Patients and samples
The study population consisted of women referred to Reproductive Medicine Center of Shanxi Women and Infants Hospital, Taiyuan, China between June 2016 and December 2016. All subjects were Han ethnic, from the Shanxi Province, in north China. The study was approved by the Ethics Committee of Shanxi Women and Infants Hospital, and all the participants signed a written informed consent for participation in this study. The blood and follicular fluid samples were obtained from 26 PCOS patients and 30 healthy controls. Diagnosis of PCOS was based on Rotterdam Criteria , including oligo-ovaluation and/ or anovulation, excess androgen activity, and ultrasound image of polycystic ovaries. Patients with endometriosis, congenital adrenal hyperplasia, hypothyroidism, androgen-secreting tumors, Cushing’s syndrome and other systemic diseases were excluded from the study. The controls were infertility patients due to tubal and/or male factors with regular menstrual cycle. All participants accepted endocrine tests and other routine checks and the results are listed in Table1.
Controlled ovarian hyperstimulation protocol
Both PCOS patients and control patients received in vitro fertilization and embryo transfer (IVF-ET) treatments, following standard operation procedure (SOP). All women underwent controlled ovarian hyper-stimulation (COH) with gonadotropin releasing hormone (GnRH) agonist long protocol commenced pituitary suppression with leuprolide acetate (Dipherelin 0.1mg, GenSci Company, Chang Chun, China) at a dose of 0.05mg/d, during the mi-luteal phase of the preceding cycle. Complete pituitary suppression was comfired by a serum FSH level<5mIU/ml, LH level<5mIU/ml, E2 level<50pg/ml, bilateral antral follicle diameter <5mm, endometrial thickness ≤5mm. Urofollitropin (LiZHu, China) were used at doses ranging between 75IU/day and 300IU/day in accordance with patient age, body mass index, size and number of antral follicles, and serum basic FSH level. The dosage of urofollitropin was adjusted according to ovarian reponse, which was assessed by ultrasound and serum E2 levels. Recombinant human horogonadotropin-afa solution (hCG, Merck Serono S.p.A, Italy) was administered subcutaneously at the 250μg dosing level when at least two follicles with ≥18mm average diameter were detected. Oocyte retrieval was performed under the guidance of transvaginal ultrasounds 34 to 36 hours after the hCG injection. Human granulosa cells (GCs) were obtained from follicular fluid at the same time.
After oocyte retrieval, all follicular fluids from each patient were pooled and stored in a tube. The GCs were parade and cultured as described previously . Briefly, the aspirated follicular fluid was centrifuged at 1000g for 10 minmutes after removal of oocytes. The cell pellet was resuspended in 1ml phosphate buffer saline (PBS). Then, the suspension was overlayed on 1ml Ficoll, and centrifuged at 800g for 30 min. GCs were aspirated from the interface and washed a few times with PBS. Next, the isolated and purified GCs were cultured in Dulbecco’s modified Eagle medium/nutrient mixture F12 Ham medium (DMEM/F12, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, TBD, Tianjing, China), at 37℃ with 5% CO2 and 95% humidity.
Human granulosa-like tumor cell line, KGN cells were from the American Type Culture Collection (ATCC, VA, USA), which maintained the physiological characteristics of ovarian cells. The cells were grown in (DMEM/F12, Invitrogen, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, TBD, Tianjing, China), at 37℃ with 5% CO2 and 95% humidity. The miRNA-132 mimics, inhibitor, and negative control (NC) were designed and synthesized by Qiagen (Valencia, California). Foxa1-siRNA, Foxa1-pcDNA3.1, pcDNA3.1 empty vector and negative control (si-NC) were designed and synthesized from Gene-Pharma (Shanghai, China). The cells were seeded in 6-wel plates (2×105 cells/well) 1day before transfection to reach the confluency of 90%, and then the medium was replaced by serum- and antibiotic-free medium. Then, miR-132 mimic, miR-132 inhibitor, and siFoxa1 were transfected at a final concentration of 50 nM using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, California) following the manufacturer’s protocols. At 36 hours after transfection, the cells were collected for the following assays.
Cell proliferation assay. Cell proliferation was assessed using the CCK-8 method. For the effect of miRNA-132 or Foxa1 on proliferation, cells transfected with miRNAs or siRNAs were plated in 96-well plates at 5×103 cells/well. Cell proliferation was detected at 24, 48, and 72h after transfection using CCK-8 at 45nm according to the protocol of the manufacturer’s instructions (Beyotime Institute of Biotechnology, Shanghai, China) at 37℃ for 4h.
Tunel Assay. Granulosa cell were cultured directly on coverslips. After the treatment, the granulosa cells’ apoptosis was determined by TUNEL staining using an TUNEL cell apoptosis detection kit (KeyGEN BioTECH, Nanjing, China), according to the manufacturer’s recommendations. Images were taken using an Olympus microscope (BX40, Tokyo, Japan). Five photos (magnification ×200) were taken randomly for each sample. Data are reported as the percentage of TUNEL-positive cells among the total number of cells as described. Each experiment was performed in triplicate.
RNA isolation and qRT-PCR assay. Total RNA of GCs was extracted using the RNeasy/miRNeasy Mini kit (Qiagen, Limburg, The Netherlands) according to the manufacturer’s instructions. Total RNA (2ng) was used for reverse transcription, using the one-step RT-PCT kit (Qiagen, Limburg, The Netherlands) following the manufacturer’s instructions. The primers for miR-132 were the exact sequence of mature miR-132. U6 was used as the internal control. They were purchased from Qiagen (Limburg, The Netherlands). The conditions for miRNAs as follows (40 cycles): 95℃ for 15min, followed 95℃ for 15seconds, and 60℃ for 1min. The primer sequences for Foxa1 and GAPDH were as follows: Foxa1 (sense, 5’-AGGGCTGGATGGTTGTAT TG-3’; antisense, 5’-GCCTGAGTTCATGTTGCTGA-3’); GAPDH (sense, 5’-GAAGGTGAAG GTCGGAGTC-3’; antisense, 5’-GAAGATGGTGATGGGATTTC-3’) was used as an internal control. All reactions were run in triplicate and gene expression was determined by 2-△△Ct.
Dual-Luciferase Reporter Assay. The Foxa1-3’ UTR and the Foxa1-mutated-3’UTR fragment were amplified by PCR. Then, the fragment was inserted into the pGL3 luciferase promoter vector (Promega, Madison, WI, USA) to develop the Luc-pGL3-Foxa1-3’UTR and Luc-pGL3-Foxa1-mut-3’UTR vectors. Cells in 24-well plates were contransfected with Luc-pGL3-Foxa1-3’UTR or Luc-pGL3-Foxa1-mut-3’UTR vector and miR-132 mimics or miR-NC using Lipofectamine 2000 regent (Invitrogen) according to the instructions of the manufacturer. The Renilla luciferase reporter vector was transfected as an internal control in each assay. At 48h post-transfection, firefly and Renilla luciferase activities were detected using a dual-luciferase reporter system (Promega, Madison, WI, USA). The results are expressed as relative luciferase activity (Firefly Luc/Renilla Luc). All experiments were performed 3 times in triplicate.
Western blot analysis. Western blot analysis was applied to evaluate levels of Foxa1. Briefly, the cells were collected and lysed on ice in RIPA lysis buffer (Beyotime, Beijing, China) with protease inhibitor according to the manufacturer’s instructions. The protein concentration of cell lysates was determined using BCA kit (Boster, China). Equal amounts of protein lysates (30μg each lane) was resolved by 10% SDS-PAGE, and then electrotransferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked with TBST containing 5% non-fat milk for 2h at room temperature, and then incubated with the specific antibodies at 4℃ overnight, including mouse anti-Foxa1 (1:1000; ab40868) monoclonal antibodies and mouse anti-GAPDH (1:200; ab8245) monoclonal antibodies (All from Abcam, Cambridge, MA, USA). After washing with TBST, The membranes were further incubated with HRP-labelled goat anti-mouse IgG (1:2000; cat. no. BA1051; Boster biological engineering co., LTD; Wuhan; China) at 37℃ for 1h, followed by chemiluminescence for visualization with an ECL kit (KeyGEN BioTECH, Nanjing, China).
Statistical analysis. All statistical analyses were performed using GraphPad Prism 6.0 (GraphPad software, Inc., USA). For the analysis of endocrine tests, results were indicated as mean±standard error of the mean (SEM). Student’s t-test was performed for comparisons of the mean values of two groups; one-way ANOVA was used to determine differences among the mean values of more than two groups because the quantitative data followed a normal distribution.. Differences between groups were considered statistically significant at a p-value of < 0.05.