Patients and samples

In our research, CC tissues and matched adjacent non-cancerous tissues were collected from twenty patients at the People’s Hospital of the Xinjiang Uygur Autonomous Region. The study received approval from the Ethics Committee of the People’s Hospital of the Xinjiang Uygur Autonomous Region, and written informed consent was obtained from all participants. None of the patients had received chemotherapy or radiotherapy before their surgical resection. Following excision, tissues were immediately snap-frozen in liquid nitrogen and stored at − 80 °C for subsequent analysis.

Cell culture and treatment

HeLa, SiHa, and normal cervical epithelial cells (HcerEpic) were obtained from Shanghai Yaji Biotechnology Co., Ltd. The cells were maintained in high-glucose DMEM (Gibco, Carlsbad, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco, Carlsbad, USA) and cultured at 37 °C in a humidified incubator with 5% CO₂ and 95% air. For the experiments, HeLa and SiHa cells were seeded at a density of 1 × 105 cells per well in a 6-well plate. In addition, cells were treated with 5 µM 5-aza-CdR (Sigma-Aldrich, St. Louis, USA) for 72 h, after which they were collected for further analysis.

Vectors and transfection

HeLa and SiHa cells were seeded at a density of 1 × 10⁵ cells per well in 6-well plates. Cells were transfected with either a negative control (NC) or siRNAs targeting BST2, DNMT1, DNMT3a, DNMT3b, and STAT1 using the siRNA Transmate reagent (Suzhou GenePharma Co., Ltd.). Transfection was performed at 37 °C for 6 h, after which the medium was replaced with fresh medium. For overexpression experiments, HeLa and SiHa cells were transfected with either the BST2 overexpression plasmid (oe-BST2; pcDNA3.1 vector, NM_004335.4, 1.5 µg/well) or a negative control plasmid (oe-NC; empty pcDNA3.1 vector, 1.5 µg/well) using Lipofectamine™ 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) at 37 °C. Cells were harvested for further experiments 24 h post-transfection. The siRNA sequences and the plasmids were obtained from Suzhou GenePharma Co., Ltd.

Lentiviral vectors targeting BST2 were produced using a second-generation system in 293T cells. Cells were transfected with GV vector (20 µg), pHelper 1.0 (15 µg), and pHelper 2.0 (10 µg) using Lipofectamine 3000. After 6 h, the medium was replaced, and supernatants were collected 48 h later, centrifuged, and concentrated by ultracentrifugation. The viral titer was 1 × 10⁸ TU/ml. SiHa cells were transduced at a multiplicity of infection (MOI) of 50 in the presence of polybrene (8 µg/ml), followed by puromycin selection (2 µg/ml). Finally, viral titers and knockdown efficiency were validated.

RNA extraction and RT-qPCR

Total RNA was extracted from cells or tissues using the UNIQ-10 column Trizol total RNA extraction kit (Sangon Biotech). Complementary DNA (cDNA) was synthesized from 1 µg of total RNA using PrimeScript RT Master Mix (Takara Bio) according to the manufacturer’s instructions. The reverse transcription reaction was performed at 37 °C for 15 min, followed by inactivation at 85 °C for 5 s and cooling to 4 °C using a PCR Veriti thermal cycler (Applied Biosystems).

Quantitative PCR (qPCR) was performed using the QuantStudio®5 Real-Time PCR System (Applied Biosystems) with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories). The thermal cycling conditions included an initial denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Primer sequences are listed in Table 1. Relative gene expression was normalized to GAPDH and calculated using the 2−ΔΔCt method.

Table 1 Sequences of primers for RT-qPCRWestern blot analysis

Tissues or cells were lysed in RIPA buffer containing PMSF (100:1) at 4 °C for 30 min. Subsequently, the lysate was subjected to centrifugation at 10,000×g for 10 min at 4 °C to obtain the supernatants. The protein concentration was determined using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). A total of 20 µg of protein per lane was separated by SDS-PAGE and transferred to PVDF membranes (Invitrogen; Thermo Fisher Scientific, Inc.). Membranes were blocked with 5% non-fat milk for 2 h at room temperature and incubated overnight at 4 °C with primary antibodies, including anti-BST2 (1:1,000; cat. no. ab243230; Abcam), anti-DNMT1 (1:1,000; cat. no. sc-514784; Santa Cruz Biotechnology), anti-DNMT3a (1:1,000; cat. no. 3598; Cell Signaling Technology), anti-DNMT3b (1:1,000; cat. no. sc-393845; Santa Cruz Biotechnology), anti-STAT1 (1:1,000; cat. no. ab234400; Abcam), anti-cleaved Caspase-3 (1:1000, cat. no. 9661; Cell Signaling Technology), anti-γH2AX (1:1000, cat. no. 2577; Cell Signaling Technology), and anti-GAPDH (1:1,000; cat. no. sc-137179; Santa Cruz Biotechnology). Following washing with TBST, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000; Bioworld Technology, Inc.) for 1 h at 25 °C. Protein bands were visualized using the Amersham Imager 680 (Cytiva) along with ECL reagent (MilliporeSigma), and the quantification of protein expression was performed using ImageJ software (version 1.46; National Institutes of Health), with GAPDH serving as the loading control.

Cell proliferation assay

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; Beyotime Institute of Biotechnology). After transfection, cells were seeded into 96-well plates at a density of 5 × 10³ cells per well and cultured at 37 °C in a humidified incubator with 5% CO₂. At 0, 24, 48, 72, and 96 h post-seeding, 10 µl of CCK-8 solution was added to each well. The plates were incubated for 2 h at 37 °C, and the absorbance was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

Cell apoptosis assay

Cell apoptosis was evaluated 48 h post-transfection utilizing the Annexin V-FITC/PI Apoptosis Detection Kit (Beyotime Biotechnology, Shanghai, China). Briefly, treated cells were collected and washed twice with cold phosphate-buffered saline (PBS). The resulting cell pellet was then resuspended in 1× Binding Buffer at a concentration of 1 × 10⁶ cells/ml. A volume of 100 µl of this cell suspension was placed into a flow cytometry tube and stained with 5 µl of Annexin V-FITC and 10 µl of propidium iodide (PI) for a duration of 15 min at room temperature in the absence of light. After staining, 400 µl of 1× Binding Buffer was added to the cells. Apoptosis was subsequently assessed using a flow cytometer (BD FACSCalibur), and the data were processed with FlowJo software (Tree Star, Inc.). Early apoptotic cells were characterized as Annexin V-FITC-positive and PI-negative, whereas late apoptotic and necrotic cells were identified as Annexin V-FITC-positive and PI-positive.

Animal studies

Female BALB/c nude mice (6 to 8 weeks old) were utilized for the xenograft experiment. SiHa cells, which had undergone stable BST2 knockdown (kd-BST2) or this controls (kd-NC), were collected and subcutaneously injected into the right flank (3 × 106 cells/100 µl). After a period of 28 days, the mice were euthanized, and the tumors were removed for subsequent analysis. Tumor volume was determined using the formula: volume = (length × width²)/2.

DNA methylation analysis

Genomic DNA was isolated from HeLa and SiHa cell lines and subsequently quantified utilizing a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). The bisulfite conversion of the genomic DNA was conducted using the EpiTect Bisulfite Kit (Qiagen, Germany), following the guidelines provided by the manufacturer. For methylation-specific PCR (MSP), the converted DNA was amplified with primers specifically designed to target the region of interest. The resulting PCR products were subjected to separation on a 2% agarose gel (Invitrogen, USA) and visualized through staining with GelRed (Biotium, USA) under ultraviolet light. The methylation status was assessed by analyzing the presence or absence of distinct bands that corresponded to either methylated or unmethylated alleles.

Dual-luciferase reporter assay

The promoter region of BST2 was amplified via PCR with specific primers and subsequently cloned into the pGL3-Basic luciferase reporter vector (Promega, USA). Bioinformatics analysis was employed to identify the STAT1-binding sites within the BST2 promoter, and mutations were introduced utilizing the QuikChange Site-Directed Mutagenesis Kit (Agilent, USA). The sequences of both the wild-type (WT) and mutant (MUT) promoters were verified through Sanger sequencing. Cells were plated at a density of 1 × 10⁴ cells per well in 96-well plates. Co-transfection of cells was performed with 100 ng of either BST2-WT or BST2-MUT promoter constructs alongside 10 ng of the pRL-TK Renilla luciferase vector (Promega, USA), using Lipofectamine 3000 reagent (Invitrogen, USA). The total amount of DNA was adjusted with an empty pGL3-Basic vector to maintain consistent transfection efficiency. After a 48-h incubation, cells were lysed, and luciferase activity was assessed using the Dual-Luciferase Reporter Assay System (Promega, USA). Firefly luciferase activity was normalized against Renilla luciferase activity to correct for variations in transfection efficiency and cell viability.

Chromatin Immunoprecipitation (ChIP)

Chromatin immunoprecipitation (ChIP) assays were conducted on HeLa and SiHa cell lines, as well as on clinical and nude mouse tissues, utilizing the Magna ChIP™ G Kit (MilliporeSigma, USA) in accordance with the manufacturer’s guidelines. In summary, the samples were subjected to crosslinking with 1% formaldehyde for 10 min at room temperature to ensure the stabilization of protein-DNA interactions, followed by a quenching step with 0.125 M glycine. Subsequently, the samples were lysed and sonicated with a Bioruptor® Plus sonication device (Diagenode, Belgium) to fragment the chromatin into pieces measuring approximately 200–500 bp. The resulting lysates were centrifuged, and the supernatants were retained for immunoprecipitation, which was carried out overnight at 4 °C using either an anti-STAT1 antibody (Cell Signaling Technology, USA) or IgG as a negative control. Immune complexes were isolated using protein G magnetic beads, followed by washing and elution. The crosslinks were then reversed, and DNA was purified through a spin column. The enrichment of the BST2 promoter region was assessed via quantitative PCR (qPCR) employing specific primers designed for the BST2 promoter.

Statistical analysis

Data obtained from three separate experiments were evaluated utilizing GraphPad Prism 9 and SPSS 19.0. The results are presented as mean ± standard deviation (SD). For statistical comparisons, either Student’s t-test or one-way ANOVA with Tukey’s test was employed, with nonparametric tests applied when necessary. A p-value of less than 0.05 was considered statistically significant.