Cell lines and cell culture

Human ovarian cancer cell lines SKOV3 was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2.

Reagents and antibodies

Sempervirine (purity ≥ 98%) was obtained from Yuanye Biotechnology (Shanghai, China). 5-Fluorouracil (5-Fu) was purchased from Sigma-Aldrich. Primary antibodies against apelin, CD34 and GAPDH were purchased from Cell Signaling Technology. Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology.

Cell proliferation assay

The effect of sempervirine on ovarian cancer cell proliferation was determined by CCK8 assay. SKOV3 cells were seeded into 96-well plates at a density of 5 × 103 cells/well and cultured overnight. Cells were then treated with various concentrations of sempervirine (0, 0.1, 0.5, 1, 5, 10, 25, 50, 100 µM) for 6 h, 24 h and 48 h. CCK8 solution (Beyotime Institute of Biotechnology) was added to each well followed by 2 h incubation at 37 °C. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad). Cell viability was calculated as the percentage of control cells without drug treatment.

Transwell invasion assays

The effects of sempervirine on ovarian cancer cell invasion were evaluated using transwell chambers (8 μm pore size, Corning). For invasion assay, the upper chamber was precoated with Matrigel (BD Biosciences). SKOV3 cells were pretreated with sempervirine (0, 1, 5, 10 µM) for 24 h, resuspended in serum-free medium and then seeded into the upper chambers at a density of 5 × 104 cells/chamber. The lower chambers were filled with medium containing 10% FBS as chemoattractant. After 24 h incubation, the non-migrating or non-invading cells on the upper surface were removed gently with a cotton swab. The cells on the lower surface were fixed with 4% paraformaldehyde, stained with 0.1% crystal violet and photographed under a microscope. Five random fields were captured and the number of migrated or invaded cells was counted.

Colony formation assay

The effect of sempervirine on SKOV3 cell proliferation was assessed using a colony formation assay. Cells were trypsinized and seeded at a density of 500 cells per well in 6-well plates. Following a 24-hour incubation, cells were treated with different concentrations of sempervirine (2.5 µM, 5 µM, 10 µM) or with vehicle (control group) for 48 h. After treatment, the drug-containing medium was replaced with drug-free medium, and the cells were cultured for an additional 7 days, with the medium being refreshed every 2 days. After 7 days, cells were fixed with 4% paraformaldehyde for 30 min at room temperature, followed by staining with 0.1% crystal violet for 30 min. Stained plates were washed with sterile water and allowed to dry, and colonies were counted manually.

Apoptosis analysis by flow cytometry

Apoptosis induction by sempervirine was assessed using an Annexin V-APC/PI double staining method. SKOV3 cells were seeded at a density of 1.5 × 10⁵ cells per well in 6-well plates. After adherence, cells were treated with sempervirine at various concentrations (2.5 µM, 5 µM, 10 µM) for 24 h. Cells were then harvested using trypsin without EDTA, washed twice with PBS, and resuspended in 500 µL of Binding Buffer. Next, 5 µL of Annexin V-APC and 5 µL of PI staining solution were added to the cell suspension, followed by gentle mixing. The cells were incubated at room temperature for 5–10 min in the dark. Apoptosis was analyzed within 1 h using flow cytometry (BD Biosciences) with excitation at 633 nm and emission detection at 660 nm. Fluorescence compensation was performed using apoptosis-induced cells as a control.

Cell cycle analysis

To investigate the effect of sempervirine on the cell cycle, SKOV3 cells were seeded at a density of 1.5 × 10⁵ cells per well in 6-well plates and treated with sempervirine at 2.5 µM, 5 µM, and 10 µM concentrations for 24 h. Cells were then harvested by trypsinization, washed twice with PBS, and fixed with 70% pre-chilled ethanol overnight at 4 °C. Fixed cells were centrifuged at 500 g for 5 min, the supernatant was discarded, and cells were washed with pre-chilled PBS three times. Cells were resuspended in propidium iodide (PI) staining solution and incubated at room temperature for 30 min. Cell cycle distribution was analyzed by flow cytometry, and the proportions of cells in the G0/G1, S, and G2/M phases were determined.

RNA extraction, library preparation and sequencing

Total RNAs were extracted from SKOV3 cells using TRIzol Reagent (Invitrogen, cat. NO 15596026)following the protocol. Post-extraction, genomic DNA contamination was removed by treatment with DNase I. RNA quality and purity were assessed by measuring the A260/A280 ratio using a NanoDrop™ OneC spectrophotometer (Thermo Fisher Scientific). RNA integrity was further confirmed through 1.5% agarose gel electrophoresis, and RNA concentration was accurately quantified using the Qubit™ RNA Broad Range Assay Kit (Life Technologies, Cat. No. Q10210) on a Qubit 3.0 fluorometer.

For stranded RNA sequencing library preparation, 2 µg of total RNA was processed using the KC-Digital™ Stranded mRNA Library Prep Kit for Illumina® (Cat. No. DR08502, Wuhan Seqhealth Co., Ltd., China) according to the manufacturer’s instructions. This kit minimizes PCR and sequencing duplication bias by incorporating an 8-base unique molecular identifier (UMI) to label pre-amplified cDNA molecules. Library fragments between 200 and 500 bp were enriched, quantified, and subsequently sequenced using the DNBSEQ-T7 sequencer (MGI Tech Co., Ltd., China) with paired-end 150 bp reads.

RNA-Seq data analysis

Raw sequencing data were first processed using Trimmomatic (version 0.36) to remove low-quality reads and trim adaptor sequences. Clean reads were further refined using custom scripts to eliminate duplication bias introduced during library preparation and sequencing. Specifically, clean reads were clustered based on unique molecular identifiers (UMIs), grouping reads with identical UMI sequences into the same cluster. Pairwise alignment was then performed within each cluster, and reads with greater than 95% sequence identity were grouped into sub-clusters. For each sub-cluster, multiple sequence alignment was used to generate a single consensus sequence, effectively correcting for errors and biases from PCR amplification and sequencing. These deduplicated consensus sequences were used for downstream RNA-seq analysis.

Subsequently, the deduplicated reads were mapped to the human reference genome using STAR (version 2.5.3a) with default parameters. Reads aligned to the exon regions of each gene were quantified using feature Counts (Subread-1.5.1, Bioconductor), and gene expression levels were calculated as reads per kilobase of transcript per million mapped reads (RPKM). Differentially expressed genes between experimental groups were identified using the edgeR package (version 3.12.1), with a p-value cutoff of 0.05 and a fold-change threshold of 2 considered statistically significant.

Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis for differentially expressed genes were conducted using KOBAS software (version 2.1.1), with a p-value threshold of 0.05 indicating significant enrichment. Alternative splicing events were identified using rMATS (version 3.2.5), with a false discovery rate (FDR) cutoff of 0.05 and an absolute Δψ threshold of 0.05.

Western blot analysis

Total protein was extracted from ovarian cancer cells using RIPA lysis buffer containing protease and phosphatase inhibitors. The protein concentration was determined by BCA assay. Equal amounts of protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked in 5% non-fat milk for 1 h and incubated with primary antibodies at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were visualized using ECL reagents and band intensity was quantified by ImageJ software.

In vivo xenograft model

All animal experiments were approved by the Institutional Animal Care and Use Committee of shanghai tongren hospital. Female BALB/c nude mice (5–6 weeks old) were purchased from Vital River Laboratory (Beijing, China). SKOV3 cells (2 × 106) in 100 µLPBS were subcutaneously injected into the right flank of each mouse to establish the ovarian cancer xenograft model. When the tumor volume reached about 100 mm [3], the mice were randomized into three groups (n = 6 per group) and treated as follows: (1) Control group: 100 µL normal saline by intraperitoneal injection every two days; (2) 5-Fu group: 5-Fu at 1 mg/kg by intraperitoneal injection every two days; (3) Sempervirine group: sempervirine at low-dose1mg/kg; mediun-dose 3 mg/kg, high-dose10 mg/kg by intraperitoneal injection every two days. After 15 days treatment, mice were sacrificed and tumors were excised and weighed. Tumor tissues were collected for further analysis.

Immunohistochemistry (IHC)

Tumor tissues were fixed, embedded in paraffin and sectioned at 5 μm thickness. The sections were deparaffinized, rehydrated and subjected to antigen retrieval. After blocking with bovine serum albumin (BSA), the slides were incubated with Ki-67 primary antibody overnight at 4 °C, followed by incubation with biotinylated secondary antibody and HRP-conjugated streptavidin. The signaling was developed using DAB solution and counterstained with hematoxylin.

Statistical analysis

All experiments were performed in triplicate. Data were expressed as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 7 software. Differences between groups were analyzed by one-way ANOVA or Student’s t-test. p < 0.05 was considered statistically significant.