Cell culture

The human ovarian cancer cell lines SKOV3 and ES2 were purchased from the National Collection of Authenticated Cell Cultures (Shanghai, China). SKOV3 and ES2 cells were cultured in McCoy’s 5a medium (Gibco, 16600082) supplemented with 10% fetal bovine serum (FBS, Gibco, 10091155), 100 U/ml penicillin and 100 µg/ml streptomycin (HyClone, SV30010). The culture condition was 37 °C and a humidified atmosphere with 5% CO2. The cells were subcultured every 2–4 days according to cell confluence.

RNA sequencing (RNA-seq) analysis

ES2 cells were treated with 40 µM ART (MCE, HY-N0193) or DMSO (vehicle control) for 24 h. Total RNA was extracted using TRIzol reagent (Invitrogen, c#15596026). RNA quality was tested by examining the A260/A280 ratio with NanoDrop (Thermo Fisher Scientific, USA). RNA-seq libraries were constructed using the Invitrogen Collibri Stranded RNA Library Prep Kit (Invitrogen Life Technologies, A39117024) according to the manufacturer’s instructions. Paired-end sequencing was performed on an Illumina HiSeq 4000 platform (Illumina, USA). Differentially expressed genes (DEGs) between the control and ART treatment groups were analyzed using the DESeq2 R package. Significant differences in gene expression were determined according to the criteria of |log2FC| > 1 and p value < 0.01. Functional annotation and enrichment analysis were performed using the Gene Ontology (GO) database (http://www.geneontology.org/) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Mapper (http://www.genome.jp/kegg/mapper.html), respectively. The clusterProfiler R package was used for enrichment analysis.

Quantitative real-time polymerase chain reaction (qRT‒PCR) analysis

Total RNA was extracted from cells using TRIzol reagent (Yeasen, 10606ES60). RNA concentration and purity were checked with NanoDrop. cDNA was synthesized using the HiScript IV 1st Strand cDNA Synthesis Kit (Vazyme, R412-01) according to the manufacturer’s protocol. qRT-PCR was performed with QuantStudio 12 K (Thermo Fisher Scientific, USA) using Hieff® qPCR SYBR Green Master Mix (Low Rox) (Yeasen, 11202ES08). The PCR program was as follows: denaturation at 95 °C for 5 min, followed by 40 cycles of 95 °C for 10 s, 60 °C for 20 s and 72 °C for 20 s. GAPDH was used as an internal control, and the data were analyzed using the 2−ΔΔct method. The primers used for amplification were listed in Table 1.

Table 1 Primer sequences for qRT-PCRWestern blot analysis

Cells or tissues from each experimental group were harvested and washed with PBS. Total protein was extracted with RIPA Lysis Buffer (Beyotime, P0013) containing protease and phosphatase inhibitor cocktail (Beyotime, P1045). Protein concentration was detected using a BCA assay kit (Beyotime, P0012). Equal amounts of protein (30 µg per sample) were separated by SDS-PAGE and transferred to an NC membrane. Membrane was blocked with 5% nonfat milk and then incubated with primary antibody at 4 °C overnight. The dilution ratios for primary antibodies were as follows: GAPDH (Proteintech, 10494-1-AP) at 1:10000; Bax (Proteintech, 50599-2-Ig), GPX4 (Proteintech, 67763-1-Ig), and Bcl-2 (Proteintech, 12789-1-AP) at 1:5000; AKT (Proteintech, 60203-2-Ig), p-AKT (Proteintech, 66444-1-Ig), and PIK3CG (Abcam, ab140307) at 1:2000; HOXC11 (Novus Biologicals, OTI3E10), PROM2 (Affinity, DF14670), and p-PI3K (CST, 17366) at 1:1000. The dilutions of the goat anti-rabbit and goat anti-mouse secondary antibodies were 1:5000. Proteins were visualized using BeyoECL Plus (Beyotime, P0018S), and detected with a ChemiDoc™ XRS + system (Bio-Rad, USA). Relative protein levels were measured by ImageJ (Rasband, USA) and normalized to GAPDH.

Dual-luciferase reporter assay

The coding sequence (CDS) of HOXC11 was inserted into the pcDNA3.1 plasmid, and the promoter sequence of PROM2 was inserted into the upstream of a luciferase reporter gene in the pGL3-basic plasmid. pcDNA3.1-HOXC11 and pGL3-PROM2 were co-transfected with pRL-TK into HEK-293T cells using Lipofectamine 2000 (Invitrogen, 11668019) according to the manufacturers’ protocols. The empty pcDNA3.1 and pGL3-basic vectors were used as controls. After 48 h of transfection, cells were lysed, and a Dual-Luciferase Reporter Assay System (Promega, E1910) was used for the luciferase activity assay according to the manufacturers’ protocols. Relative luciferase activity was calculated as the ratio of firefly to Renilla luciferase activity.

Electrophoretic mobility shift assay (EMSA)

The full-length sequence of HOXC11 was inserted into the pET-28b vector and transformed into Escherichia coli BL21 (DE3). Isopropyl β-d-1-thiogalactopyranoside (IPTG, 1 mM) was used to induce recombined protein at 37°C for 5 h. The recombinant HOXC11 protein was purified using Ni-NTA-agarose resin (Qiagen). DNA probes were labeled with biotin at the 3’ end using an EMSA Probe Biotin Labeling Kit (Beyotime, GS008). The EMSA was carried out using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher, 20148) following the instruction.

Recombinant lentivirus particles and cell line transduction

Recombinant lentivirus particles were obtained from GenePharma (Shanghai, China). ES2 and SKOV3 cells were transduced with lentivirus (MOI = 10) and 8 µg/ml polybrene for 2–3 days, followed by 2 µg/ml puromycin treatment until puromycin-resistant cells predominated. The overexpression of HOXC11 and knockdown efficiency of PROM2 were verified by western blot analysis.

CCK-8 assay

ES-2 and SKOV-3 cells were seeded in 96-well plates at a density of 9000 cells per well overnight. Cells were treated with 40 µM ART for 24 h. Cell viability was measured using the Cell Counting Kit-8 reagent (Dojindo, CK04).

Flow cytometry analysis

ES-2 and SKOV-3 cells were seeded in 24-well plates at a density of 8 × 104 cells per well overnight. An Annexin V-FITC Apoptosis Detection Kit (Beyotime, C1062M) was used according to the manufacturer’s instructions for the apoptosis assay. Briefly, cells were suspended in 200 µL of Annexin binding buffer containing 5 µL of Annexin V-FITC and 10 µL of propidium iodide (PI). After incubating in the dark at room temperature for 10–20 min, cell apoptosis was tested by flow cytometry (Agilent, USA). Lipid peroxides were tested using BODIPY 581/591 C11 (Invitrogen, D3861) staining. Cells were incubated with 5 µM BODIPY for 30 min at 37℃and analyzed by flow cytometry.

Wound healing assay

ES-2 and SKOV-3 cells were seeded in 6-well plates at a density of 6 × 105 cells per well overnight. When cell confluence exceeded 90%, a 10 µL pipette tip was used to make a scratch in the bottom of the plates. Cells were washed with PBS and incubated in serum-free medium. Images of wound healing were taken by microscope (Olympus, Japan) at 0 h, 24 h and 48 h post-scratch.

Reactive oxygen species (ROS) detection

ROS assay was performed using ROS Assay Kit (Beyotime, S0033S). Cells were incubated with 10 µM DCFH-DA at 37 °C for 20 min. The cells were washed with serum-free medium for 3 times, and images of green fluorescence intensity were tested by microscope.

Biochemical index detection

Cells or tissues from the indicated groups were collected. Superoxide dismutase (SOD) activity, glutathione (GSH) concentration, and malondialdehyde (MDA) concentration were examined using a SOD activity assay kit (Nanjing Jiancheng Bioengineering Institute, A001-3), a GSH assay kit (Nanjing Jiancheng Bioengineering Institute, A006-2-1) and an MDA assay kit (Beyotime, S0131S), respectively, according to the manufacturer’s guidelines.

Transmission electron microscope (TEM) analysis

ES-2 and SKOV-3 cells were fixed in 2.5% glutaraldehyde solution at room temperature for 2 h. Then, the cells were dehydrated and embedded in resin. Ultrathin sections were stained with uranyl acetate and lead citrate. The cell ultrastructure was observed under a transmission electron microscope (Hitachi H-7650, Japan).

Xenograft mouse model

5–6 weeks old female BALB/c nude mice were purchased from SiPeiFu (Beijing) Biotechnology Co., Ltd. A total of 5 × 106 SKOV3 cells were injected subcutaneously into the right flank of the mice to establish a xenograft model. Mice with approximately 100 mm3 SKOV3 tumor xenografts were randomly divided into two groups (n = 6 per group). Mice were intraperitoneally injected with vehicle (5% DMSO + 95% PBS) or 70 mg/kg ART for 25 days. Tumor volume and body weight were measured every three days. Tumor volume was calculated using the following formula: volume (mm3) = 1/2 × (tumor length) × (tumor width)2, length > width. Mice were euthanized at the end of the treatment period, and tumor tissues were collected for further analysis. All animal experiments were performed under the institutional guidelines and were approved by the Ethics Committees of North Sichuan Medical College (NSMC2023 (114)).

Immunohistochemistry (IHC) assay

Tumor tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 5 μm slices. The slides were deparaffinized and rehydrated. After incubating with methanol containing 3% H2O2 and heat-inducing antigen retrieval by boiling in sodium citrate, the slides were blocked with goat serum for 30 min and incubated with primary antibodies at 4 °C overnight. The primary antibodies used were against GPX4 (1:1000, Proteintech, 67763-1-Ig), HOXC11 (1:100, Novus Biologicals, OTI3E10), and PROM2 (1:100, Affinity, DF14670). After incubation with a secondary antibody, proteins of interest were visualized with DAB solution (Beyotime, P0202), and the nuclei were stained with hematoxylin (Beyotime, C0107). Images were taken using a microscope (Olympus, Japan).

Statistical analysis

All data were analyzed using GraphPad Prism 8.0.2. The data were shown as mean ± standard deviation (SD). Two-tailed Student’s t test was used for comparisons between two independent groups, and one-way ANOVA was used for comparisons multiple independent groups. P < 0.05 was considered to indicate statistical significance.