The Ethics Committee of the first Affiliated Hospital of Jinzhou Medical University approved the current study. All procedures were carried out in conformity with the 1964 Declaration of Helsinki. The subjects were informed of the project and signed an informed consent form. Ovarian cancer tissue and adjacent tissue were gathered from 15 patients. Inclusion criteria: age ≥ 18 years; no anti-tumor therapy prior to enrollment. Exclusion criteria: combined with tumors in other regions, systemic infections, serious disorders of vital organs, and autoimmune diseases. Total RNA and proteins were extracted from these samples followed by RT-qPCR and immunoblotting as detailed below.

Ovarian epithelial cells IOSE-80 (EK-Bioscience, Shanghai), cancer cell lines OVCAR3 (ATCC), HEY (ATCC), and A2780 (ECACC) were cultured in RPMI-1640 (Gibco, ThermoFisher). Cancer cell line SKOV3 (ATCC) and cisplatin-resistant cell line SKOV3/DDP were cultured in McCoy’s 5a (Gibco). SKOV3/DDP was created utilizing the previously reported concentration-increasing approach [31] with cisplatin (C2210000, Merck). They were all supplemented with 10% fatal bovine serun and 1% penicillin-streptomycin (Gibco). Cells were grown at 37 °C with 5% CO2/95% air.

Specific CARD9 or HOXB5 shRNAs and scrambled shRNAs as negative controls (NC; GenePharma, Shanghai) were transfected into SKOV3 or SKOV3/DDP cells to achieve knockdown. To achieve overexpression, HOXB5 plasmids and empty plasmids (GenScript, Nanjing) as the NC were transfected into SKOV3 or SKOV3/DDP cells. The assay was operated following the product instruction manual of Lipofectamine 3000 transfection reagent (Invitrogen). The transfection mixture was replaced with fresh medium after 6 h of transfection, and efficacy was determined using RT-qPCR and immunoblotting as detailed below.

TRIzol® reagent (Invitrogen) and LabScript RT kit (Biolab, Beijing) were used to extract total RNA from cells or tissues and generate cDNA. The QuantiTect SYBR Green PCR kit (Qiagen) was used for RT-qPCR. The relative mRNA levels were quantified using the ∆∆Ct method following normalization against GAPDH. Primer sequences are listed in Table 1.

Protein was extracted from cells or tissues following RIPA lysis buffer (WellBio, Shanghai) treatment and quantified using Nano 300 protein detector (Allsheng, Hangzhou). The proteins were apart on the polyacrylamide gel through sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to PVDF membranes (Roche). The membranes were incubated with skimmed milk for 1 h and then they were cut into strip-shaped blots. The blots were incubated with primary antibodies at 4 °C overnight followed by horse radish peroxidase-conjugated antibody for 1.5 h. Blots were visualized with an enhanced chemiluminescence solution (Sbjbio, Nanjing) and results were analyzed with ImageJ 1.52 software. Information for antibodies are listed in Table 2.

Cells in the logarithmic growth phase were trypsinized with 0.25% trypsin (Gibco) before being seeded into a 96-well plate for 24 h. CCK-8 reagent (Beyotime) was added to the wells and the cells were incubated at 37 °C for 1 h. Optical density was measured at 450 nm using a microplate reader (Perlong, Beijing).

Cells were seeded into culture dishes at a density of 500 cells/dish. They were cultured for 2 weeks, with the medium changed every 3 days. Following that, the cells were rinsed twice with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (TCI, Shanghai), and stained with 0.5% crystal violet (Macklin, Shanghai). A colony was defined as a cluster of ≥50 cells.

A wound healing assay was used to analyze migration. Cells were cultivated until they formed a confluent monolayer, then a wound in the center was created with a sterile pipette tip followed by another 24 h of culture. Results were observed under a microscope (magnification × 100, Olympus).

The cell pellet was gently mixed with pre-chilled 70% ethanol and incubated overnight at 4 °C. Then cells were centrifuged and washed in ice-cold PBS. Propidium iodide stock solution and RNase A solution were added to the staining buffer provided with the kit (Yeasen, Shanghai) and mixed. The above-prepared staining solution was added to cells followed by incubation at 37 °C for 30 min in the dark. Flow cytometer (BD FACS Calibur, USA) and FlowJo7.6.1 software was used to determine the cell cycle distribution.

CARD9 wild-type and mutant-type luciferase expression vectors containing HOXB5 binding sites were created and co-transfected into cells along with HOXB5 plasmids and empty plasmids. The Dual-luciferase Reporter Assay System (Promega) was used to measure luciferase activity, which was normalized to Renilla luciferase activity.

The association between CARD9 and HOXB5 was examined using a ChIP kit (Amylet, Wuhan, China). The cells were treated with 16% methanol, lysis buffer, and sonicated. The cells were then incubated overnight with an anti-HOXB5 or lgG antibody. Following incubation, protein A/G beads were used to harvest the protein-DNA complex and 5 mmol/l NaCl was used to retrieve the DNA. The level of CARD9 was determined using PCR.

The levels of CARD9 and HOXB5 in ovarian cancer, as well as their association with overall survival, were investigated using GEPIA. The correlation analysis between CARD9 and HOXB5 gene levels was based on the TCGA database. The binding location between CARD9 promotor and HOXB5 was obtained using the JASPAR database. GraphPad Prism 8.0 was utilized for statistical analysis. The data is presented as the mean ± SD (n ≥ 3). Statistical significance was determined via Student’s t-test and ANOVA with Tukey’s post-hoc test. P < 0.05 was considered to be statistically significant.