1, Patients specimens
Human ovarian GCs were collected from six PCOS patients (mean age, 29 ± 2.4 years old) and six control patients (mean age, 29.8 ± 3.6 years old). All patients with PCOS met the Rotterdam diagnostic criteria of 2003. Women in the control group had regular menstrual cycles and no abnormal reproductive endocrine diseases. These patients did not receive any medical treatment for three months. Informed consent was obtained and signed by all participants in this study.
2, Isolation of GCs and peripheral blood mononuclear cells (PBMC) isolation
Follicular fluid (FF) from 12 patients was collected and GCs were purified by density gradient centrifugation as follows: FF 300 g centrifugation for 10 min, and supernatant was discarded. Phosphate-bufered saline (PBS; servicebio, Wuhan,China) resuspended lower GCs. 50% Percoll (Biosharp, Wuhan, China) was slowly added to the cell suspension at a ratio of 1:1, centrifuged at 1800 r/min for 20 min, the cell layer was sucked with Pasteur pipette. Then incubated with red blood cell lysate (Biosharp) for 5 minutes, and cleaned with PBS.
To isolate PBMC, 4ml peripheral blood from healthy women was collected, and PBMC were extracted by density gradient method on Ficoll-Paque (GE Life, Sweden).
3, Cell culture, transfection, and treatment
Human granulosa cell-like tumor cells (KGN), acquired from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, were cultured in Dulbecco modified Eagle Medium/Nutrient mixture F-12 (DMEM/F12; Gibco, Grand Island, NE, USA) with 10% fetal bovine serum (FBS; Gibco) and 1% antibiotics (mixture penicillin, streptomycin; Gibco). Human mononuclear Cell line THP-1 obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 (Gibco) medium supplemented with 10% FBS and 1% antibiotics. Human PBMC was also cultured in RPMI-1640 medium with 10%FBS, 1% antibiotics and purified anti-human CD3 (5 ug/ml, BD Farmingen, USA), anti-human CD28 (1 ug/ml, BD) and human IL-2 (10 ng/ml, BD). The cells were cultured at 37℃ under a humidifed atmosphere with 5% CO2.
For AOC4P overexpression, KGN cells were infected with AOC4P overexpression lentivirus and empty vector designed and constructed by GeneChem (shanghai, China). After 48h, the cells were selected with puromycin (2 ug/ml, Biosharp) for 2 weeks to establish stable expression cell lines.
The cells were treated with dihydrotestosterone (DHT, 500 ng/ml; Sigma) for 24 h to stimulate a high androgen environment as a PCOS model. This is a common model used in some previous studies[21–23].
4, Total RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)
A SteadyPure Universal RNA Extraction Kit AG21017 (Accurate Biotechnology (Hunan) Co.,Ltd.) was used to extract total RNA according to the instructions. The RNA was reversed into cDNA by Bio-Rad PCR instrument with reverse kit (Accurate). QPCR Green Premix Pro Taq HS qPCR reagent (Accurate) was used for real-time fluorescence quantification in the Roche RT PCR system. PCR amplification conditions: 95 ℃ 60 s, 95 ℃ 5 s, 60 ℃ 30 s, a total of 40 cycles. Gene expression was quantified by 2−△△Ct method, GAPDH was selected as the reference gene. Primer sequences are shown in Table S1.
5, Western blotting assay
Protein extraction and western blottig assay were performed as described previously[24]. Total proteins were extracted from cells using RIPA lysates (Beyotime, Shanghai, China) containing protease inhibitors (MedChemExpress, NJ, USA) and phosphorylase inhibitors (MedChemExpress). Protein concentration was measured with BCA kit (Beyotime). About 30 µg protein was electrophoretized in SDS polyacrylamide gel and transferred to 0.45 µm PVDF membrane (Millipore, MA, United States). After blocked membrane with 5% skim milk, it was incubated overnight with primary antibody at 4 ℃. Then incubated with HRP-linked secondary antibody at room temperature for 1 h and analyzed by the Chemiluminescence Western Detection System (Bio-Rad, Hercules, CA, USA). Antibodies against JNK(1:1000; abmart, shanghai, China), p-JNK (1:1000; abmart), Bcl2 (1:1000; Proteintech, Wuhan, China), Bax(1:1000; Proteintech), caspase3 (1:1000; Proteintech), P65༈1:1000; abmart༉,p-P65 (1:1000; Cell Signaling Technology, Boston, USA), ERK1/2 (1:1000; Proteintech), p-ERK1/2 (1:1000; Affinity, Jiangsu, China), IL1-β༈1:1000; Proteintech༉, IL18༈1:1000; Proteintech༉, β-actin (1:5000; abmart), NLRP3༈1:1000; abmart༉were used according to the manufacturer's instruction.
6, Co-culture model
THP-1 cells or PBMC were inoculated into 12-well plates, and THP-1 cells were treated with 100 ng/mL phorbol 12-myristate 13-acetate (PMA; Sigma-aldrich) for 24h to differentiate into M0 macrophages. Transwell cells were placed in 12-well plates to form upper and lower chambers, which were connected by 0.45µm polycarbonate membrane. KGN cells overexpressing AOC4P or NC group were laid in the upper chamber. After co-culture for 48 h, lower compartment cells were collected for flow cytometery analysis, and culture supernatant was collected for the determination of inflammatory factors.
7, Cell proliferation assay
KGN cells from the overexpressing AOC4P group or the NC group were inoculated into 96-well plates with 3000 cells/well, and cell proliferation was determined according to CCK-8 assay kit (Biosharp). After 24, 48, and 72 h, adding 10µL CCK-8 solution to each well of the 96-well plate. After incubation at 37℃ for 1 h, the absorbance of the cells was detected at 450 nm by a microplate reader.
8, Flow-cytometery assay (FCM)
When detecting macrophages, the cells were digested by trypsin (Biosharp) and washed once with buffer, and the cell precipitation was suspended with 200ul buffer. 5ul PerCP/Cyanine5.5 anti-human CD86 (BD Famingen, USA) and PE anti-human CD163 (BD Famingen, USA) antibodies were added to each well, and incubated at 4℃ for 30 min. Then, the buffer was washed once. Using 200ul buffer to resuspend the cells and analyzing it with Beckman Coulter Cytofex Flow cytometer.
Th1, Th2 and Treg cells were tested separately, and Th1 and Th2 were tested using a leukocyte activated cocktail (BD Famingen, USA) for 6 hours in a CO2 incubator humidified at 37°C. Cells were then stained with FITC anti-human CD4 antibody and PE anti-human CD25 antibody in darkness at 4°C (each 5 µL/Test, BD Farmingen, USA). The cells were fixed and permeated with Fix/Perm (BD Pharingen, USA) according to the reagent manufacturer's instructions, and the intracellular factors were stained 30 minutes later. APC anti-human IL-4, BB515 anti-human IFN-γ, and APC anti-human foxp3 antibody (each 5 µL/Test, BD Farmingen, USA) were added and incubated in darkness at 4℃ for 30 min. After buffer washing and re-suspension, detection was performed. FITC, PE, APC and BB515 mouse IgG were used as homotypic controls.
All datas were analyzed using FlowJo Software (Version 10.6.2)
9, Enzymelinked immunosorbent assay (ELISA)
The levels of IL10, transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α) in cell culture supernatant were determined by ELISA kits (abclonal, Shanghai, China) for human. Each sample was measured in duplicate.
10, Statistical analysis
SPSS 25.0 was used for statistical analysis. Student's T test was used to analyze statistical significance. A value of P < 0.05 was considered statistically significant and data from 3 independent samples were shown in the figure and expressed as mean ± standard deviation (SD).