2.1 Study Subjects
We recruited nine PCOS patients and nine patients with no-PCOS under the age of 35 who underwent in vitro fertilization and embryo transfer (IVF-ET) in the Huai'an Second People's Hospital between January 2018 and April 2019. We measured the subjects’ weight, height, and then calculated body mass index (BMI). Selection criteria for the PCOS group (refer to Rotterdam criteria (11)):1) rare ovulation and/or anovulation; 2) clinical manifestations of hyperandrogenism and/or biochemical changes; 3) polycystic ovary was found by ultrasound examination. Those who met 2 of the above 3 items were enrolled in the PCOS group. The no-PCOS group was subjects with normal ovaries function, no PCOS, no history of ovarian adverse reactions, no endometriosis, and receiving IVF-ET due to male factors. All patients voluntarily participated in this research project and signed informed consent. This study was approved by the Ethics Committee of the Huai'an Second People's Hospital.
2.2 Collection of serum
5 mL of fasting venous blood was collected on the morning of the third day of the menstrual cycle. Blood samples were centrifuged at 1500 r/min for 5 min to draw the upper layer of serum, aliquoted into a 0.5 mL EP tube, and stored in the refrigerator at -80 °C for testing.
2.3 Acquisition of follicular fluid
All subjects induced ovulation according to the GnRH-a/FSH/ HMG/HCG protocol. The dosage and time were adjusted based on the individual situation. 36 hours after HCG injection, the follicles of both ovaries were sucked through the posterior vaginal foramen under the guidance of ultrasound. The first tube of clear follicular fluid without blood staining was collected and centrifuged at 400x g for 10 min at room temperature. The supernatant was stored at -80 °C until detecting.
2.4 Collection, separation and purification of ovarian granulosa cells
We collected the discarded follicle puncture fluid after the separation of the ovarian crown complex, centrifuged it at 2000 rpm for 10 min, and then discarded the supernatant. The cells were resuspended in PBS and digested using 0.25% trypsin solution at 37℃ for 5 ~ 10min. After termination of digestion, the cell suspension was centrifuged at 2000 rpm for 10min and resuspended in PBS. Next, we added an equal volume of human lymphocyte separation solution (Tianjin Med Pacific Technology, China) to cells. After centrifugation at 2000 rpm for 20 min, the intermediate cell layer was drawn. The cells were washed with DMEM (Gibco, USA) containing 1% Pen-Strep (Invitrogen, USA) and 10% FBS, centrifuged at 1000 rpm for 10 min, and resuspended with DMEM. Trypan blue staining proved that the cell survival rate was greater than 90%. The cell density was adjusted to 2 × 105 / mL and fostered in a 37 °C, 5% CO2 incubator. After the cells adhered to the growth, they were rinsed once or twice every 24h and then replaced with fresh culture medium to further purify the mixed blood cells and other impurities.
2.5 Cell transfection
Ovarian granulosa cells in the PCOS group were randomly divided into three groups: si-negative control (NC) group (transfected with NC-siRNA), si-circ_0058063-1 group (transfected with circ_0058063-1-siRNA), si-circ_0058063-2 group (transfected with circ_0058063-2-siRNA). Cells were transfected by LipofectamineTM 2000 (Invitrogen, USA) reagent according to the manufacturer’s instructions.
2.6 Hormone levels detection
The concentrations of follicle stimulating hormone (FSH), testosterone (T), luteinizing hormone (LH), progesterone (P4) and estradiol (E2) were measured by radioimmunoassay. The concentration of aromatase was detected using an ELISA kit. The kits were provided by Xiamen Huijia Biological Technology (Xiamen, China).
After 24-hour transfection, the culture medium was replaced, and cells were continued to foster for 48 hours. The total RNA was extracted by Trizol (Invitrogen, USA), and the RNA quality was detected and quantified by ultraviolet spectrophotometry. The cDNA was synthesized using PrimeScript™ RT Master Mix (Takara, China), and qPCR was performed utilizing TB Green® Premix Ex Taq™ (Takara, China) according to the standard protocol. U6 and β-actin were used as internal reference controls. The relative expression of circ_0058063 and Aromatase mRNA was calculated using the 2−ΔΔCt method.
2.8 Western blotting
The total protein of the cell was extracted using RIPA buffer (Biyuntian, China). The protein concentration was assessed via a BCA kit (Biyuntian, China). Next, 50μl protein samples were taken for SDS-polyacrylamide gel electrophoresis to separate the target protein, and the membrane was transferred by the wet transfer method. We sealed the membrane with 5% skimmed milk powder for 2 hours, washed the membrane 3 times with PBST solution, and added primary antibodies (1:1000, CST, USA), including Cyclin A, Cyclin D1, Bcl-2, Bax, and GAPDH. Primary antibodies were incubated at 4 ℃ overnight. Then a secondary antibody (1: 3000) was added and incubated at 37 °C for 2 hours. The ECL kit obtained from Amersham Biosciences (Piscataway, NJ) was applied to visualize the immunocomplexes.
2.9 Colony formation assay
Cells were seeded at colony forming density (200 cells/ dish) in a 60-mm tissue-culture dish carrying 5 mL medium at 37 °C under 5% CO2. After 14 days, the colony formation rate was observed in each group. Cells were fixed with 4% paraformaldehyde for 15 min after discarding the medium. Next, cells were stained with 0.1% crystal violet for 20 min. In each well, five randomly fields were selected to calculate the number of colonies. Colony formation rate = (number of colonies/number of inoculated cells) × 100% (12).
2.10 Cell-Counting Kit-8 Assay
The cell viability was detected using CCK-8 (Biyuntian, China) based on the instructions. In short, after transfection for 48 hours, 10 μl CCK8 reagent was added to ovarian granulosa cells in each well. The cells were incubated at 37 °C for 4 hours. The microplate reader (Bio-Rad, USA) was used to detect the optical density (OD) at a wavelength of 450 nm.
2.11 Flow cytometry
Cell apoptosis was measured utilizing the Annexin V-FITC Apoptosis Detection Kit (Abcam, USA) as the manufacturer’s protocol. In short, we added pre-chilled 1xbinding buffer to resuspend the cells, and then added 10µl PI and 5µl Annexin V-FIT. Cells were stained for 15 ~ 25min in the darkroom. Flow cytometry (BD, USA) was used to detect the apoptosis rate.
2.12 The establishment of PCOS mouse model
Fifty 25-day immature female BALB/c mice were purchased from Wuhan University Center for Animal Experiment. Mice were randomly divided into PCOS group (n=40) and control group (n=10). Mice in the PCOS group were injected with 6 mg DHEA per 100 g body weight (purchased from Hangzhou Aladdin Information Technology Co., Ltd., China) every day for 20 consecutive days to establish a PCOS mouse model. The control group was injected subcutaneously with 0.4 mL of normal saline daily for 20 days. The fasting blood glucose and fasting insulin levels were measured 3 weeks after the injection. HOMA-IR = FI (mU/L) × FG (mmol/L) / 22.5 (13). Insulin resistance is defined as HOMA-IR> 2.6 (14). Thirty PCOS mice with HOMA-IR> 2.6 were randomly divided into si-NC group, si-circ_0058063-1 group and si-circ_0058063-2 group, with 10 mice in each group. Then 5 × 108 PFU/mL lentivirus containing si-circ_0058063 (GenePharma, China) or negative control vector was injected to the mice ovary. Mice were detected IR and then sacrificed 2 weeks after infection. Mice ovaries were collected for the further experiment. All animal experiments are in compliance with the ethical requirements of laboratory animals and laboratory animal management regulations of the Huai'an Second People's Hospital.
2.13 Immunohistochemistry assay
The Bax protein expression of mice ovaries was examined via immunohistochemistry. The ovarian tissue was taken for sectioning, deparaffinization, and hydration. After washing 3 times with PBS for 5 min each time, 3% H2O2 were added in the ovarian tissue to block endogenous peroxidase for 10 min at room temperature. Then Bax antibody (Wuhan Boster Biological Technology., Ltd, China) were added in the tissue after washing with PBS (3 times, 5 min each) and incubated overnight at 4 °C. Next, peroxidase-labeled goat anti-rabbit immunoglobulin (Ig) G secondary antibody (Wuhan Boster Biological Technology., Ltd, China) were added. Finally, DAB staining was conducted, and the results were observed using a microscope.
2.14 Statistical analysis
SPSS 21.0 software was utilized for statistical analysis. The data were expressed as mean ± standard deviation. The differences between multiple groups were compared with one-way ANOVA followed by LSD multiple comparisons. P<0.05 was considered statistically significant.