Abstract Background Cervical cancer, prevalent in low- and middle-income countries, is primarily caused by high-risk HPV16. Vesicle-Associated Membrane Protein 8 (VAMP8), involved in vesicle trafficking and autophagy, may influence HPV16-related cervical cancer progression. Methods VAMP8 expression was evaluated in cervical tissue specimens from patients with HPV16-positive lesions (including low- and high-grade squamous intraepithelial lesions and cancer) and HPV-negative normal controls using proteomics, qPCR, and immunohistochemistry. A Cox proportional hazards model for prognosis was developed using immunohistochemical data from a cohort of cervical cancer patients. The clinical significance of VAMP8 was further assessed using RNA-seq and clinical data from The Cancer Genome Atlas-Cervical Cancer (TCGA-CESC) cohort. The effects of VAMP8 on autophagy and tumor progression were examined in HPV16 E6/E7-immortalized cervical epithelial cells (Ect1/E6E7) and cervical cancer cell lines (SiHa, HeLa, C-33A) in vitro, and in a SiHa xenograft model in vivo. Transcriptomic analysis of Ect1/E6E7 and SiHa cells identified VAMP8-regulated pathways. Chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays in SiHa cells were used to confirm the regulation of the HIF-1 pathway. Results VAMP8 was upregulated in HPV16-positive samples, particularly in low-grade squamous intraepithelial lesions (LSIL). Elevated VAMP8 correlated with poor survival outcomes and advanced tumor stages. VAMP8 enhanced autophagy and reduced proliferation and invasiveness in HPV16-positive cervical cells but increased in established cancer cell lines. In vivo, VAMP8 overexpression promoted tumor growth and autophagy. The HIF-1 pathway emerged as a key regulatory axis of VAMP8, enhancing hypoxic responses and angiogenesis. Conclusion Elevated VAMP8 in HPV16-associated cervical cancer promotes tumor progression by enhancing autophagy via the HIF-1 pathway, suggesting its potential as a diagnostic and prognostic biomarker. Supplementary Information The online version contains supplementary material available at 10.1186/s12916-025-04378-3. Keywords: Cervical cancer, HPV16, VAMP8, Autophagy, HIF-1 pathway, Biomarker Highlights 1. VAMP8 is upregulated in HPV16-positive tissues, especially in LSIL. 2. Elevated VAMP8 correlates with poor survival and advanced tumor stages. 3. VAMP8 enhances autophagy and promotes tumor progression in cervical cancer. 4. VAMP8 interacts with HIF-1 pathway, enhancing hypoxic response and angiogenesis. Supplementary Information The online version contains supplementary material available at 10.1186/s12916-025-04378-3. Background Cervical cancer remains a significant global health challenge, ranking as the fourth most common cancer among women, particularly in low- and middle-income countries [[36]1–[37]3]. The link between high-risk human papillomavirus (HPV) infections and cervical cancer is well-established, with HPV16 responsible for approximately 60% of cases [[38]4, [39]5]. HPV16 contributes to carcinogenesis by integrating its DNA into the host genome, leading to the expression of viral oncogenes E6 and E7, which inactivate tumor suppressor proteins p53 and Rb, promoting unchecked cellular proliferation and evasion of apoptosis [[40]6, [41]7]. The progression from HPV infection to cervical cancer involves a series of histopathological changes, from low-grade squamous intraepithelial lesions (LSIL) to high-grade squamous intraepithelial lesions (HSIL) and ultimately invasive carcinoma [[42]8, [43]9]. Understanding the molecular mechanisms behind these transitions is crucial for developing effective diagnostic, prognostic, and therapeutic strategies. The integration of molecular markers into clinical management has revolutionized cervical cancer diagnosis, prognosis, and treatment. These markers provide insights into the biological behavior of cervical lesions, facilitating early malignancy detection, disease progression prediction, and identification of patients at higher risk for poor outcomes [[44]10, [45]11]. Current clinical markers, such as p16INK4a, Ki-67, and HPV DNA, enhance the accuracy of cytological and histopathological evaluations [[46]12–[47]14]. p16INK4a is overexpressed in HPV-associated neoplasia and serves as a surrogate marker for oncogenic HPV activity, while Ki-67 indicates cellular proliferation [[48]15, [49]16]. HPV DNA testing, particularly for high-risk types like HPV16 and HPV18, is vital in screening programs due to its higher sensitivity compared to traditional cytology [[50]17–[51]19]. However, limitations exist; for instance, p16INK4a and Ki-67 do not comprehensively elucidate the molecular pathways driving carcinogenesis, and HPV DNA positivity does not always correlate with disease severity, as many infections are transient [[52]20–[53]22]. This highlights the pressing need for novel biomarkers that improve diagnostic precision and offer prognostic value and therapeutic potential, ideally reflecting dynamic changes within the tumor microenvironment. Vesicle-Associated Membrane Protein 8 (VAMP8), a member of the SNARE family, plays a crucial role in intracellular vesicle trafficking, including exocytosis, endocytosis, and autophagy [[54]23–[55]25]. VAMP8 is primarily involved in vesicle fusion, essential for maintaining cellular homeostasis and responding to physiological stimuli [[56]26, [57]27]. Recent studies suggest VAMP8’s significance in cancer biology, with roles in tumor cell proliferation, migration, invasion, and stress response [[58]28]. For example, VAMP8 enhances enzyme and cytokine secretion in pancreatic cancer, promoting tumor growth and metastasis, and facilitates autophagy in breast cancer, aiding survival under nutrient deprivation [[59]29]. Despite these findings, the role of VAMP8 in cervical cancer, particularly in HPV16-associated carcinogenesis, remains poorly understood. Given VAMP8’s critical cellular functions and emerging links to oncogenic processes, it is hypothesized that it may significantly influence cervical cancer pathophysiology. Given VAMP8’s emerging roles in vesicle trafficking and autophagy in other cancers, we hypothesized that VAMP8 expression might be altered during HPV16-mediated transformation and could play a critical role in cervical carcinogenesis. Therefore, this study was designed to systematically investigate the expression patterns, clinical significance, and functional role of VAMP8 in the progression of HPV16-associated cervical cancer. Although HPV16’s role in cervical cancer etiology is established, the specific molecular mechanisms mediating the progression from infection to invasive carcinoma are not fully understood. This study aims to explore VAMP8’s functional significance in cervical cancer, particularly as a diagnostic and prognostic biomarker. Additionally, investigating VAMP8’s role may uncover novel therapeutic targets, especially if it is implicated in critical oncogenic pathways such as autophagy, proliferation, and immune modulation. We aim to elucidate VAMP8’s expression patterns, functional mechanisms, and potential as a diagnostic and prognostic biomarker. VAMP8 expression levels will be quantified in HPV16-positive and -negative cervical tissues across various lesion stages using proteomic analysis, qPCR, and immunohistochemistry. The correlation between VAMP8 expression and clinical parameters, such as tumor stage, grade, and patient outcomes, will be assessed. Functional impacts of VAMP8 on autophagy, proliferation, migration, invasion, and apoptosis will be explored through in vitro experiments and validated in vivo using nude mouse models. Transcriptomic sequencing and bioinformatics will identify VAMP8-regulated genes and pathways, focusing on its interaction with the HIF-1 signaling pathway and its role in the tumor immune microenvironment. By integrating experimental data with clinical findings, this research aims to provide a holistic view of VAMP8’s involvement in cervical cancer, potentially identifying it as a therapeutic target. Methods Study population and clinical specimens This study was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University, ensuring ethical compliance and informed consent from all participants. Isolated samples were obtained from patients who underwent surgery for cervical lesions or benign uterine diseases at our hospital from 2014 to 2018. Specimens were categorized into five groups, each comprising 30 cases: (1) control group with normal cervical tissue (HPV-negative), (2) HPV16-positive cervical tissue, (3) HPV16-positive low-grade squamous intraepithelial lesions (LSIL), (4) HPV16-positive high-grade squamous intraepithelial lesions (HSIL), and (5) HPV16-positive cervical cancer tissue (CA). Immunohistochemical samples were collected from paraffin-embedded sections, which included 30 cases each of HPV-negative normal tissue, HPV16-positive tissue, HPV16-positive LSIL, HPV16-positive HSIL, and 68 cases of cervical cancer tissues (30 HPV16-positive, 22 HPV18-positive, and 16 HPV-negative samples). Inclusion criteria were designed to ensure a representative sample, while exclusion criteria ruled out patients who had received preoperative chemotherapy or radiotherapy to avoid confounding effects. All diagnoses were confirmed via histopathological examination to ensure the reliability of clinical specimens. The paraffin-embedded sections used for immunohistochemical analysis were derived from the same cohort of surgical patients from whom fresh or frozen tissue samples were procured for proteomic and qPCR analyses, ensuring consistency in the patient population across different analytical platforms. Cell lines and culture conditions The study utilized the human cervical epithelial cell line HCK1T, and Ect1/E6E7 transformed with HPV16 E6/E7, obtained from the American Type Culture Collection (ATCC). Human cervical cancer cell lines SiHa, HeLa, and C-33A were acquired from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere containing 5% CO[2] at 37 °C. For experiments, cells were seeded in 24-well plates at a density of 1 × 10^5 cells per well and allowed to adhere for 18–24 h. Transfections were performed using lentiviral vectors encoding VAMP8 shRNA or overexpression plasmids, along with controls, including non-targeting shRNA and GFP-expressing vectors. The transfection medium was supplemented with polybrene (6 µg/ml) to enhance transduction efficiency. Following a 4-h incubation at 37 °C, the medium was replaced with fresh DMEM containing 10% FBS. After 72 h, stable transfected cell lines were selected using DMEM with 2 µg/ml puromycin until resistant colonies were established, which were then maintained in DMEM with puromycin for subsequent experiments. Proteomic analysis For proteomic analysis, proteins were extracted from clinical tissue samples using a lysis buffer containing 8 M urea, 1% protease inhibitor cocktail, and 2 mM EDTA. Lysates were sonicated on ice and centrifuged at 14,000 × g for 15 min at 4 °C to remove debris. The supernatants were collected, and protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, USA). Absorbance was measured at 562 nm using a Synergy HTX Multi-Mode Reader (BioTek Instruments, Winooski, VT, USA). Equal amounts of protein were reduced with 5 mM dithiothreitol (DTT) for 30 min at 37 °C, followed by alkylation with 11 mM iodoacetamide (IAA) for 15 min at room temperature in the dark. Samples were diluted with 100 mM ammonium bicarbonate to reduce the urea concentration and digested with sequencing-grade trypsin (Promega, USA) at a 1:50 enzyme-to-substrate ratio overnight at 37 °C. Post-digestion, peptide mixtures were desalted using C18 Sep-Pak columns (Waters, USA) and dried by vacuum centrifugation. Dried peptides were resuspended in 0.1% formic acid and analyzed via liquid chromatography-tandem mass spectrometry (LC–MS/MS) on an EASY-nLC 1000 coupled to a Q Exactive HF mass spectrometer (Thermo Scientific, USA). Peptides were separated on a reversed-phase analytical column (75 µm × 25 cm, Thermo Scientific) using a linear gradient of 5–35% acetonitrile in 0.1% formic acid over 120 min at a flow rate of 300 nl/min. The mass spectrometer operated in data-dependent acquisition mode, performing full scans (350–1600 m/z) at a resolution of 70,000, followed by higher-energy collisional dissociation (HCD) fragmentation of the top 20 most intense ions. Raw data were processed using MaxQuant software (version 1.6.0.1) and searched against the UniProt human protein database. Search parameters included fixed carbamidomethylation on cysteine residues, variable methionine oxidation, and a false discovery rate (FDR) of less than 1% at both peptide and protein levels. Quantification utilized the label-free quantification (LFQ) algorithm integrated in MaxQuant. Differentially expressed proteins were identified based on a fold change of ≥ 2 and statistical significance (p < 0.05) using Perseus software (version 1.6.0.7). Identified proteins underwent pathway enrichment analysis using the PANTHER classification system (version 12) and the ToppFun application within the ToppGene suite. Network analysis was conducted using the R package “igraph,” calculating key topological parameters (degree, betweenness, closeness, and clustering coefficient) to evaluate protein importance within identified modules. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from clinical tissue samples and cultured cell lines using TRIzol reagent (Ambion, USA) as per the manufacturer’s instructions. RNA quality and concentration were assessed with a NanoDrop spectrophotometer (Thermo Scientific, USA), ensuring an A260/A280 ratio between 1.8 and 2.0. Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the PrimeScript™ RT Reagent Kit (TaKaRa, Japan). qRT-PCR was performed using the SYBR® Premix Ex Taq™ II PCR Kit (Takara, Japan) on an Applied Biosystems 7500 Real-Time PCR System (Thermo Scientific, USA). Reactions were set up in a 20 µL volume containing 10 µL SYBR Green master mix, 0.4 µL each of forward and reverse primers (10 µM), 2 µL cDNA, and 7.2 µL nuclease-free water. The thermal cycling conditions included initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 34 s. Each reaction was performed in triplicate for accuracy. Relative expression levels of target genes were normalized to the housekeeping gene β-actin using the comparative Ct method (ΔΔCt). The fold change in expression was calculated using 2 − ΔΔCt. Melt curve analysis was conducted to validate amplification specificity. The specific sequences of the primers used for qRT-PCR, including both forward and reverse primers for each target gene, are provided in Table S1. Western blot Protein extraction was performed using RIPA lysis buffer (Thermo Scientific, USA) supplemented with a protease inhibitor cocktail (Roche, Switzerland) and phosphatase inhibitors (Sigma-Aldrich, USA). The lysates were incubated on ice for 30 min and then centrifuged at 14,000 × g for 15 min at 4 °C to remove cellular debris. Supernatants were collected, and protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, USA). Absorbance was measured at 562 nm using the same microplate reader mentioned above. Equal amounts of protein (20 µg per sample) were separated on 4–20% gradient SDS-PAGE gels (Bio-Rad, USA) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked in 5% non-fat dried milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature to prevent non-specific binding. Following blocking, the membranes were incubated overnight at 4 °C with primary antibodies diluted 1:1000 in blocking buffer. After three washes in TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (diluted 1:5000 in blocking buffer) for 1 h at room temperature. Following three additional washes in TBST, protein bands were visualized using an enhanced chemiluminescence (ECL) detection system (Bio-Rad, USA) and imaged with the FluorChem M imaging system (ProteinSimple, USA). The intensity of the protein bands was quantified using ImageJ software (National Institutes of Health, USA), with β-actin used as the internal control to normalize the expression levels of target proteins. Relative protein expression levels were calculated by dividing the intensity of the target protein band by the intensity of the β-actin band. The primary antibodies used in this study were purchased from Abcam and included ab76021 (anti-VAMP8/EDB), ab1 (anti-HIF-1 alpha), ab46154 (anti-VEGFA), ab22797 (anti-ENO1), and ab8227 (anti-β-actin). The original, uncropped images for all blots are provided. Immunohistochemistry Tissue Section (4 µm thick) were dewaxed in xylene and rehydrated through graded ethanol. Antigen retrieval was performed by heating sections in citrate buffer (pH 6.0) in a microwave for 20 min. After cooling, sections were washed in phosphate-buffered saline (PBS) and incubated in 3% hydrogen peroxide to quench endogenous peroxidase activity. Non-specific binding was blocked with 5% bovine serum albumin (BSA) for 30 min at room temperature. Sections were incubated overnight at 4 °C with primary antibodies diluted 1:50 in PBS. After washing with PBS, sections were incubated with a biotinylated secondary antibody (1:200) for 30 min, followed by streptavidin–horseradish peroxidase (HRP) conjugate for 30 min. Immunoreactivity was visualized using 3,3'-diaminobenzidine (DAB) as a chromogen, and sections were counterstained with hematoxylin. Slides were dehydrated through graded alcohols, cleared in xylene, and mounted with coverslips. Immunostaining was evaluated by two independent pathologists, blinded to clinical data, using the histochemical score (H-score) system, calculated by multiplying the percentage of positive cells (0–100%) by staining intensity (0, negative; 1, weak; 2, moderate; 3, strong), resulting in scores from 0 to 300. Prognostic prediction model construction A prognostic prediction model was constructed using comprehensive follow-up data from cervical cancer surgery patients, integrating immunohistochemical staining data from postoperative specimens. Patients were categorized into high and low VAMP8 expression groups based on immunohistochemical scores. The primary endpoints were overall survival (OS) and disease-free survival (DFS). Statistical analysis employed the Cox proportional hazards regression model to assess the impact of VAMP8 expression on patient outcomes. The model’s discriminative ability was evaluated using the concordance index (C-index), and Kaplan–Meier survival curves were generated to visualize differences in OS and DFS between expression groups, with the log-rank test used for significance. Hazard ratios (HR) with 95% confidence intervals (CI) quantified risks associated with high VAMP8 expression, and calibration plots compared predicted and observed survival probabilities. The development and validation of the prognostic prediction model were reported in accordance with the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) guidelines. Bioinformatics analysis of the TCGA-CESC database Bioinformatics analysis was performed on the TCGA-CESC (The Cancer Genome Atlas-Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma) database to examine VAMP8 expression and its clinical correlations in cervical cancer. The dataset included RNA sequencing data, clinical information, and pathological details of patients [[60]30]. Differential expression analysis compared VAMP8 levels between cancer and normal cervical tissues, with statistical significance determined using the DESeq2 package in R, applying a fold change threshold of ≥ 2 and adjusted p-value < 0.05. Diagnostic efficacy was evaluated via receiver operating characteristic (ROC) curve analysis, calculating the area under the curve (AUC) for diagnostic accuracy. Correlations of VAMP8 expression with clinical parameters were assessed using Spearman’s rank correlation coefficient. Immune cell infiltration analysis To analyze immune cell infiltration in cervical cancer tissues, data from the TCGA-CESC database were utilized to explore the relationship between VAMP8 expression and the tumor immune microenvironment. Bioinformatics tools such as CIBERSORT and TIMER were employed to estimate the relative proportions of various immune cell types within the tumor samples. Immune cell infiltration profiles were compared between high and low VAMP8 expression groups to identify significant differences [[61]31]. Statistical analyses were conducted using the Mann–Whitney U test to assess the significance of differential immune cell infiltration. Correlation analyses were performed using Spearman’s rank correlation coefficient to evaluate associations between VAMP8 expression and specific immune cell subsets, including dendritic cells (DCs), activated dendritic cells (aDCs), central memory T cells (Tcm), and natural killer (NK) cells. Scatter plots were generated to visualize these correlations. Additionally, single-sample Gene Set Enrichment Analysis (ssGSEA) was used to validate the association of VAMP8 with immune cell activity and infiltration [[62]32]. In vitro functional assays In vitro functional assays were performed to investigate the role of VAMP8 in cellular processes such as autophagy, proliferation, migration, invasion, and apoptosis in cervical cell lines. Human cervical epithelial cell line Ect1/E6E7 and cervical cancer cell lines SiHa, HeLa, and C-33A were utilized. Autophagy was assessed using transmission electron microscopy (TEM) to visualize autophagic vesicles. Treatment with autophagy inducers or inhibitors served as controls. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded in 96-well plates at a density of 1 × 10^4 cells per well and incubated for 24, 48, and 72 h, with absorbance at 450 nm recorded using an iMark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA). Migration and invasion assays were conducted using Transwell chambers. For migration assays, cells were seeded in the upper chamber with serum-free medium, while the lower chamber contained medium with 10% FBS. After 24 h, cells that migrated to the lower surface were stained with crystal violet and counted. In invasion assays, the upper chamber was coated with Matrigel. Cells were harvested, stained according to the manufacturer’s instructions (BD Biosciences, USA), and analyzed using a BD FACSCanto™ II flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Cell cycle analysis was performed using flow cytometry, with cells fixed in 70% ethanol, stained with propidium iodide (PI) and RNase, and analyzed for cell cycle distribution. Apoptosis was analyzed using flow cytometry with Annexin V-APC/PI staining. Colony formation assays were performed by seeding 500 cells per well in 6-well plates, incubating for 14 days, staining the formed colonies with crystal violet, and counting them under a microscope. Angiogenesis was assessed using tube formation assays, where human umbilical vein endothelial cells (HUVECs) were seeded on Matrigel-coated plates and treated with conditioned media from cervical cancer cells, with capillary-like structures quantified after 6 h of incubation. In vivo tumor formation assays All animal procedures were approved by the Animal Care and Use Committee of the Obstetrics and Gynecology Hospital of Fudan University. Female BALB/c (nu/nu) nude mice (4–6 weeks of age, weighing 18–22 g) were procured from Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China and housed under specific-pathogen-free (SPF) conditions. The housing environment was maintained at a constant temperature (22 ± 2 °C) and humidity (55 ± 10%) with a 12-h light/dark cycle. Animals had ad libitum access to standard chow and sterile water. To evaluate the pro-tumorigenic and pro-autophagic effects of VAMP8 in vivo, a xenograft tumor model was established. A total of 16 mice were used, allocated into four experimental groups (n = 4 mice per group): (1) shNC group (injected with SiHa cells transfected with a negative control shRNA vector); (2) shVAMP8 group (injected with SiHa cells with stable VAMP8 knockdown); (3) OE-NC group (injected with SiHa cells transfected with a negative control overexpression vector); and (4) OE-VAMP8 group (injected with SiHa cells with stable VAMP8 overexpression). Animals were assigned to experimental groups upon arrival, without the use of formal randomization. SiHa cells (5 × 10⁶) suspended in 100 µL of sterile phosphate-buffered saline (PBS) were injected subcutaneously into the right subaxillary region of each mouse. Tumor formation was monitored every 7 days by measuring the perpendicular length (L) and width (W) of the tumors with Vernier calipers. Tumor volume was calculated using the formula: Volume = (L × W^2)/2. The study was not conducted in a blinded manner. The inclusion criterion was healthy female BALB/c (nu/nu) mice within the specified age and weight range. No other pre-defined exclusion criteria were established. During the experiment, no animals died or were removed from the study, and all 16 animals were included in the final analysis. Animal welfare was monitored daily. Humane endpoints were established based on signs of distress (e.g., > 20% weight loss, ulceration of tumor, or impaired mobility), which would have resulted in immediate euthanasia. The primary experimental endpoint was 28 days post-implantation, at which point all mice were humanely euthanized by CO₂ asphyxiation followed by cervical dislocation. Tumors were then excised, photographed, and weighed. Tumor tissues were subsequently fixed in 10% neutral-buffered formalin and embedded in paraffin for immunohistochemical (IHC) analysis, or were processed for transmission electron microscopy (TEM) to quantify autophagic vesicles. RNA sequencing and transcriptomic analysis Total RNA was extracted from Ect1/E6E7 and SiHa cells overexpressing VAMP8, as well as from their control counterparts, using TRIzol reagent (Ambion, USA) per the manufacturer’s protocol. RNA quality and integrity were assessed with an Agilent 2100 Bioanalyzer (Agilent Technologies, USA), ensuring RNA integrity number (RIN) values greater than 7. Libraries were prepared using the Illumina TruSeq RNA Sample Preparation Kit (Illumina, USA) and sequenced on an Illumina HiSeq 2500 platform, generating 150 bp paired-end reads. Raw sequencing data underwent quality control with FastQC and were trimmed using Trimmomatic to remove low-quality bases and adapter sequences. The cleaned reads were aligned to the human reference genome (GRCh38) using HISAT2 (version 2.1.0), and gene-level expression counts were generated using featureCounts from the Subread package (version 1.6.0). Differential expression analysis was conducted using the DESeq2 package in R, with a threshold of fold change ≥ 2 and an adjusted p-value < 0.05 to identify significantly differentially expressed genes (DEGs). Additionally, transcriptomic data were integrated with the TCGA-CESC dataset to validate findings and identify clinically relevant DEGs. Single-gene differential expression and correlation analyses were performed on the TCGA-CESC dataset, focusing on VAMP8 expression. The intersection of DEGs from RNA-seq analysis and significant genes from TCGA-CESC single-gene analyses yielded a refined set of VAMP8-regulated genes. Gene Ontology (GO) and KEGG pathway enrichment analysis VAMP8-regulated genes were subjected to functional annotation using the DAVID Bioinformatics Resources (version 6.7). The DEGs were input into the DAVID platform to categorize them into Gene Ontology (GO) terms, including biological processes (BP), molecular functions (MF), and cellular components (CC). Enrichment analysis determined the overrepresentation of specific GO terms among the DEGs, with statistical significance set at an adjusted p-value < 0.05. For KEGG pathway analysis, DEGs were mapped to known biological pathways using the KEGG database. Enrichment of KEGG pathways was determined by comparing the distribution of DEGs in each pathway to the background distribution of all genes in the genome, utilizing Fisher’s exact test to calculate p-values. Pathways with an adjusted p-value < 0.05 were considered significantly enriched. The enriched GO terms and KEGG pathways were visualized using bubble plots and bar charts generated by the ggplot2 package in R. Chromatin immunoprecipitation (ChIP) assays Cells were cultured to approximately 80% confluence in 10-cm dishes and cross-linked with 1% formaldehyde for 10 min at room temperature to preserve protein-DNA interactions. The cross-linking reaction was halted by adding glycine to a final concentration of 0.125 M. Cells were washed with cold PBS, harvested by scraping, and lysed in SDS lysis buffer (50 mM Tris–HCl pH 8.1, 10 mM EDTA, 1% SDS) supplemented with protease inhibitors (Roche, Switzerland). The chromatin was sonicated on ice to shear DNA into fragments of 200–500 bp. Lysates were centrifuged at 14,000 × g for 10 min at 4 °C to remove debris, and the supernatants containing sheared chromatin were collected. ChIP was performed using the ChIP-IT High Sensitivity Kit (Active Motif, USA) following the manufacturer’s protocol. The chromatin was pre-cleared with protein G agarose beads for 1 h at 4 °C with gentle agitation. Pre-cleared chromatin was incubated overnight at 4 °C with 5 µg of primary antibodies specific or a control IgG antibody. The antibody-chromatin complexes were captured by incubation with protein G agarose beads for 2 h at 4 °C. The beads were washed sequentially with low-salt wash buffer, high-salt wash buffer, LiCl wash buffer, and TE buffer to remove non-specifically bound proteins and DNA. Immune complexes were eluted from the beads with elution buffer, and cross-links were reversed by incubation at 65 °C for 4 h in the presence of 200 mM NaCl. Proteins were digested with proteinase K, and the DNA was purified using spin columns. Quantitative real-time PCR (qRT-PCR) was performed to quantify the immunoprecipitated DNA fragments using specific primers targeting the promoter regions of genes of interest, including HIF1A, VEGFA, and ENO1. PCR reactions were carried out in a 20 µL volume containing SYBR Green master mix (Takara, Japan), 0.4 µL of each primer (10 µM), and 2 µL of ChIP DNA. Thermal cycling conditions included an initial denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 34 s. Each reaction was performed in triplicate. The enrichment of specific DNA sequences in the immunoprecipitated samples was calculated relative to input chromatin and normalized to control IgG using the comparative Ct method (ΔΔCt). Luciferase reporter assays Cells were seeded in 24-well plates at a density of 1 × 10^5 cells per well and allowed to adhere for 18–24 h. Cells were then co-transfected with a luciferase reporter plasmid containing the promoter region of interest linked to the firefly luciferase gene, alongside a Renilla luciferase plasmid as an internal control for transfection efficiency. Transfections were performed using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. Cells were also co-transfected with either VAMP8 overexpression plasmids or VAMP8 shRNA plasmids, along with their respective control vectors. After 24 h, cells were lysed using Passive Lysis Buffer (Promega, USA), and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA). Luminescence was detected using a microplate reader (BioTek, USA) that measured both firefly and Renilla luciferase activities. Firefly luciferase activity was normalized to Renilla luciferase activity to account for variations in transfection efficiency and cell number, and the normalized luciferase activity was compared between experimental groups to assess the impact of VAMP8 on transcriptional regulation. Statistical analysis Data were collected from at least three independent experiments, and results were presented as mean ± standard deviation (SD) to ensure robustness and reproducibility. All statistical analyses were performed using SPSS 25.0 software. For comparisons between two groups, independent samples t-tests were utilized, while one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test was employed for comparisons involving more than two groups. A p-value of less than 0.05 was considered statistically significant for all analyses. In quantitative analysis figures, statistical significance was indicated by asterisks: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. Sample sizes for clinical specimens and animal experiments were determined based on previous similar studies in the field of cervical cancer research and VAMP8 biology, aiming to provide sufficient statistical power to detect meaningful biological effects while adhering to ethical considerations for animal use. Post hoc power analyses confirmed adequate power for the primary endpoints. Results VAMP8 expression is significantly elevated in HPV16-positive tissues, with highest levels in low-grade lesions Proteomic analysis and quantitative PCR (qPCR) assays revealed significant upregulation of VAMP8 expression in HPV16-positive cervical tissues compared to HPV16-negative tissues (Fig. [63]1A, B). This finding was consistently supported by elevated VAMP8 mRNA levels detected in HPV16-positive cervical cell lines (Fig. [64]1C). Immunohistochemical staining of postoperative cervical specimens further confirmed these results, showing pronounced VAMP8 overexpression specifically in HPV16-positive tissues (Fig. [65]1D). Fig. 1. [66]Fig. 1 [67]Open in a new tab VAMP8 Expression analysis in cervical tissues and cells. A, B Proteomic analysis (A) and qPCR (B) of VAMP8 in HPV16-positive and HPV16-negative tissues. C qPCR measurement of VAMP8 mRNA levels in cervical cell lines. D Immunohistochemical staining of VAMP8 in postoperative specimens A differential expression pattern of VAMP8 was noted across various stages of cervical lesions. Notably, VAMP8 expression was markedly higher in tissues with low-grade squamous intraepithelial lesions (LSIL) compared to those with high-grade squamous intraepithelial lesions (HSIL) and cervical cancer. However, all pathological stages—including LSIL, HSIL, and cervical cancer—exhibited significantly elevated VAMP8 levels relative to normal HPV16-negative cervical tissues. These results suggest a dynamic role for VAMP8 in the early stages of cervical lesion development, potentially influencing cellular mechanisms that distinguish low-grade from high-grade lesions. The consistent overexpression of VAMP8 in HPV16-positive tissues indicates its involvement in the pathogenesis and progression of HPV16-associated cervical lesions. The observed differential expression across lesion stages underscores VAMP8’s potential as a biomarker for diagnosing and monitoring cervical pathology progression. High VAMP8 expression correlates with poor prognosis in cervical cancer patients A prognostic prediction model was constructed by integrating comprehensive follow-up data from cervical cancer surgery patients with immunohistochemical staining data from postoperative specimens. This analysis aimed to elucidate the impact of VAMP8 expression levels on patient outcomes. The constructed model, utilizing a Cox proportional hazards approach, revealed a significant association between high VAMP8 expression and adverse prognostic indicators (Fig. [68]2A,B). Specifically, patients with elevated VAMP8 levels demonstrated markedly reduced overall survival (OS) and disease-free survival (DFS) compared to those with lower expression levels. The predictive accuracy of the model was robust, evidenced by a concordance index (C-index) of 0.81, indicating strong discriminative ability between patients with different risk profiles. Kaplan–Meier survival analysis further corroborated these findings, illustrating that high VAMP8 expression is significantly correlated with shorter OS and DFS (p < 0.05). Hazard ratios (HR) for high VAMP8 expression were calculated to be 1.87 (95% CI 1.10–3.18) for OS and 1.85 (95% CI 1.09–3.14) for DFS, underscoring the elevated risk associated with increased VAMP8 levels. Fig. 2. [69]Fig. 2 [70]Open in a new tab Prognostic analysis of low/high VAMP8 expression based on immunohistochemical staining of postoperative cervical cancer specimens. A, B Kaplan–Meier curves for overall survival (OS) (A) and disease-free survival (DFS) (B) in patients with low/high VAMP8 expression The findings from this prognostic model underscore VAMP8’s potential as both a biomarker for identifying high-risk patients and a target for therapeutic intervention. High VAMP8 expression is associated with significantly worse prognostic outcomes, including reduced survival times and increased recurrence risk, suggesting that patients with elevated VAMP8 levels may benefit from more aggressive and targeted treatment strategies. Consequently, further research is warranted to explore the mechanistic pathways involving VAMP8 and validate its clinical utility in improving patient outcomes. VAMP8 serves as a highly accurate diagnostic biomarker with elevated expression in cervical cancer Analysis of the TCGA-CESC dataset revealed a significant elevation of VAMP8 expression in cervical cancer tissues compared to normal tissues. The expression of VAMP8 in tumor tissues was markedly higher than in normal tissues (p < 0.001) (Fig. [71]3A). The receiver operating characteristic (ROC) curve analysis further demonstrated the diagnostic capability of VAMP8, with an area under the curve (AUC) of 0.956, indicating a high level of accuracy in distinguishing cervical cancer from normal tissues (Fig. [72]3B). Fig. 3. [73]Fig. 3 [74]Open in a new tab Analysis of VAMP8 expression in cervical cancer tissues and its clinical correlation based on the TCGA-CESC database. A Comparison of VAMP8 expression between normal and tumor tissues. B Receiver operating characteristic (ROC) curve illustrating the diagnostic performance of VAMP8. C VAMP8 expression across different histological subtypes (adenocarcinoma vs. squamous cell carcinoma). D-F VAMP8 expression in relation to pathological T stage (D), pathological M stage (E), and clinical stage (F) Further clinical correlation analysis highlighted significant associations between VAMP8 expression and key clinical parameters. Patients with squamous cell carcinoma exhibited significantly higher VAMP8 expression levels compared to those with adenocarcinoma (p < 0.01) (Fig. [75]3C). Patients in the T1 stage had significantly higher VAMP8 expression than those in the T2 and T3/T4 stages (p < 0.05 and p < 0.001, respectively), indicating a negative correlation between VAMP8 expression and tumor progression in terms of T stage (Fig. [76]3D). VAMP8 expression was significantly higher in patients without distant metastasis (M0) compared to those with distant metastasis (M1) (p < 0.05), while patients with unknown metastatic status (MX) displayed intermediate expression levels (Fig. [77]3E). Patients in clinical stages I and II exhibited higher VAMP8 expression compared to those in stages III and IV (p < 0.05), further supporting a negative association between VAMP8 expression and advanced clinical stage (Fig. [78]3F). In summary, these findings suggest that VAMP8 not only holds promise as a diagnostic biomarker but also correlates with clinical and pathological features of cervical cancer. Higher VAMP8 expression is more pronounced in early-stage disease, indicating its potential utility in early detection and in stratifying patients for personalized therapeutic approaches. VAMP8 expression positively correlates with dendritic cell infiltration and negatively with NK cell infiltration The analysis of the TCGA-CESC database demonstrated a significant relationship between VAMP8 expression and immune cell infiltration in cervical cancer tissues. This highlights the potential role of VAMP8 in modulating the tumor immune microenvironment. Specifically, VAMP8 expression was positively correlated with the infiltration of various immune cells, including dendritic cells (DC), activated dendritic cells (aDC), central memory T cells (Tcm), and natural killer (NK) cells. Correlation analysis revealed that dendritic cells (DC) exhibited the highest positive correlation with VAMP8 expression (R = 0.272, p < 0.001), followed by activated dendritic cells (aDC) (R = 0.260, p < 0.001), cytotoxic cells (R = 0.225, p < 0.001), and NK CD56dim cells (R = 0.212, p < 0.001) (Fig. [79]4A). This suggests that VAMP8 may enhance the recruitment of these cells into the tumor microenvironment, potentially facilitating antigen presentation and immune activation. Conversely, certain immune cell types, such as NK cells and Tcm cells, displayed negative correlations, with NK cells showing the most pronounced negative correlation (R = − 0.166, p = 0.004) (Fig. [80]4A). Fig. 4. [81]Fig. 4 [82]Open in a new tab Correlation analysis between VAMP8 expression and immune cell infiltration in cervical cancer. A Correlation between VAMP8 expression and various immune cell types. The size and color of the circles represent the strength of correlation and significance, respectively. B Boxplots comparing immune cell enrichment scores for DC, aDC, Tcm, and NK cells between high and low VAMP8 expression groups. C, D Scatter plots illustrating the relationship between VAMP8 expression and the enrichment of DCs and NK cells Enrichment score analysis further supports these findings, showing that tumors with high VAMP8 expression exhibited significantly higher enrichment of DCs, aDCs, Tcm, and NK cells compared to those with low VAMP8 expression (p-values ranging from p < 0.05 to p < 0.001) (Fig. [83]4B). These results indicate that VAMP8 expression levels play a crucial role in shaping the immune landscape within cervical cancer. Scatter plots demonstrated a positive correlation between VAMP8 expression and the enrichment of dendritic cells (Spearman R = 0.272, p < 0.001) (Fig. [84]4C), while a negative correlation was seen with NK cell enrichment (Spearman R = − 0.166, p = 0.004) (Fig. [85]4D). These results underline VAMP8’s selective influence on immune cell populations, potentially driving differential immune responses in the tumor microenvironment. These findings suggest that VAMP8 contributes to immune cell recruitment, particularly enhancing the infiltration of antigen-presenting cells like dendritic cells and activated dendritic cells, which are essential for initiating and sustaining anti-tumor immune responses. The differential correlation with various immune cells, including central memory T cells and NK cells, implies that VAMP8’s role in modulating the immune response is multifaceted, potentially affecting both innate and adaptive immunity. VAMP8 enhances autophagy and exhibits stage-dependent effects on cervical cell proliferation and metastasis The qPCR results confirmed the successful establishment of VAMP8 knockdown and overexpression cell lines across four cervical cell lines, including Ect1/E6E7, SiHa, HeLa, and C-33A (Fig. [86]5A). Transmission electron microscopy analysis revealed a significant increase in the number of autophagic vesicles in the VAMP8 overexpression groups, while a marked reduction was observed in the knockdown groups across all four cell lines (Fig. [87]5B). Notably, Ect1/E6E7 cells exhibited a particularly pronounced increase in autophagic vesicles following VAMP8 overexpression (Fig. [88]5B). These findings indicate a close correlation between VAMP8 expression levels and autophagic activity. Fig. 5. [89]Fig. 5 [90]Open in a new tab VAMP8 modulation and autophagic vesicles in cervical cell lines. A qPCR analysis confirming VAMP8 knockdown and overexpression in Ect1/E6E7, SiHa, HeLa, and C-33A cell lines. shNC: negative control for knockdown; shVAMP8: VAMP8 knockdown; OE-NC: negative control for overexpression; OE-VAMP8: VAMP8 overexpression. B Transmission electron microscopy images of autophagic vesicles in VAMP8 knockdown and overexpression cell lines across the four cervical cell lines. Scale bar = 2 μm The CCK-8 proliferation assay demonstrates that VAMP8 knockdown significantly promotes cell proliferation in Ect1/E6E7 cells, while its overexpression inhibits proliferation, indicating a suppressive role in early HPV16-associated cervical transformation (Fig. [91]6A). In contrast, overexpression of VAMP8 promotes proliferation in cervical cancer cell lines, including SiHa, HeLa, and C-33A (Fig. [92]6B–D). These findings suggest that VAMP8 exerts distinct effects on cell proliferation depending on the cellular context and stage of transformation. Fig. 6. [93]Fig. 6 [94]Open in a new tab Effects of VAMP8 knockdown and overexpression on cell proliferation measured by CCK-8 assay. A–D Cell proliferation was assessed at 0, 24, 48, and 72 h in Ect1/E6E7, SiHa, HeLa, and C-33A cell lines, respectively Transwell migration and invasion assays revealed that in Ect1/E6E7 cells, VAMP8 knockdown significantly increased migration and invasion, while VAMP8 overexpression suppressed these processes (Fig. [95]7A). In contrast, in SiHa, HeLa, and C-33A cells, VAMP8 overexpression markedly promoted migration and invasion, whereas knockdown reduced these capacities (Fig. [96]7B–D). These findings suggest that VAMP8 acts as a suppressor of migration and invasion during early HPV-mediated cervical transformation in Ect1/E6E7 cells, while in more advanced cervical cancer cells like SiHa, VAMP8 shifts its function, potentially due to the altered tumor microenvironment or signaling pathways, to promote metastatic behavior, indicating a stage-dependent regulatory role of VAMP8 in cervical carcinogenesis. Fig. 7. [97]Fig. 7 [98]Open in a new tab Effects of VAMP8 knockdown and overexpression on cell migration and invasion in cervical cell lines. A-D Transwell migration and invasion assays were performed in Ect1/E6E7, SiHa, HeLa, and C-33A cell lines, with relative cell numbers quantified Flow cytometry analysis revealed that VAMP8 overexpression in Ect1/E6E7 cells increased the proportion of cells in the G1 phase, accompanied by elevated apoptosis rates, as shown by Annexin V-APC/PI staining (Fig. [99]8A,B). In contrast, in SiHa, HeLa, and C-33A cells, VAMP8 overexpression decreased apoptosis rates and enhanced the proportion of cells in the G2/M phase, indicating promoted cell cycle progression and survival (Fig. [100]8A,B). These results demonstrate the opposite effects of VAMP8 overexpression on cell fate determination at different stages of cervical cancer progression. Fig. 8. [101]Fig. 8 [102]Open in a new tab Flow cytometry analysis of cell cycle distribution and apoptosis in cervical cell lines. A Cell cycle distribution analysis after VAMP8 knockdown or overexpression. B Annexin V-APC/PI staining for apoptosis detection with VAMP8 knockdown or overexpression The contrasting effects of VAMP8 overexpression between early HPV16-transformed cells and established cervical cancer cells may be attributed to stage-specific interactions with autophagy-related signaling pathways, where VAMP8 enhances autophagic cell death in early transformation but shifts to facilitating autophagy-mediated survival mechanisms in advanced cancer cells. This functional switch could be due to differential regulation of downstream effectors or alterations in the tumor microenvironment that reprogram VAMP8’s role from promoting apoptosis to supporting cellular proliferation and metastasis. Collectively, these findings highlight VAMP8’s complex duality, acting as a guardian against early malignant transformation while fostering tumor growth and metastasis in established cancers. The precise molecular determinants underlying this stage-dependent functional switch of VAMP8 remain to be fully elucidated and represent an important avenue for future investigation. It is plausible that this dichotomy arises from context-specific interactions within the evolving tumor microenvironment, alterations in epigenetic landscapes during tumor progression, or differential engagement with HPV oncogenes such as E6/E7 at various stages of neoplastic transformation. Future studies incorporating comparative transcriptomic and proteomic analyses of early-stage versus advanced-stage cervical lesions, coupled with detailed investigation of VAMP8’s interactome in these distinct cellular contexts, will be crucial for unraveling the complex regulatory networks governing its dual role. Overexpression of VAMP8 promotes tumor growth and autophagy in vivo The in vivo tumor formation assays utilizing nude mice demonstrate that overexpression of VAMP8 significantly increases tumor volume and weight compared to controls, while knockdown of VAMP8 reduces tumor growth (Fig. [103]9A–C). Statistical analysis of tumor volumes shows a marked acceleration of tumor growth in the VAMP8 overexpression group, particularly by day 28 (Fig. [104]9B). Similarly, tumor weights are significantly higher in the VAMP8 overexpression group than in both the control and knockdown groups (Fig. [105]9C). Transmission electron microscopy of tumor tissues reveals a higher number of autophagic vesicles in the VAMP8 overexpression group, indicating enhanced autophagic activity (Fig. [106]9D). Fig. 9. [107]Fig. 9 [108]Open in a new tab Effects of VAMP8 on tumor growth and autophagy in vivo. A Representative images of excised tumors from nude mice (n = 4 per group) at day 28 post-injection. B Tumor volume growth curve measured over 28 days. C Violin plots showing tumor weight comparison across groups. D Transmission electron microscopy images of tumor tissues displaying autophagic vesicles in different experimental conditions These observations suggest that VAMP8 may drive cervical cancer progression by enhancing autophagic flux, which in turn supports tumor cell survival and growth under stress conditions within the tumor microenvironment. VAMP8 regulates genes involved in HIF-1 signaling and cytoplasmic translation pathways RNA sequencing of VAMP8-overexpressing Ect1/E6E7 and SiHa cells, along with their controls, identified differentially expressed genes (DEGs) in both cell lines (Fig. [109]10A). A single-gene differential expression analysis in the TCGA-CESC dataset further confirmed VAMP8-regulated DEGs (Fig. [110]10B). Correlation analysis of VAMP8 expression revealed a set of clinically significant genes in cervical cancer (Fig. [111]10C). The intersection of these analyses resulted in the identification of 845 common genes, as shown by the Venn diagram (Fig. [112]10D). GO and KEGG enrichment analyses highlighted the involvement of cytoplasmic translation and ribosomal pathways, with the HIF-1 signaling pathway being notably enriched (Fig. [113]10E). Fig. 10. [114]Fig. 10 [115]Open in a new tab Analysis of VAMP8-regulated genes and enriched pathways in cervical cancer. A Volcano plots showing differentially expressed genes between VAMP8-overexpressing and control Ect1/E6E7 and SiHa cells. B Volcano plot of single-gene differential expression analysis for VAMP8 in the TCGA-CESC dataset. C Heatmap showing the correlation analysis between VAMP8 expression and other genes in the TCGA-CESC dataset. D Venn diagram depicting the overlap of genes identified through differential expression and correlation analyses across the two cell lines and TCGA data. E GO and KEGG enrichment analysis of the common genes, highlighting key biological processes and pathways Given VAMP8’s impact on key components like HIF1A, VEGFA, and ENO1, it is hypothesized that VAMP8 enhances tumor survival and growth under hypoxic conditions by regulating the HIF-1 pathway, making it a potential therapeutic target for disrupting hypoxia-driven tumor progression. VAMP8 activates HIF-1 signaling, enhancing hypoxic survival and angiogenesis in SiHa cells Overexpression of VAMP8 in SiHa cells significantly increased mRNA and protein levels of HIF-1α, VEGFA, and ENO1, highlighting its role in upregulating key hypoxia-inducible genes (Fig. [116]11A, B). Chromatin immunoprecipitation (ChIP) assays confirmed that VAMP8 overexpression enhanced HIF-1α binding to VEGFA and ENO1 promoters (Fig. [117]11C). Luciferase reporter assays further demonstrated increased HRE-linked luciferase activity in VAMP8-overexpressing cells (Fig. [118]11D). Functionally, VAMP8 overexpression promoted hypoxic cell viability over time, as shown by the CCK-8 assay (Fig. [119]11E). Colony formation assays revealed a significant increase in the number of colonies formed under hypoxia in VAMP8-overexpressing cells (Fig. [120]11F). Conditioned media from these cells also promoted capillary-like tube formation in HUVECs, increasing both the number of tubes and total tube length (Fig. [121]11G). Fig. 11. [122]Fig. 11 [123]Open in a new tab Effects of VAMP8 overexpression on HIF-1 signaling, cell viability, and angiogenesis in SiHa cells. A Relative mRNA expression levels of HIF-1α, VEGFA, and ENO1 in VAMP8-overexpressing cells compared to controls. B Relative protein expression levels of HIF-1α, VEGFA, and ENO1 in VAMP8-overexpressing cells. C ChIP assay showing the relative enrichment of HIF-1α binding to the VEGFA and ENO1 promoters. D Luciferase reporter assay of HRE-linked activity in VAMP8-overexpressing cells. E Relative hypoxic cell viability at 24 h, 48 h, and 72 h. F Colony formation assay showing the relative number of colonies formed under hypoxia. G HUVEC tube formation assay displaying the total number of tubes and total tube length formed in the presence of conditioned media from VAMP8-overexpressing cells The results suggest that VAMP8 may act as a critical modulator of the HIF-1 signaling pathway, facilitating hypoxia-induced cellular adaptation by enhancing transcriptional activity of genes involved in angiogenesis and metabolic reprogramming. This mechanistic role positions VAMP8 as a potential therapeutic target, where inhibition could disrupt HIF-1α-mediated survival and angiogenic processes in hypoxic tumor microenvironments, thereby impairing tumor progression in cervical cancer. Discussion This study elucidated critical findings regarding VAMP8’s role in HPV16-associated cervical cancer. Proteomic analysis and quantitative PCR assays revealed significant upregulation of VAMP8 expression in HPV16-positive cervical tissues compared to HPV16-negative tissues, corroborated by elevated VAMP8 mRNA levels in HPV16-positive cell lines. Immunohistochemical staining of postoperative specimens confirmed pronounced VAMP8 overexpression specifically in HPV16-positive tissues. Notably, VAMP8 expression varied across cervical lesion stages, with higher levels in low-grade squamous intraepithelial lesions (LSIL) compared to high-grade squamous intraepithelial lesions (HSIL) and cervical cancer. However, all pathological stages, including LSIL, HSIL, and cervical cancer, exhibited significantly elevated VAMP8 levels relative to normal HPV16-negative tissues. This dynamic expression pattern suggests VAMP8’s crucial role in early cervical lesion development, potentially influencing mechanisms that differentiate low-grade from high-grade lesions. Additionally, a prognostic prediction model using follow-up data indicated that high VAMP8 expression is significantly associated with reduced overall survival (OS) and disease-free survival (DFS), underscoring its value as an independent prognostic factor. Analysis of the TCGA-CESC database further supported the diagnostic efficacy of VAMP8 expression, showing a significant correlation with tumor stages, distant metastasis, and clinical stages. Moreover, VAMP8 was significantly associated with increased immune cell infiltration, including dendritic cells, central memory T cells, and natural killer cells, suggesting its role in modulating the tumor immune microenvironment. In vitro and in vivo experiments demonstrated that VAMP8 enhances autophagy, proliferation, migration, and invasion in cervical cancer cells, promoting tumor growth and autophagy in mouse models. Finally, transcriptomic and bioinformatics analyses identified the HIF-1 signaling pathway as a major axis of VAMP8 regulation, highlighting its role in angiogenesis and metabolic adaptation under hypoxic conditions. The findings of this study align with and expand upon existing literature implicating VAMP8 in various oncogenic processes across cancer types. Previous research has highlighted VAMP8’s role in promoting tumor growth and metastasis in pancreatic and breast cancers, primarily through its regulation of autophagy and vesicle trafficking [[124]28, [125]33, [126]34]. These studies demonstrate that VAMP8 facilitates the secretion of pro-tumorigenic factors and enhances cancer cell survival under stress, reinforcing its role as a key mediator in tumor progression [[127]24, [128]35]. In cervical cancer, our study provides novel insights by showing significant VAMP8 upregulation in HPV16-positive tissues, consistent with its function in other malignancies. The differential expression of VAMP8 across cervical lesion stages—higher in LSIL than in HSIL and invasive cancer—suggests a nuanced role in early versus advanced disease stages, contrasting with the uniform overexpression observed in other cancers and indicating a unique regulatory mechanism in HPV16-associated carcinogenesis. Additionally, the association of high VAMP8 expression with adverse prognostic indicators, such as reduced overall survival (OS) and disease-free survival (DFS), aligns with findings in other cancers, reinforcing VAMP8’s potential as a prognostic biomarker. The integration of TCGA-CESC database analysis identifies VAMP8 as a marker for poor prognosis and tumor stage. Our study extends these observations by linking VAMP8 to immune cell infiltration, suggesting an immunomodulatory role not extensively explored in other contexts. This connection provides deeper insights into how VAMP8 may influence the tumor microenvironment and immune evasion strategies in cervical cancer. The seemingly contradictory findings regarding VAMP8 expression and its prognostic implications between our surgical cohort/murine model data and certain aspects of the TCGA-CESC analysis warrant careful consideration. Our study demonstrated that high VAMP8 expression in established cervical cancer patients correlated with poorer OS and DFS, and VAMP8 overexpression promoted tumor growth in vivo. Conversely, TCGA data indicated higher VAMP8 expression in earlier clinical stages (e.g., Stage I/II vs. III/IV, T1 vs. T2/T3/T4) and a positive correlation with the infiltration of certain immune cells, such as dendritic cells, which could imply a potential anti-tumor role or, at least, a more complex involvement in early tumorigenesis. This apparent discrepancy may reflect the multifaceted and stage-dependent roles of VAMP8 in cancer progression. Several factors could contribute to this. Firstly, the expression of VAMP8 in very early lesions (our IHC data also showed VAMP8 highest in LSIL) and early-stage cancers (TCGA) might reflect its involvement in initial cellular stress responses, active vesicle trafficking during early neoplastic changes, or an early host immune interaction. The positive correlation with dendritic cell infiltration, for instance, might represent an initial, potentially beneficial, immune surveillance or antigen presentation process that is more prominent in less advanced tumors. However, as the tumor progresses and establishes a more complex, often immunosuppressive microenvironment, the functional consequences of sustained high VAMP8 expression may shift. In established cancers, as represented by our surgical cohort with follow-up and our in vivo models using established cancer cell lines, the pro-autophagic and HIF-1 pathway-activating roles of VAMP8 likely become dominant, conferring survival advantages, promoting angiogenesis, and ultimately contributing to a more aggressive phenotype and poorer prognosis. This is consistent with our in vitro findings showing VAMP8 having a suppressive role in early HPV16-transformed cells but a promotional role in established cancer cell lines (section “VAMP8 enhances autophagy and exhibits stage-dependent effects on cervical cell proliferation and metastasis”), highlighting a context-dependent functional switch. Furthermore, while immune cell infiltration can be beneficial, the specific functional state (e.g., activated, exhausted, or regulatory) of these cells, which is not fully detailed by bulk transcriptomic deconvolution methods like CIBERSORT, is critical. VAMP8 might contribute to the recruitment of immune cells, but other factors within the advanced tumor microenvironment could subvert their anti-tumor functions. Therefore, the interpretation of VAMP8’s role must consider the specific stage of carcinogenesis, the cellular context, and the intricate dynamics of the tumor microenvironment. Future studies focusing on the temporal evolution of VAMP8 expression and its functional interactions at different stages of cervical cancer development, including its impact on distinct immune cell subtype functions, are needed to fully reconcile these observations. The mechanistic insights from this study illuminate the multifaceted role of VAMP8 in HPV16-associated cervical cancer pathogenesis, particularly through its modulation of autophagy, proliferation, migration, invasion, and apoptosis. Our in vitro experiments demonstrated that VAMP8 overexpression in HPV16-positive cervical epithelial cells significantly enhances autophagic activity, evidenced by increased autophagic vesicles and elevated levels of autophagy markers LC3-II and p62. This indicates that VAMP8 facilitates autophagy, which can either promote cell survival under stress or contribute to cell death, depending on the context. Moreover, VAMP8’s impact on cell proliferation was context-dependent; it acted as a suppressor in early-stage HPV16-transformed cells but promoted proliferation in fully transformed cervical cancer cell lines. This dualistic behavior underscores VAMP8’s complex role in tumor progression, potentially serving as a tumor suppressor in the initial stages of oncogenesis while promoting growth in established cancers. Additionally, VAMP8 overexpression reduced the migratory and invasive capabilities of early-stage HPV16-positive cells but enhanced these properties in cervical cancer cell lines, indicating a stage-specific regulatory function. The significant reduction in apoptosis rates and the shift in cell cycle distribution toward the G2/M phase in cancer cells further highlight VAMP8’s role in enhancing cell survival and proliferation. Crucially, our study identifies the HIF-1 signaling pathway as a major downstream target of VAMP8, with VAMP8 overexpression leading to increased HIF-1α stability and activity under hypoxic conditions. This interaction promotes the transcription of key HIF-1 target genes involved in angiogenesis, glycolysis, and cell survival, such as VEGFA and ENO1 [[129]36, [130]37]. The enhanced binding of HIF-1α to hypoxia-responsive elements in these genes’ promoters underscores the mechanistic link between VAMP8 and the hypoxic response. While our findings robustly demonstrate that VAMP8 overexpression leads to increased HIF-1α levels and activity, thereby promoting downstream hypoxia-responsive gene expression, the precise nature of this interaction—whether VAMP8 directly modulates HIF-1α stability or transcriptional activity, or if its effects are indirectly mediated through other cellular components—warrants further investigation. Future studies employing techniques such as co-immunoprecipitation (Co-IP) or proximity ligation assays (PLA) would be instrumental in determining a direct physical interaction between VAMP8 and HIF-1α or identifying potential intermediate regulatory proteins. These findings suggest that VAMP8-mediated activation of the HIF-1 pathway may provide a survival advantage to cervical cancer cells in the hypoxic tumor microenvironment, promoting angiogenesis and metabolic adaptation. The findings of this study carry significant clinical implications for enhancing diagnostic, prognostic, and therapeutic strategies for HPV16-associated cervical cancer. The consistent overexpression of VAMP8 in HPV16-positive cervical tissues, along with its differential expression across various stages of cervical lesions, underscores its potential as a diagnostic biomarker. VAMP8’s elevated levels in early lesions, such as low-grade squamous intraepithelial lesions (LSIL), compared to more advanced stages like high-grade squamous intraepithelial lesions (HSIL) and cervical cancer, suggest it could serve as an early indicator of disease progression, facilitating timely intervention. Moreover, the prognostic value of VAMP8, evidenced by its association with reduced overall survival (OS) and disease-free survival (DFS), highlights its potential in stratifying patients based on risk profiles [[131]38]. This could allow clinicians to identify high-risk patients who may benefit from more aggressive monitoring and tailored therapeutic regimens. Additionally, the correlation of VAMP8 expression with critical clinical parameters such as tumor stage further reinforces its relevance in clinical assessments. Beyond diagnostic and prognostic applications, VAMP8’s role in modulating key oncogenic pathways presents promising therapeutic opportunities. Targeting VAMP8 could disrupt its regulatory effects on autophagy, proliferation, migration, and invasion, thereby inhibiting tumor growth and metastasis. The mechanistic link between VAMP8 and the HIF-1 signaling pathway, vital for tumor adaptation to hypoxic conditions, suggests that VAMP8 inhibitors could impair the tumor’s ability to thrive in low-oxygen environments [[132]39, [133]40]. Furthermore, the association between VAMP8 and immune cell infiltration indicates that modulating VAMP8 activity might enhance the effectiveness of immunotherapeutic approaches by altering the tumor immune microenvironment [[134]41, [135]42]. VAMP8 emerges as a promising therapeutic target in HPV16-associated cervical cancer, offering multiple avenues for intervention. Its dualistic nature—acting as a tumor suppressor in early stages of HPV16-induced transformation while promoting tumor growth in established cancer—creates unique therapeutic opportunities. Inhibiting VAMP8 in advanced cervical cancer could potentially hinder its pro-tumorigenic activities, such as enhancing autophagy, proliferation, migration, and invasion. Specific therapeutic strategies could include the development of small-molecule inhibitors that selectively bind to VAMP8 and disrupt its function or its interaction with essential components of the SNARE machinery. Alternatively, nucleic acid-based therapies, such as RNA interference (RNAi) using siRNAs or shRNAs, or gene editing approaches like CRISPR/Cas9-mediated knockout of VAMP8, could be employed to downregulate its expression in tumor cells. Preclinical studies evaluating these approaches in relevant cervical cancer models would be essential to validate their efficacy and safety. Targeted therapies could also be designed to disrupt VAMP8’s interactions with key oncogenic pathways, particularly the HIF-1 signaling pathway. Given VAMP8’s role in stabilizing and activating HIF-1α under hypoxic conditions, VAMP8 inhibitors might impair the tumor’s ability to adapt to hypoxic stress, reducing angiogenesis and metabolic adaptation critical for tumor growth and survival [[136]43–[137]45]. Additionally, modulating VAMP8 could affect immune cell infiltration within the tumor microenvironment, potentially enhancing immunotherapy efficacy. By altering the immune landscape, VAMP8-targeted treatments could improve the recruitment and activation of immune cells, such as dendritic cells and natural killer cells, thus boosting the anti-tumor immune response [[138]46, [139]47]. The development of these targeted agents could provide a novel therapeutic approach, either as monotherapy or in combination with existing treatments like chemotherapy, radiotherapy, and immunotherapy. This targeted approach could significantly reduce morbidity and mortality associated with cervical cancer. To translate these therapeutic potentials into clinical practice, further preclinical studies and clinical trials are necessary to validate the efficacy and safety of VAMP8 inhibitors. Despite the significant findings and potential clinical implications, this study has several limitations that must be acknowledged. Firstly, the sample size, particularly for in vivo and clinical data, was relatively small, which may limit the generalizability of the results. Larger cohorts and multi-center studies are needed to validate the findings and ensure applicability across diverse populations. Secondly, although the study utilized a comprehensive range of methodologies, including proteomic analysis, qPCR, immunohistochemistry, and bioinformatics, the cross-sectional nature of the clinical data restricts the ability to infer causality. Longitudinal studies would provide more robust evidence of the temporal relationship between VAMP8 expression and cervical lesion progression. Thirdly, the focus on HPV16-positive cervical cancer, while relevant, does not encompass the entire spectrum of HPV-associated cervical malignancies. Future research should include other high-risk HPV types to determine whether the findings related to VAMP8 are universally applicable or specific to HPV16. Additionally, while the in vitro and in vivo experiments offered valuable insights into VAMP8’s mechanistic role, the complex interactions within the tumor microenvironment in clinical settings may not be fully represented by these models. Advanced techniques, such as organoid cultures and patient-derived xenografts, could provide more accurate depictions of tumor biology. Another limitation is the reliance on existing databases like TCGA-CESC, which, although comprehensive, may introduce biases related to sample selection and data annotation. Future research should build upon this study’s findings to further elucidate VAMP8’s role in cervical cancer and explore its potential as a therapeutic target. A critical direction is validating VAMP8 as a biomarker across larger, more diverse patient cohorts and in prospective studies to establish its utility in predicting disease progression and patient outcomes. Additionally, expanding research to include other high-risk HPV types will help determine whether the observed effects of VAMP8 are consistent across different HPV-driven cervical cancers. Mechanistic studies should delve deeper into the pathways regulated by VAMP8, particularly its interaction with the HIF-1 signaling pathway and its influence on the tumor microenvironment, to uncover the precise molecular mechanisms underlying its dualistic role in tumor suppression and promotion. High-throughput screening of small molecules and rational drug design approaches could identify effective compounds targeting VAMP8. Combination therapy studies, integrating VAMP8 inhibitors with existing treatments like chemotherapy, radiotherapy, and immunotherapy, could reveal synergistic effects and optimal therapeutic strategies [[140]48, [141]49]. Investigating the impact of VAMP8 modulation on the immune landscape within the tumor microenvironment will also be crucial, potentially enhancing immunotherapy efficacy. Finally, translating these preclinical findings into early-phase clinical trials will be a key step in assessing the safety, tolerability, and preliminary efficacy of VAMP8-targeted therapies in patients. The mechanistic diagram presented in Fig. [142]12 illustrates how persistent HPV16 infection drives cervical epithelial cells toward tumorigenesis through dysregulated VAMP8 expression, which differentially influences cellular processes such as proliferation, migration, invasion, and apoptosis. VAMP8 overexpression induces autophagy by enhancing the formation of autophagosomes and promoting lysosomal degradation, contributing to both cell survival and tumor progression. Furthermore, VAMP8 activation stabilizes HIF-1α under hypoxic conditions, facilitating increased transcription of pro-angiogenic and glycolytic genes, such as VEGFA and ENO1, thereby supporting tumor adaptation to the hypoxic microenvironment. Collectively, these findings highlight VAMP8’s role in mediating the crosstalk between autophagy and hypoxia, thereby orchestrating a microenvironment conducive to tumor growth and metastasis in HPV16-positive cervical cancer. Fig. 12. [143]Fig. 12 [144]Open in a new tab Schematic representation of VAMP8’s role in HPV16-associated cervical cancer progression It shows its involvement in autophagy, hypoxia adaptation, and modulation of tumor microenvironment factors that contribute to tumorigenesis and metastasis. Conclusions This study demonstrates that VAMP8 is significantly upregulated in HPV16-positive cervical tissues and cell lines, especially in early-stage lesions, underscoring its critical role in HPV16-associated cervical carcinogenesis. High VAMP8 expression correlates with decreased overall and disease-free survival, serving as an independent prognostic factor, and mechanistically enhances autophagy, proliferation, migration, invasion, and activates the HIF-1 signaling pathway, promoting tumor adaptation under hypoxic conditions. Additionally, VAMP8 is associated with increased immune cell infiltration, suggesting its involvement in modulating the tumor immune microenvironment. These findings identify VAMP8 as a potential diagnostic biomarker and therapeutic target in HPV16-associated cervical cancer. Supplementary Information [145]12916_2025_4378_MOESM1_ESM.docx^ (13.1KB, docx) Additional file 1. Table S1. Sequences of primers used for quantitative real-time PCR (qPCR) analysis. [146]12916_2025_4378_MOESM2_ESM.docx^ (19.1KB, docx) Additional file 2. TRIPOD Checklist for Prediction Model Development. [147]12916_2025_4378_MOESM3_ESM.pdf^ (153.8KB, pdf) Additional file 3. Original, Uncropped Western Blot Images. [148]12916_2025_4378_MOESM4_ESM.docx^ (23.5KB, docx) Additional file 4. ARRIVE Guidelines 2.0 Checklist. Acknowledgements