Clinical samples

According to the Rotterdam criteria (2004), patients were diagnosed as PCOS with at least two of the following criteria; (1): polycystic ovaries, (2): anovulation and/or oligo-ovulation, (3): signs of clinical and/or biochemical hyperandrogenism with the exclusion of congenital adrenal hyperplasia, Cushing's syndrome and androgen-secreting tumors. During the period of April 2019 and December 2020, a total of forty-five patients (fourteen patients from Yantai Yuhuangding Hospital and thirty-one patients from Qingdao Women and Children’s Hospital) who satisfied the above criteria were classified as PCOS, and sixty-five women (eighteen patients from Yantai Yuhuangding Hospital and forty-seven patients from Qingdao Women and Children’s Hospital) were recruited as the non-PCOS group who sought assisted reproductive techniques primarily due to male factors or tubal problems. The participants were treated with in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). This study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (Qingdao Women and Children’s Hospital and Yantai Yuhuangding Hospital). We obtained written informed consent from all participants. The clinical characteristics of all patients are exhibited in Table 1 and Table 2.

Table 1 Comparison of clinical characteristics and endocrine parameters in PCOS and non-PCOS patients for mRNA analysisTable 2 Comparison of clinical characteristics and endocrine parameters in PCOS and non-PCOS patients for miRNA analysisCumulus granulosa cell isolation

The follicles were aspirated by transvaginal puncture under ultrasound echo-guidance 36 h after hCG injected, and then the cumulus-oocyte complex (COC) was obtained. Cumulus granulosa cells surrounding a single oocyte were discreetly isolated by using a sharp needle. After rinsing in phosphate-buffered saline (HyClone, USA) three times, the GCs were stored at -80℃ until RNA extraction. The GCs isolated from the 14 PCOS and 18 control patients were used in miRNA qRT-PCR. The cumulus granulosa cells isolated from other patients (31 PCOS and 47 control patients) were used in mRNA qRT-PCR.

Cell lines and cell culture

KGN cells (RCB1154, RIKEN, Japan) were cultured in DMEM/F-12 medium (SparkJade Science, China) supplemented with 10% (v/v) fetal bovine serum (FBS; PAN Biotech, Germany) and 1% penicillin-streptomycin solution (SparkJade Science, China). IOSE80 cells (BNCC340318) and COV434 cells (BNCC338036, BNCC, China) were cultured in DMEM (SparkJade Science, China). 293 T cells were kindly donated by Professor Shen Wei from Qingdao Agricultural University. Cells were cultured in a humidified atmosphere with 5% CO2 at 37℃.

Target prediction and bioinformatics analysis

The targeted miRNA of circ_0043532 was predicted by Circinteractome (https://circinteractome.nia.nih.gov/mirna_target_sites.htmL) and starBase (http://starbase.sysu.edu.cn/index.php). And the targeted mRNA of miRNAs were predicted by miRWalk 2.0 (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/) which included miRNA-mRNA interaction information produced by several established miRNA prediction programs (i.e., RNA22 (https://cm.jefferson.edu/rna22/Interactive/), miRWalk, TargetScan (http://www.targetscan.org/), miRanda (http://www.microrna.org/microrna/home.do), RNAhybrid (https://bibiserv.cebitec.uni-bielefeld.de/rnahybr)) on 3' UTRs of all known human genes (p ≤ value 0.05) [21].

Cell transfection

The mimics, inhibitors and the negative controls (NCs) were designed and synthesized by Genepharma (Shanghai, China). Circ_0043532 sequence was cloned and inserted into vector pEX-3 (GenePharma, Shanghai, China) to over-express circ_0043532. We seeded about 5 × 104 KGN cells/well in a 6-well plate with DMEM/F12 added with 5% FBS. The KGN cells reached 70%-90% confluence after 24 h, then starved for 12 h with only DMEM/F12. And then the miRNAs, inhibitors and NCs were transfected into the KGN cells by Lipofectamine 2000 reagent (Invitrogen, USA) based on the manufacturer’s protocols. The Sequence of mimics, inhibitor and NC were listed in Table S1. Next, the transfected cells were incubated for 24 h until the transfection efficiency was determined by RT-qPCR.

Estradiol assays

The transfected-KGN cells were cultured in DMEM/F-12 supplemented with 5% FBS, plus androstenedione (100 nM) as a substrate. After cultivating for 48 h, cell incubation medium (1 × 105 cells) was collected and centrifuged, the supernatant was extracted. The secretion of estradiol was determined by enzyme-linked immunosorbent assays (ELISA) kit (KGE014, R&D Systems, USA). The ELISA was performed as the manufacturer’s protocol. Next, the absorbance was measured at 450 nm and 570 nm through a microplate reader (BioTek Instruments, USA). Each experiment was repeated three independent times. Four parameter logistic (4-PL) curve fit was used to calculate the results of the samples.

RNA extraction and qRT-PCR

Total RNA was extracted from the cultured cell lines or granulosa cells using miRcute miRNA Isolation Kit (TIANGEN, China) following the manufacturer’s instructions. The quality and concentration of RNAs were determined by QuickDrop (Molecular Devices, USA). Only final products with absorbance ratios A260/A280 between 1.8 and 2.0 were recruited to be eligible for further experiments. Specific divergent primer spanning the back-splicing sites of circRNA was designed in Primer-BLAST (NCBI, USA). The SPARKscript II RT Plus Kit (SparkJade Science, China) and qPCR SYBR Green Pro Taq HS (Accurate Biotechnology, China) were used for mRNA and circRNA qRT-PCR. The miRcute Plus miRNA First-Strand cDNA Kit (TIANGEN, China) and miRcute Plus miRNA qPCR Kit (TIANGEN, China) were used to detecting the expression level of miRNAs. qRT-PCR was performed with QuantStudioTM5 (Applied Biosystems, USA). GAPDH was chosen as the internal reference for the detection of mRNA and circRNA expression levels, while miRNA levels were normalized by U6. Each sample in each group of qRT-PCRs was run in three times and the relative expression of each gene was calculated by 2−ΔΔCT method. All primers used in this study were compounded by TSINGKE Biological Technology (Beijing, China). The sequences of paired primer were listed in Table S2.

Dual-luciferase reporter assay

The wild type circ_0043532 containing the predicated target sites for predicted miRNAs (miR-576-5p, miR-1270, miR-142-5p) (WT-circ_0043532) and the mutant sequences (MUT-circ_0043532) were inserted into pmirGLO luciferase reporter vectors (GenePharma, Shanghai, China), respectively. The 3’-untranslated region (3’UTR) of CYP19A1 containing the predicted binding site for predicted miRNA (miR-1270) (CYP19A1-3’UTR-WT) and the mutant sequences (CYP19A1-3’UTR-MUT) were also inserted into pmirGLO luciferase reporter vectors (GenePharma, Shanghai, China), respectively. 293 T cells were seeded in 48-well plates and cultured overnight then co-transfected with a luciferase reporter vector and miRNA mimics (GenePharma, Shanghai, China) by using Lipo2000 reagent (Invitrogen, Carlsbad, USA). After 48 h incubation at 37℃ with 5% of CO2, luciferase activity was detected using the Dual-luciferase Reporter Assay Kit (Vazyme, Nanjing, China) following the manufacturer’s protocol. The results were expressed as relative luciferase activity (Firefly Luciferase/ Renilla Luciferase).

Western blotting

After transfection for 48 h, KGN cells were harvested and lysed on ice with RIPA lysis buffer (Beyotime, China) complemented with Phenylmethanesulfonyl fluoride (PMSF, Beyotime, China). Each protein sample was detached using 8% SDS-PAGE gel and then transferred onto a PVDF membrane. These membranes were blocked in 5% BSA (Solarbio, China) for 1.5 h at room temperature and incubated with the following primary antibodies specific against CYP19A1 (ab18995, Abcam, Cambridge, MA, USA) or β-actin (Boster, China) all night at 4℃. Next these membranes were washed and incubated with secondary antibody (HRP-labeled Goat Anti-Rabbit IgG(H + L), A0208, Beyotime, China) at room temperature for 1.5 h. After washing, the bands were visualized by BeyoECL Plus (Beyotime, China). β-actin was as the endogenous control.

Statistical analysis

All data in this study were presented as mean ± standard deviation (SD). GraphPad Prism version 8 was employed for data analysis. The Gaussian distribution of the continuous variables was tested by the Kolmogorov-Smirnov statistic. Student’s t-test was applied to analyze the differences, and a p-value < 0.05 was considered as statistically significant.