervical diseases by modulating VAMP8.

[0008] In summary, current technologies present many limitations and challenges in the diagnosis and treatment of cervical diseases. A deeper understanding of the mechanisms of HPV16 infection and cervical diseases, particularly the occurrence of autophagy, has a significant impact on viral infection and disease progression. This is of great scientific and clinical importance for developing more effective diagnostic and treatment methods for cervical diseases. Identifying new molecular markers, such as VAMP8, and developing targeted therapies against these markers will be key to improving the accuracy and effectiveness of diagnosis and treatment, reducing side effects, and better addressing potential drug resistance in patients to existing treatments.

[0009] Basic information about the VAMP8 gene and protein

[0010] Gene name: VAMP8

[0011] Gene also known as: EDB; VAMP-8

[0012] Gene ID:8673

[0013] Species: Homo sapiens

[0014] Gene sequence number: NM_003761.5

[0015] Gene description: Homo sapiens vesicle associated membrane protein 8 (VAMP8), mRNA

[0016] DNA coding region:

[0017] atggaggaagccagtgaaggtggaggaaatgatcgtgtgcggaacctgcaaagtgaggtggagggagttaagaatattatgacccagaatgtggagcggatcctggcccggggggaaaacttggaacatctccgcaacaagacagaggatc tggaagccacatctgagcacttcaagacgacatcgcagaaggtggctcgaaaattctggtggaagaacgtgaagatgattgtccttatctgcgtgattgtttttatcatcatcctcttcattgtgctctttgccactggtgccttctcttaa

[0018] Protein sequence number: NP_003752.2

[0019] Protein description: Vesicle-associated membrane protein 8 [Homo sapiens]

[0020] Protein size: 100 aa

[0021] Protein sequence:

[0022] MEEASEGGGNDRVRNLQSEVEGVKNIMTQNVERILARGENLEHLRNKTEDLEATSEHFKTTSQKVARKFWWKNVKMIVLICVIVFIIILFIVLFATGAFS Summary of the Invention

[0023] The purpose of this invention is to provide the application of VAMP8 in the preparation of products for the diagnosis and treatment of cervical diseases related to HPV16 virus infection, in order to overcome the shortcomings of the prior art.

[0024] The present invention adopts the following technical solution:

[0025] The first aspect of this invention provides the application of VAMP8 as a diagnostic biomarker for HPV16 infection.

[0026] A second aspect of the present invention provides the application of VAMP8 in the preparation of diagnostic products related to HPV16 infection, said products including reagents, chips or kits.

[0027] A third aspect of the present invention provides the use of VAMP8 as a diagnostic biomarker for HPV16 infection-related cervical diseases.

[0028] A fourth aspect of the present invention provides the use of VAMP8 in the preparation of products for assessing the severity of HPV16 infection-related cervical diseases, said products including reagents, chips, or kits.

[0029] The fifth aspect of this invention provides the use of VAMP8 as a prognostic biomarker for cervical cancer.

[0030] The sixth aspect of this invention provides the use of VAMP8 in the preparation of products related to assessing the prognosis of cervical cancer, said products including reagents, chips, or kits.

[0031] The seventh aspect of the present invention provides the use of VAMP8 overexpression in the preparation of a product for treating early HPV16 infection, said product comprising a drug.

[0032] The eighth aspect of the present invention provides the use of VAMP8 activators in the preparation of products for treating early HPV16 infection, said products comprising a medicament.

[0033] The ninth aspect of the present invention provides the use of VAMP8 inhibition in the preparation of a product for treating cervical cancer, said product comprising a medicament.

[0034] The tenth aspect of this invention provides the use of VAMP8 inhibitors in the preparation of products for treating cervical cancer, said products comprising pharmaceuticals.

[0035] The beneficial effects of this invention are:

[0036] By gaining a deeper understanding of the role of VAMP8 in HPV16 infection-related cervical diseases, we aim to find new and more precise methods for the prevention, diagnosis, and treatment of cervical cancer, thereby improving patients' quality of life and prognosis.

[0037] 1. The Dual Role of VAMP8 in HPV16 Infection and Cervical Disease: This study clearly revealed the core role of vesicle-associated membrane protein 8 (VAMP8) in the progression of HPV16 infection and cervical disease. In the early stages of HPV16 infection, high expression of VAMP8 inhibits cell proliferation, migration, and invasion by promoting autophagy, maintaining cellular homeostasis and preventing disease progression. However, in the cervical cancer stage, high expression of VAMP8 promotes tumor cell proliferation, migration, and invasion. This demonstrates the dual role of VAMP8 in the development of cervical disease.

[0038] 2. VAMP8-based Early Diagnosis and Personalized Treatment of Cervical Diseases: A novel diagnostic and treatment strategy is proposed. By detecting VAMP8 expression, the development of HPV16-related cervical diseases can be more accurately assessed and predicted, enabling early diagnosis and personalized treatment. Targeting VAMP8 regulation aims to control cell proliferation, migration, and invasion in the early stages of HPV16 infection, while simultaneously inhibiting cell proliferation, migration, and invasion in the cancer stage, providing patients with more precise and personalized treatment options and reducing side effects and recurrence rates.

[0039] 3. VAMP8 as a prognostic biomarker for cervical cancer: According to bioinformatics analysis of the TCGA database, high VAMP8 expression is closely related to poor prognosis and the degree of immune infiltration in cervical cancer. Therefore, VAMP8 can be used as a prognostic biomarker for cervical cancer to help doctors assess patients' treatment response and prognosis.

[0040] 4. Deepening the understanding of HPV16 cervical disease mechanisms and optimizing treatment: The research results contribute to a deeper understanding of the molecular mechanisms by which HPV16 infection causes cervical diseases, providing new theoretical basis for disease prevention and treatment. Compared with existing technologies, this invention has higher sensitivity and specificity, playing an important role in improving the screening and treatment effects of HPV16 infection-related cervical diseases.

[0041] In summary, this invention, by studying the expression and function of VAMP8 in HPV16 infection and the progression of cervical disease, provides a new potential biomarker and therapeutic target for the prevention and treatment of HPV16-related cervical diseases, and has significant clinical application value. Compared with existing technologies, this invention can more precisely target HPV16 infection and induced cervical diseases, reduce side effects, and improve treatment efficacy. Attached Figure Description

[0042] Figure 1 To detect the expression level of VAMP8 in the cervix. (A) Mean and relative expression levels of proteomics in different types of cervical tissues, HPV_negative represents normal HPV16-negative cervical tissue, HPV_16_positive represents HPV16-positive cervical tissue, LSIL represents low-grade cervical squamous intraepithelial lesion, HSIL represents high-grade cervical squamous intraepithelial lesion, and CA represents cervical cancer. (B) mRNA expression levels of VAMP8 in different cervical cell lines in qPCR experiments, Ect1 / E6E7 represents HPV16-positive cervical cell lines, HeLa, SiHa, and C-33A represent different cervical cancer cell lines, and Primary represents primary cervical cells.

[0043] Figure 2 Immunohistochemical staining was used to verify the expression level of VAMP8 in different types of cervical tissue. HPV_negative represents normal HPV16-negative cervical tissue, HPV_16_positive represents HPV16-positive cervical tissue, LSIL represents low-grade cervical squamous intraepithelial lesion, HSIL represents high-grade cervical squamous intraepithelial lesion, and CA represents cervical cancer.

[0044] Figure 3 To perform bioinformatics analysis of the VAMP8 gene in the TCGA database. (A, B) VAMP8 expression levels in unpaired / paired pan-cancer samples from the TCGA_GTEx database. (C) VAMP8 expression levels in unpaired cervical cancer samples from the TCGA-CESC database. (D) Receiver operating characteristic (ROC) analysis to calculate the diagnostic efficacy of VAMP8 as a cervical cancer biomarker. (EG) Survival analysis to calculate the impact of VAMP8 as a prognostic biomarker on survival in cervical cancer.

[0045] Figure 4Clinical correlation analysis was used to show the differences in VAMP8 expression levels among different clinical groups of cervical cancer. (A) T stage: T1 / T2 / T3 / T4. (B) N stage: N0 / N1. (C) M stage: M0 / M1 / MX. (D) Clinical stage: I / II / III / IV. (E) Treatment efficacy: Complete remission (CR), partial remission (PR), stable disease (SD), and progressive disease (PD). (F) Pathological type: Squamous cell carcinoma, Adenocarcinoma, Adenosquamous carcinoma. (G) Pathological grade: G1 / G2 / G3 & G4. (H) Menopausal status: Pre- / Per- / Post-.

[0046] Figure 5 To illustrate the correlation between VAMP8 and immune cells in immune infiltration analysis. (A) Lollipop plot showing the correlation between VAMP8 and various immune cells. (B) Significant differences in enrichment scores among immune cells. (C) Scatter plot showing the correlation between NK cells and VAMP8. (D) Scatter plot showing the correlation between DC cells and VAMP8.

[0047] Figure 6 To observe the changes in the morphology and number of autophagosomes after knockdown / overexpression of VAMP8 in different cervical cell lines using transmission electron microscopy.

[0048] Figure 7 To detect changes in cell proliferation capacity after knockdown / overexpression of VAMP8 in different cervical cell lines in a CCK-8 cell proliferation assay.

[0049] Figure 8 To detect changes in cell migration / invasion ability after knockdown / overexpression of VAMP8 in different cervical cell lines using Transwell assays.

[0050] Figure 9The changes in the proportion of different phases of the cell cycle after knockdown / overexpression of VAMP8 in different cervical cell lines were detected by flow cytometry PI staining. G0 / G1 phase: G0 phase: This is a quiescent phase of the cell cycle. When a cell is not ready to divide or grow, it enters the G0 phase. Some cells, such as nerve cells, may remain permanently in the G0 phase. G1 phase: This is the first growth phase of the cell cycle. During this period, the cell grows and synthesizes the necessary proteins and other molecules to prepare for DNA replication. S phase: This is the synthetic phase of the cell cycle. During this period, the cell replicates its DNA. In this way, both daughter cells receive a complete copy of the genetic information during cell division. G2 / M phase: G2 phase: This is the second growth phase of the cell cycle. During this period, the cell continues to grow and prepares for the upcoming cell division, while carrying out some necessary biosynthetic activities. M phase: This is the mitotic phase of the cell cycle. During this period, the nucleus and cytoplasm of the cell divide to form two new daughter cells. This process includes four sub-phases: prophase, metaphase, anaphase, and telophase.

[0051] Figure 10 The change in apoptosis rate after knockdown / overexpression of VAMP8 in different cervical cell lines was detected by flow cytometry and Annexin V staining.

[0052] Figure 11 To verify the changes in tumorigenicity of the cervical cancer cell line SiHa after knockdown / overexpression of VAMP8 in vivo using a subcutaneous injection tumorigenesis experiment in nude mice. (A) Macroscopic appearance of tumors; (B) Changes in tumor volume in each group; (C) Final weight of tumors in each group.

[0053] Figure 12 To observe the changes in autophagosomes that form tumors in cervical cancer cell lines after SiHa knockdown / overexpression of VAMP8 using transmission electron microscopy. Detailed Implementation

[0054] The present invention will be further explained below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0055] Example 1

[0056] Vesicle-associated membrane protein 8 (VAMP8) is a membrane-bound protein involved in intracellular vesicle transport and fusion, playing a crucial role in physiological and pathological processes such as immune responses, neurotransmitter release, and pathogen invasion. The potential association between VAMP8 and cervical disease is not as well-studied as its basic functions and interactions. This study primarily focuses on the impact of VAMP8 in HPV16 infection-related cervical disease.

[0057] 1. VAMP8 as a diagnostic biomarker

[0058] 1.1. Sample Collection

[0059] ① Cervical tissue samples, both HPV16 positive and negative, were collected from the hospital. Privacy was protected for all participants based on their consent forms.

[0060] ② Divide the positive and negative samples into two groups respectively, and ensure that the number of samples in each group is equal to ensure statistical validity.

[0061] ③ Clinically classify the samples: normal, low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), and cervical cancer.

[0062] 1.2. Proteomics Analysis

[0063] ① Sample collection and processing: The collected tissue samples are mechanically fragmented and chemically broken down to release the proteins in the tissue.

[0064] Mechanical fragmentation can be achieved through one of the following methods: (1) Friction / tissue grinder: Tissue is physically fragmented into powder or liquid by grinding with high-speed rotating blades. (2) Ultrasound: Cell structure is destroyed by high-frequency vibration of ultrasound, causing proteins to be released. The commonly used equipment is the ultrasonic cell disruptor. (3) Vibrating ball grinding: Cell walls and cell membranes are destroyed by high-speed vibration of steel balls. It is often used for protein extraction from plants and solid tissues. (4) Cryo-grinding: Tissue is rapidly frozen in liquid nitrogen and then physically ground to rupture cells and release proteins.

[0065] Chemical disruption can be achieved through one of the following methods: (1) Using detergents: such as Triton X-100, NP-40, etc. These detergents can disrupt cell membranes and organelle membranes, releasing proteins. (2) Using solvents: such as urea, ammonium sulfate, etc., which can disrupt the structure of proteins and dissolve them. (3) Treating with acids or alkalis: Disrupting cell structure and releasing proteins by changing the pH value.

[0066] To ensure high-quality protein samples, mechanical fragmentation and chemical disruption processes often need to be carried out under freezing or low-temperature conditions to prevent protein degradation or inactivation. During mechanical fragmentation, protease inhibitors and other chemical reagents are often added to ensure protein stability and integrity.

[0067] ② Protein Extraction and Purification: Extract proteins from tissue samples using an appropriate protein extraction buffer. Purify the extracted proteins using a protein purification kit.

[0068] Protein extraction buffer is a specially formulated liquid for dissolving and extracting proteins from tissues or cells. This liquid typically contains certain ions, such as Tris, EDTA, and NaCl, as well as surfactants such as Triton X-100 or SDS that can disrupt protein-protein or protein-lipid interactions. In addition, extraction buffers usually contain protease inhibitors to protect proteins from degradation.

[0069] Protein extraction process: a. Place the tissue sample in a homogenizer and add an appropriate amount of protein extraction buffer; b. Use the homogenizer to physically break down the tissue, releasing the protein into the buffer; c. Centrifuge the homogenized material and transfer the supernatant to a new centrifuge tube; d. The supernatant contains the extracted protein.

[0070] Protein purification process: a. Binding: The protein solution first contacts a specific affinity column, allowing the target protein to bind to the column material; b. Washing: The column is washed with buffer to remove non-specifically bound proteins and impurities; c. Elution: The target protein is eluted from the column material using a specific elution buffer; d. Concentration: If necessary, the eluted protein solution can be concentrated using methods such as ultrafiltration; e. Further purification: Such as gel permeation chromatography, ion exchange chromatography, etc., to further improve the purity of the protein.

[0071] ③ Protein mass spectrometry analysis: The purified protein was analyzed by liquid chromatography coupled with mass spectrometry (LC-MS / MS). The VAMP8 protein was identified based on the mass spectrum, and its abundance was quantified.

[0072] a. Liquid chromatography-LC-MS conditions:

[0073] Column: C18 reverse phase column (e.g., 2.1 mm x 150 mm, 1.9 μm particle size).

[0074] Solvent A: 0.1% formic acid aqueous solution.

[0075] Solvent B: 0.1% formic acid acetonitrile solution.

[0076] Gradient: From 5%B to 40%B, 40 minutes; From 40%B to 80%B, 10 minutes.

[0077] Flow rate: 0.3 mL / min.

[0078] Injection volume: 5 μL.

[0079] b. Mass spectrometry (MS) conditions:

[0080] Mass spectrometer: Q-Exactive HF, or similar instrument.

[0081] Electrospray source: cationic mode.

[0082] Range: m / z 375-1600.

[0083] Resolution: 30000.

[0084] Automatic gain control (AGC): 3e6.

[0085] Maximum spray time: 50ms.

[0086] Secondary mass spectrometry fragmentation mode: High collision energy dispersion (HCD).

[0087] c. Data Analysis:

[0088] Use software such as MaxQuant, Proteome Discoverer, or Mascot to analyze raw data and identify and quantify peptides and proteins.

[0089] For the identification of VAMP8 protein, there should be at least two unique, high-confidence peptide matches.

[0090] Quantification was performed using standardized peptide internal standard strategies or tag-free quantification methods such as MaxLFQ. First, a specific representative VAMP8 peptide was selected and quantified by peak area. Relative or absolute quantification was then performed using an internal standard of known concentration or peak area relative to other reference proteins.

[0091] 1.3. Quantitative PCR (qPCR) Analysis

[0092] ① RNA extraction: Total RNA was extracted from the sample using an RNA extraction kit (TRIzol, TaKaRa, RT reagent Kit).

[0093] ②Reverse transcription: RNA was converted into cDNA using a reverse transcription kit (Novizan, ChamQ SYBR qPCR Master Mix).

[0094] ③ Quantitative PCR: Specific primers targeting the VAMP8 gene were designed (VAMP8: forward 5'-tgtgcggaacctgcaaagt-3'; reverse 5'-cttctgcgatgtcgtcttgaa-3'), and qPCR reactions were performed using SYBR Green or TaqMan probes. Simultaneously, one or more internal reference genes were selected for normalization (ACTB / β-actin: forward 5'-catgtacgttgctatccaggc-3'; reverse 5'-ctccttaatgtcacgcacgat-3').

[0095] ④ Data analysis: Use data analysis software for qPCR instruments, such as the 2^-ΔΔCT method, to perform data analysis.

[0096] 1.4. Data Analysis and Results Interpretation

[0097] ① Statistical analysis was performed on the results of proteomics and qPCR to determine the expression differences of VAMP8 in different categories of samples.

[0098] ②Analyze the association between VAMP8 expression levels and the severity of cervical diseases by combining clinical data.

[0099] ③ Discuss whether VAMP8 can serve as a potential biomarker for diagnosing HPV16 infection and assessing the severity of cervical disease.

[0100] 2. VAMP8 as a prognostic biomarker

[0101] 2.1 Data Acquisition and Preprocessing

[0102] ① We used gene expression datasets for cervical cancer from the Cancer Genome Atlas (TCGA) database, including clinical information data and gene expression level data.

[0103] Clinical information data: such as the patient's age, gender, date of diagnosis, survival time, clinical stage, pathological type, pathological stage, treatment method, etc.

[0104] Gene expression data: Shows the expression level or activity of each gene in the sample.

[0105] ② Perform quality control and preprocessing on these data, including data cleaning, formatting, standardization, batch effect correction, and data integration, to improve the accuracy of subsequent analysis.

[0106] Data cleaning: First, incomplete or obviously erroneous records need to be removed. For example, samples or genes with a large number of missing values ​​may be excluded. Records that are clearly inconsistent with other data also need to be corrected or deleted.

[0107] Formatting: To ensure all data uses the same format and standard, it may be necessary to convert the data format. For example, convert all gene expression data to the same standardized form, or convert all dates to the same format.

[0108] Standardization: Because data may come from different laboratories or platforms, their scope and distribution may vary. The standardization process aims to ensure that all data have the same proportions and distribution. For example, Z-score transformation or max-min standardization can be performed on gene expression data.

[0109] Batch effect correction: When integrating multiple batches of data, systematic differences may be observed due to technical reasons. Commonly used methods, such as ComBat, can be used to correct these batch effects to ensure that the data between different batches are comparable.

[0110] Data integration: If multiple types of data (such as mRNA expression and miRNA expression) are used simultaneously, it may be necessary to integrate them into a consistent format for overall analysis.

[0111] 2.2. Differential Expression Analysis

[0112] ① The expression level of VAMP8 was differentially analyzed using the limma or DESeq2 package in R language, and the differences between the cervical cancer group and the normal group were compared.

[0113] ② Perform multiple hypothesis testing to correct the results in order to reduce the occurrence of false positives.

[0114] 2.3. ROC Curve Analysis

[0115] ① The ROC curve (Receiver Operating Characteristic Curve) is a comprehensive indicator reflecting sensitivity and specificity, often used to evaluate the accuracy of diagnostic tests. Given different classification thresholds, the ROC curve depicts the relationship between the true positive rate (TPR, sensitivity) and the false positive rate (FPR, 1-specificity). The area under the curve (AUC) is the area between the ROC curve and the horizontal axis, which quantifies the overall performance of the classifier. An AUC value of 0.5 indicates a random classifier, and a value of 1 indicates a perfect classifier.

[0116] ②Specific analysis method: For binary classification problems, determine the labels of positive and negative examples; calculate the probability of a positive example for each sample; sort the samples according to the probability values; gradually change the classification threshold and calculate TPR and FPR; plot the ROC curve on the coordinate graph, with FPR on the horizontal axis and TPR on the vertical axis; calculate the AUC value to quantify the performance of the classifier.

[0117] 2.4. Survival Analysis

[0118] ① Based on the expression level of VAMP8, cervical cancer cases were divided into high-expression and low-expression groups. The median VAMP8 expression level was used as a threshold; those above or equal to this threshold were considered the high-expression group, while those below this threshold were considered the low-expression group.

[0119] ② Survival analyses (such as the Kaplan-Meier method) were used to investigate the relationship between VAMP8 expression levels and overall survival (OS) and disease-free survival (DFS) in cervical cancer patients. Overall survival (OS): refers to the time from randomization or the start of treatment until death from any cause. This is a direct indicator of patient survival, regardless of the cause of death. Disease-free survival (DFS): refers to the time from randomization or the start of treatment until disease recurrence, progression, or death from the disease. DFS focuses on whether the patient is in a disease-free state, i.e., without disease recurrence or progression. OS focuses on the event of death, while DFS focuses on disease recurrence, progression, or death from the disease.

[0120] ③ The Cox proportional hazards model was used to further verify the independent association between VAMP8 expression level and cervical cancer prognosis.

[0121] 2.5. Clinical Relevance Analysis

[0122] ① Data preparation: Collect patients' clinical data and biomarkers or model predictions.

[0123] ② Select appropriate statistical tests for different clinical indicators: based on the distribution and type of data, such as Pearson correlation, Spearman rank correlation or chi-square test.

[0124] ③ Calculate statistical significance: assess whether the observed relationship is unlikely to be caused by random factors.

[0125] ④ Effect size assessment: This measures the strength of the relationship between the prediction of a biomarker or model and the clinical outcome, such as the magnitude of the correlation coefficient.

[0126] 2.6. Analysis of the degree of immune infiltration

[0127] TIMER (Tumor Immuno Estimation Resource) is an analytical tool for calculating the level of immune cell infiltration. This tool is primarily used to analyze the penetration level of immune cells in tumor tissue, revealing the interaction between the tumor and the immune system by estimating the abundance of various immune cells in the tumor microenvironment. TIMER employs statistical analysis methods based on large-scale tumor gene expression profiling data, using gene expression characteristics of six major immune cell types (B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells). These data allow for the inference of the abundance of these cells in tumor samples. TIMER was used to analyze the correlation between VAMP8 expression and immune cell infiltration, thereby understanding the role of VAMP8 in the immune microenvironment of cervical cancer.

[0128] 2.7. Immunohistochemical Experiment

[0129] ① Sample preparation: Select tissue sections corresponding to the samples used in the bioinformatics analysis, and ensure that they have been fixed and embedded.

[0130] ② Antibody selection: Purchase or prepare specific antibodies against VAMP8, and select appropriate secondary antibodies, which are antibodies that bind to fluorescent or enzyme-linked labels.

[0131] ③ Immunostaining: The tissue sections are subjected to steps such as deacidification, peroxidase blocking, antibody incubation, rinsing, secondary antibody incubation, and staining (e.g., DAB).

[0132] ④ Results observation: The expression of VAMP8 in the tissue was observed using an optical microscope or a fluorescence microscope.

[0133] 2.8. Data Visualization

[0134] All results need to be presented clearly and accurately. Use relevant packages in R or Python (such as ggplot2, Matplotlib, etc.) to generate high-quality charts, including differential expression plots, ROC diagnostic efficacy curves, survival curves, and immune cell infiltration maps.

[0135] In conducting these analyses, the false discovery rate will be strictly controlled, and appropriate multiple test correction methods, such as the Bonferroni or Benjamini-Hochberg methods, will be used. Simultaneously, potential biases and confounding factors, such as batch effects, age, and gender, will be addressed and eliminated through normalization, batch effect correction, multivariate regression analysis, or other statistical methods.

[0136] 3. Effects of VAMP8 on cervical cell function

[0137] 3.1. Cell Culture and Processing

[0138] ① HPV16-positive cervical cell lines Ect1 / E6E7 (4. Cervical cancer cell lines HeLa, SiHa, and C-33A) were cultured in a 37°C, 5% CO2 constant temperature and humidity incubator using Dulbecco's modified Eagle's medium (DMEM), which contains 10% fetal bovine serum and 1% antibiotics (penicillin / streptomycin, 1 / 1).

[0139] ② Observe cell morphology and growth status daily using an inverted microscope. When cell coverage reaches 70%–80%, wash with sterile PBS solution and then digest and detach the cells with 0.25% trypsin-EDTA.

[0140] ③ Collect cells by centrifugation (1000 rpm, 3 minutes) (i.e., centrifugation to remove trypsin), then resuspend them in fresh complete culture medium and inoculate them into new culture plates for later use.

[0141] 3.2. Lentiviral preparation and transfection

[0142] ① Lentiviral overexpression or knockdown of the VAMP8 gene was prepared using a lentiviral packaging system. First, the lentiviral vector overexpressing VAMP8 or the lentiviral shRNA vector knockdown of VAMP8 was transfected into 293T cells using a calcium-phosphorus coprecipitation method. After 48-72 hours, the suspension was collected and filtered through a 0.45 μm filter membrane, and then the virus was precipitated using an ultracentrifuge. The lentiviral vector for VAMP8 overexpression was CMV-MCS-3Flag-Ubi-ZSGreen-IRES-Puromycin, and the selected gene CDS region sequence was: atggaggaagccagtgaaggtggaggaaatgatcgtgtgcggaacctgcaaagtgaggtggagggagttaagaatattatgacccagaatgtggagcggatcctggcccggggggaaaacttggaacatctccgcaacaagacagaggatctggaagccacatctgagcacttcaagacgacatcgcagaaggtggctcgaaaattctggtggaagaacgtgaagatgattgtccttatctgcgtgattgtttttatcatcatcctcttcattgtgctctttgccactggtgccttctcttaa. The lentiviral shRNA vector for VAMP8 knockdown was U6-MCS-CMV-zsGreen-PGK-Puromycin, and the selected interference fragment was sh-5'-gaaatgatcgtgtgcggaacc-3'.

[0143] ② For HPV16-positive cervical cells that have grown to 60%-70% (cervical cancer cells in 4.), lentivirus transfection was performed at an MOI of 10, and 6-8 μg / mL of Polybrene was added to enhance transfection efficiency. The medium was replaced with fresh complete culture medium 24 hours after transfection.

[0144] Screening and constructing stable cell lines:

[0145] ③ 72 hours after transfection, cells are screened using complete culture medium containing 2 μg / mL puromycin. Screening is usually continued for 7-10 days to select cell lines that stably express VAMP8.

[0146] 3.3. Detection of VAMP8 mRNA and protein expression

[0147] ① To detect mRNA expression, total RNA was extracted using TRIzol, then cDNA was synthesized by RT-PCR, and finally, VAMP8 mRNA expression was detected by qPCR. The specific steps are the same as in 1.3.

[0148] ② To detect protein expression, cell proteins were extracted using RIPA lysate, and protein concentrations were determined by the BCA method. Proteins were then separated by SDS-PAGE electrophoresis and Western blotting to detect the expression level of VAMP8 protein.

[0149] a. Protein extraction and SDS-PAGE electrophoresis:

[0150] Protein extraction: Total protein is extracted from HPV16-positive and HPV16-negative cervical tissue samples. RIPA buffer or other protein extraction buffers are typically used for tissue lysis and protein extraction. After lysis, centrifugation is performed to remove cellular debris; the supernatant is the total protein sample.

[0151] Protein concentration determination: Protein concentration was determined by the BCA method.

[0152] SDS-PAGE electrophoresis: The extracted protein sample is mixed with SDS loading buffer, boiled for 5 minutes, and then loaded onto a polyacrylamide gel for electrophoresis. A constant current mode is used; the initial electrophoresis is typically performed at 80-120V until the sample enters the separating gel, then the voltage is increased to 120-200V until electrophoresis is complete.

[0153] b. Transfer and immunoblotting:

[0154] Transfer: After SDS-PAGE electrophoresis, the gel is placed in a transfer apparatus. A PVDF membrane is used as the matching membrane, pre-activated with methanol and then moistened with transfer buffer. An electric current is used to transfer proteins from the gel to the PVDF membrane.

[0155] Immunoblotting: After transfer of the PVDF membrane, it is first incubated in blocking buffer containing 5% skim milk powder or BSA to prevent nonspecific binding. Then, it is incubated with diluted specific anti-VAMP8 antibody (an antibody prepared against a specific region of the VAMP8 protein for specific detection and recognition of the VAMP8 protein), followed by washing and incubation with a secondary antibody of the same species as the original antibody.

[0156] c. Signal detection and quantification:

[0157] Signal detection: If a fluorescent secondary antibody is used, signal detection can be performed directly using a bioimaging system. If a chemiluminescent secondary antibody is used, an ECL (enhanced chemiluminescence) indicator needs to be added, and exposure under specific conditions is required to detect the chemiluminescent signal.

[0158] Image acquisition and protein quantification: Detecting and acquiring signals on immunoblot membranes using bioimaging systems. This can be digital or film-captured, detecting fluorescence or chemiluminescence signals and providing relative protein quantification.

[0159] 3.4. Experimental Verification of Cellular Function

[0160] ① Cell Counting Kit-8 (CCK-8) Assay: Cell proliferation rate was determined using the CCK-8 assay kit. Cells treated or transfected with different methods were seeded in equal volumes in 96-well plates (in complete culture medium) and cultured at 37°C and 5% CO2. After 24, 48, and 72 hours of culture, the medium was replaced with fresh complete culture medium containing 10% CCK-8, and cultured for another 2 hours. The optical density (OD) value was measured at 450 nm using a microplate reader.

[0161] ② Scratch Healing Assay: To ensure consistency and reproducibility, horizontal lines were drawn at 0.5 cm intervals on the bottom of a 6-well plate, with at least four lines per well. Cells were seeded in the 6-well plates at a density of 10^6, aiming to achieve a monolayer of cells covering the bottom of each well by the next day. Using the tip of a 200 μL pipette, a straight scratch of uniform initial width was created by gently slicing through the cell layer. Cells were then washed with PBS and cultured in serum-free medium (Dulbecco's Modified Eagle's Medium (DMEM) containing 1% antibiotics (penicillin / streptomycin, 1 / 1)). Images were taken using an inverted microscope at the start of the experiment (0 hours) and 24 hours later. Horizontal lines were used for positioning to ensure observation and measurement of changes in the width of the cell scratches at the same location.

[0162] ③ Transwell cell migration and invasion assays: First, medium containing 20% ​​serum (i.e., Dulbecco's modified Eagle's medium (DMEM) containing 20% ​​fetal bovine serum and 1% antibiotics (penicillin / streptomycin, 1 / 1)) was added to the lower chamber of the Transwell apparatus. Cells from each group were resuspended at the same concentration in serum-free medium (i.e., Dulbecco's modified Eagle's medium (DMEM) containing 1% antibiotics (penicillin / streptomycin, 1 / 1)) and seeded in the upper chamber of the Transwell. After incubation at 37°C and 5% CO2 for 24 hours, cells remaining in the upper chamber were gently removed with a moistened cotton swab. In the invasion assay, the upper chamber of the Transwell was coated with Matrigel (diluted 1:2 with serum-free medium), while no Matrigel coating was required in the migration assay. Subsequently, cells that had migrated or invaded to the lower chamber and adhered were fixed with 4% paraformaldehyde for 1 hour, stained with crystal violet for 15 minutes, and then photographed under a microscope at randomly selected fields of view.

[0163] ④ Cell cycle analysis using flow cytometry:

[0164] Pretreatment: Collect the cells to be tested and then centrifuge them to remove the culture medium.

[0165] Fixation: After freezing the cells, fix them by slowly adding 70% ice-cold ethanol and storing them at 4°C overnight.

[0166] Staining: Remove the ice-cold ethanol and wash the cells at least twice with PBS. Then, add an appropriate amount of PI (Propidium Iodide) staining solution to the cells and incubate in the dark for 15-30 minutes.

[0167] Flow cytometry: Cell detection is performed using a flow cytometer, and after data collection, cell cycle analysis is performed using specialized software.

[0168] ⑤ Detection of cell apoptosis using Annexin V / PI double staining:

[0169] Pretreatment: Collect the cells to be tested and centrifuge them to remove the culture medium.

[0170] Staining: Prepare Annexin V and PI staining solutions according to the kit instructions. First, add Annexin V dye and incubate in the dark for 15 minutes, then add PI dye and incubate in the dark for 5 minutes.

[0171] Flow cytometry: Cell detection was performed using a flow cytometer. Annexin V attaches to outward-facing phosphatidylserine residues, while PI stains the DNA in dead cells. After data collection, apoptotic cells were identified and analyzed using specialized software.

[0172] Note: Experiments ④ and ⑤ require processing PI in the dark, as PI is light-sensitive. Foam formation must be avoided during the experiments to ensure the accuracy of flow cytometry detection. The sterility of equipment, tools, and reagents must be ensured before and after the experiments.

[0173] 3.5. Observation of morphological and quantitative changes of autophagosomes using transmission electron microscopy (TEM)

[0174] ① Sample Preparation: Select cells to be tested and culture them after appropriate stimulation or treatment. Fix the cells using a fixative (e.g., 2.5% glutaraldehyde). Centrifuge the cells and treat them with 2% ruthenium acetate to enhance electron density. Embed the cells in resin (e.g., EPON 812). Prepare ultrathin sections (approximately 60-90 nm thick).

[0175] ② Transmission electron microscopy operation: Place the ultrathin section on a copper grid. If necessary, the section can be contrast-stained, such as with lead citric acid or uranyl acetate. Observe under a transmission electron microscope, usually at 80-100kV.

[0176] ③ Image Acquisition and Analysis: Capture high-resolution images of different regions. Use specialized software for image analysis, labeling, and counting autophagosomes. Describe the morphology of autophagosomes, such as their integrity, size, and contents.

[0177] ④ Statistical analysis: The number of autophagosomes was statistically analyzed under multiple observation fields. Appropriate statistical methods were used to compare the differences between different experimental groups.

[0178] 4. The role of VAMP8 in cervical cancer cells

[0179] The technical solution for cell experiments is the same as in "3.".

[0180] 4.1. Tumor formation experiment in nude mice:

[0181] ① Under ethical conditions, nude mice were anesthetized and subcutaneously injected with cervical cancer cells SiHa that regulate VAMP8 expression and control cell lines from the transfection model. Approximately 1 million cells were injected into each mouse to construct a nude mouse cervical cancer xenograft model.

[0182] ② Observe and record the weight of nude mice every 2 days. At the same time, use calipers to measure the length of the longest axis and the width of the widest axis of the tumor. Calculate the tumor volume according to the formula V=0.5×length×width^2.

[0183] ③ By isolating tumor tissue, the expression level of VAMP8 in the tumor tissue was detected using IHC and qPCR experiments to observe its effect on tumor growth.

[0184] ④ After the experiment, strict procedures for nude mouse dissection and post-experimental processing were performed to meet ethical and research regulations. The dissected tumors were measured and recorded, and further pathological sections and HE staining were performed to observe the microscopic pathological changes of the tumors.

[0185] Nude mouse dissection and post-dissection: After ensuring the rodents were under anesthesia, a rigorous nude mouse dissection was performed. Post-dissection procedures were carried out in accordance with relevant ethical and research guidelines to ensure humane treatment of the rodents.

[0186] Tumor sampling and measurement: The tumor tissue was carefully dissected and placed under sterile conditions. The length and width of the tumor were measured and recorded using measuring tools such as calipers.

[0187] Pathological tissue sectioning and staining: The dissected tumor tissue was fixed in 10% neutral buffered formaldehyde. After fixation, the tissue underwent pretreatment steps including dehydration, clearing, and paraffin infiltration. Using a microtome, tissue sections with a thickness of 4-5 micrometers were prepared and placed on glass slides. Hematoxylin-eosin (HE) staining was performed on the tissue sections: first, hematoxylin was used to stain the nucleus blue, and then eosin was used to stain the cytoplasm red. After staining, the microscopic pathological changes of the tumor tissue were observed and recorded under a microscope.

[0188] Example 2

[0189] Statistical significance is a tool used to assess whether there is a significant difference between observed results and the null hypothesis. The p-value is an indicator used to represent the probability that observed data will occur under the null hypothesis. In the following technical results figures, "ns" indicates that the result is not statistically significant; different levels of statistical significance are represented by specific notations:

[0190] "*": indicates a p-value less than 0.05, implying that at the 5% significance level, the observed results differ significantly from the null hypothesis.

[0191] "**": indicates a p-value less than 0.01, implying a highly significant difference between the observed results and the null hypothesis at the 1% significance level.

[0192] "***": indicates a p-value less than 0.001, implying that at the 0.1% significance level, the observed results are extremely significant compared to the null hypothesis.

[0193] The course of HPV16-related cervical disease typically includes three stages:

[0194] Low-grade cervical intraepithelial lesion (LSIL): LSIL typically indicates mild cellular changes. These changes are often caused by human papillomavirus (HPV) infection. Most LSILs resolve spontaneously within months or years, and only a small percentage progress to HSIL.

[0195] High-grade cervical squamous intraepithelial lesion (HSIL): HSIL indicates more severe cellular abnormalities. These abnormalities have a higher risk of progressing to cervical cancer, especially if not treated appropriately or monitored for a long period of time.

[0196] Cervical cancer: If HSIL is not properly diagnosed and treated, some cases may progress to cervical cancer. Cervical cancer refers to cancer cells invading deeper tissues of the cervix, no longer confined to the epithelial layer.

[0197] LSIL and HSIL are two different degrees of abnormal proliferation of cervical cells, and cervical cancer is the result of this abnormal proliferation being left uncontrolled and progressing further. HPV infection is the main cause of these three conditions, but not all HPV infections will lead to cancer.

[0198] like Figure 1 A. Proteomics analysis revealed that VAMP8 expression in HPV16-positive cervical tissue was significantly higher than in HPV16-negative tissue. During the progression of cervical disease, VAMP8 expression in cervical LSIL (low-grade squamous intraepithelial lesion) was higher than in HSIL (high-grade squamous intraepithelial lesion) and cervical cancer, but both were significantly higher than in normal HPV16-negative cervical tissue. Figure 1 B. qPCR results from different cervical-associated cell lines showed that, except for the C-33A cervical cancer cell line where VAMP8 was almost undetectable, VAMP8 expression in HPV16-positive cells (Ect1 / E6E7) was significantly higher than in HPV16-negative primary cervical cells; VAMP8 expression in cervical cancer cell lines (HeLa and SiHa) was lower than in HPV16-positive cells (representing the advanced stage of cervical lesions) but significantly higher than in HPV16-negative cervical cells. The trends in cell qPCR results were consistent with those in proteomics results.

[0199] Proteomics analysis and qPCR technology revealed that VAMP8 expression was significantly higher in HPV16-positive cervical tissue than in HPV16-negative tissue. Particularly in low-grade cervical squamous intraepithelial lesions (LSIL), VAMP8 expression was significantly increased, exceeding that in high-grade cervical squamous intraepithelial lesions (HSIL) and cervical cancer, and was also significantly higher than in normal HPV16-negative cervical tissue. VAMP8 could serve as a potential biomarker for diagnosing HPV16 infection and assessing the severity of cervical disease. Specifically, significantly high VAMP8 expression in cervical tissue indicates a positive HPV16 infection, while a decrease in VAMP8 expression observed in regular testing of the same HPV16-positive patient suggests malignant progression of cervical disease. Considering that VAMP8 may not be the sole biomarker for HPV16 infection and cervical disease progression, combining it with other known biomarkers may increase diagnostic accuracy.

[0200] like Figure 2 As shown, immunohistochemical staining of cervical sections after surgery further confirmed the relatively high expression of VAMP8 in HPV16-positive cervical tissue.

[0201] like Figures 3-5 As shown, bioinformatics analysis of the TCGA database indicates that the VAMP8 gene is highly expressed in cervical cancer and has diagnostic efficacy, and is closely related to poor prognosis of cervical cancer. Figure 3 AC demonstrated that, in the large TCGA database, the VAMP8 gene was relatively highly expressed in cervical cancer compared to normal tissues. Figure 3 The ROC curve of D indicates that VAMP8 can serve as a sensitive diagnostic marker for cervical cancer. Figure 3 Survival analysis of EG showed that patients with high VAMP8 expression had a worse prognosis. For example... Figure 4 As shown, clinical correlation analysis of cervical cancer in TCGA revealed that high expression of the VAMP8 gene was associated with T stage, N stage, M stage, clinical stage, treatment efficacy, pathological type, pathological grade, and menopausal status. Specifically, VAMP8 expression was negatively correlated with the progression of T stage, N stage, M stage, clinical stage, and treatment efficacy; and positively correlated with the progression of pathological grade and menopausal status. Among pathological types, VAMP8 expression was higher in cervical squamous cell carcinoma than in adenocarcinoma and adenosquamous carcinoma. Figure 5 As shown, immune infiltration analysis of VAMP8 in TCGA revealed that VAMP8 is significantly associated with the infiltration of various immune cells, such as DCs, aDCs, Tcms, and NK cells.

[0202] Bioinformatics analysis of the TCGA database revealed that high VAMP8 expression is closely associated with poor prognosis and the degree of immune infiltration in cervical cancer. Therefore, VAMP8 can serve as a prognostic marker for cervical cancer.

[0203] VAMP8 gene knockdown and overexpression cell lines were constructed in four cervical-associated cell lines (Ect1 / E6E7, HeLa, SiHa, and C-33A), with the following group names: shNC – knockdown control cell line; shVAMP8 – VAMP8 gene knockdown cell line; OE_NC – overexpression control cell line; OE_VAMP8 – VAMP8 gene overexpression cell line. Pairwise control cell function experiments were conducted to verify the role of VAMP8 gene regulation in cervical cells. Figures 6 to 10 In vitro experiments showed that relatively high VAMP8 expression in HPV16-positive Ect1 / E6E7 cells led to increased autophagy flux, decreased cell proliferation, migration, and invasion, and increased apoptosis rate and interphase ratio (G0 / G1 and S phase ratio). This indicates that in early HPV16-infected cervical cells, high VAMP8 expression promotes autophagy but inhibits the growth of HPV16-positive cervical cells. In cervical cancer cell lines HeLa, SiHa, and C-33A, relatively high VAMP8 expression also enhanced autophagy, but cell proliferation, migration, and invasion were promoted, apoptosis rate decreased, and the G2 / M phase ratio increased. This means that in cervical cancer cells, high VAMP8 expression promotes autophagy and tumor cell growth. Whether in HPV16-positive cells or cervical cancer cells, high VAMP8 expression is associated with enhanced autophagy. This suggests that VAMP8 plays a crucial role in regulating autophagy in cervical cells. VAMP8 has different functions in cervical cells in the early stages of HPV16 infection and in cervical cancer cells. In cervical cells in the early stages of HPV16 infection, VAMP8 plays a protective role, helping cells fight HPV infection and suppressing malignant behavior. However, in cervical cancer cells, VAMP8 can support tumor growth and spread. These results suggest that VAMP8 plays a complex role in the biological function of cervical cells.

[0204] Figure 11 The gross appearance, volume, and mass of tumors in the nude mouse tumorigenesis experiment further validated that high expression of VAMP8 promotes the growth and metastasis of cervical cancer. Figure 12 Observation of autophagosomes in different groups of cervical cancer tumor tissues under transmission electron microscopy demonstrated the autophagy-promoting effect of VAMP8 in vivo.

[0205] Cervical cell lines with stable VAMP8 gene overexpression or knockdown were constructed via lentiviral transfection. In HPV16-positive Ect1 / E6E7 cells, relatively high VAMP8 expression led to increased autophagy flux, decreased cell proliferation, migration, and invasion, increased apoptosis, and an increased proportion of cells in G0 / G1 and S phases. In cervical cancer cell lines (HeLa, SiHa, C-33A), relatively high VAMP8 expression, on the contrary, promoted cell proliferation, migration, and invasion, reduced apoptosis, and increased the proportion of cells in G2 / M phases. In vivo experiments further confirmed that high VAMP8 expression promotes the growth and metastasis of cervical cancer.

[0206] In summary, this invention provides a new perspective for a deeper understanding of the molecular mechanisms of HPV16 infection and cervical cancer progression, while also revealing the potential of VAMP8 as a diagnostic biomarker, prognostic biomarker, and therapeutic target. High expression of VAMP8 is associated with HPV16 infection and the development of cervical cancer; therefore, VAMP8 can serve as a potential diagnostic tool to help physicians identify patients who may have cervical cancer at an early stage. Furthermore, VAMP8 expression levels can also serve as a prognostic biomarker, helping physicians assess patient treatment response and prognosis. Finally, due to the important role of VAMP8 in cervical cancer development, treatment targeting VAMP8 will help inhibit cervical cancer progression, thus providing a novel cervical cancer treatment strategy. This opens up possibilities for developing new diagnostic and therapeutic methods, as well as improving the survival rate and quality of life for cervical cancer patients.

Claims

1. The use of VAMP8 in the preparation of products for assessing the prognosis of cervical cancer, including reagents, chips, or kits.

2. The use of VAMP8 inhibition in the preparation of products for the treatment of cervical cancer, said products including pharmaceuticals.

3. The use of VAMP8 inhibitors in the preparation of products for the treatment of cervical cancer, said products including pharmaceuticals.

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