This study was approved by the Ethics Committee of the Affiliated Hospital of Guilin Medical College, and all samples were obtained with informed consent from patients. The present study included 6 women with PCOS and 6 women without PCOS who underwent in vitro fertilization-embryo transfer (IVF-ET) in the Reproductive Center of The Affiliated Hospital of Guilin Medical College from January 2020 to June 2020. For the PCOS group, the Rotterdam Consensus (2004) was used to define the phenotype of the patients with PCOS who met at least two of the following criteria: (1) sparse ovulation or anovulation (menstruation more than 35 days or menstruation less than 8 times per year); (2) signs of hyperandrogenism (evidence of increased circulating total testosterone of more than 45 ng/dL or hypertrichosis); and (3) polycystic characteristics with more than 12 follicles, all of which were less than 9 mm in diameter, as shown by ultrasonic monitoring of ovarian morphology. Other causes, such as delayed adrenocortical hyperplasia and adrenal androgen-secreting neoplasms, were excluded. The inclusion criteria of the control group were normal ovarian morphology and function, regular menstrual cycle (26–35 days), normal androgen level (< 45 ng/dL), and 6–10 antrum eggs on both sides. The exclusion criteria for both groups were as follows: age over 35 years, ovarian cysts and tumours, history of ovarian surgery or radiotherapy and chemotherapy, endometriosis, hyperprolactin, thyroid dysfunction and chromosomal abnormalities. Patients who took drugs affecting hormone levels or glucose and lipid metabolism within six months prior to treatment were also excluded.
Isolation and characterization of FF-derived exosomes
Baseline hormone levels were measured before induction of ovulation all patients, and ovulation was stimulated by a short-effect and long-duration regimen in the luteal phase. After 36 h of triggering by human chorionic gonadotropin (hCG) injection (2000 U, Lizong Medicine Factory, Zhuhai, China), serum-free FF with bilateral follicles with a diameter of > 15 mm was collected by aspiration. After centrifugation at 1500 g for 15 min, the supernatant was taken for preservation. The exosomes of FF were extracted by ultracentrifugation in strict accordance with the instructions of the Qiagen exoEasy Maxi Kit (Qiagen, Hilden, Germany). The positive marker proteins CD63 (#A19023; 1:1000 dilution, Abcam, Cambridge, UK) and CD9 (#19027; 1:1000 dilution, Abcam) were detected by Western blot assays.
The morphology of exosomes was identified by transmission electron microscopy (TEM). The phosphate-buffered saline (PBS)-dissolved exosomes were prefiltered with a 0.2 µm aqueous phase filtration membrane. The precipitation sample of 20 µl of PBS was resuspended and dropped onto the copper wire and stained with 2% phosphotungstic acid (pH 5.0) for 60 s. The morphology of exosomes was observed by transmission electron microscopy (JEOL, Tokyo, Japan). The particle size and concentration of exosomes were analysed by nanoparticle tracking (NTA), and the exosome suspension was diluted to 800 mL with PBS (1:200) and injected into the sterile sample pool. The Browne motion trajectory of captured exosomes was observed by a Nanosight NS300 on the computer, and the diameter of exosome particles was measured by Zetaview in NTA2.1.
Exosomal RNA analyses
RNA sequencing (RNA-seq) of FF-derived exosomes was performed by Aksomics (Shanghai, China). The total RNA samples were examined and quantified by agarose electrophoresis and Nanodrop, and the library quality was determined by an Agilent 2100 Bioanalyzer. Sequencing libraries of different samples were mixed and denatured into single-stranded DNA with 0.1 M NaOH. The original amplified clusters were captured on the Illumina Flow cell and cycled on an Illumina NextSeq 500 sequencer according to the instructions. The data generated by Illumina NextSeq 500 are raw sequencing data, which were evaluated by quality control to determine whether the sequencing data can be used for subsequent data analysis. Crosstalk data from crosstalk and crosstalk removed from reads were compared with Bowtie to the reference genome GRCh38 after quality control and were analysed statistically if the crosstalk results were favourable. RNAs with counts per million mean ≥ 1 were considered to be expressed in groups and statistically analysed. EdgeR was used for analysis of intergroup differences. Differentially expressed RNAs were screened by log2|fold change (FC)|≥ 0.585 and P value < 0.05 for cluster analysis. The reagents used above included the NEBNext ® Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, USA); the RiboZero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre, an Illumina Company, San Diego, USA); the NEB Multiplex Small RNA Library Prep Set for Illumina; and the TruSeq Rapid SR Cluster Kit (#GD-402-4001, Illumina, San Diego, USA). For the convenience of description, we named 6 exosome samples in the control group C1-C6 and 6 exosome samples in the PCOS group T1-T6. To explore whether the differentially expressed genes in the FF-derived exosomes between the control and PCOS groups are involved in glycolysis of GCs, we screened the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for the combined enrichment of differentially expressed miRNA and mRNA with glucose metabolism as the core via differential enrichment analysis. GO term and KEGG pathway enrichment (http://www.genome.jp/kegg/) analysis of differentially expressed genes (DEGs) was achieved by clustering Profiler R.
Cell culture and transfection
KGN cells were a generous gift from the Institute of Applied Anatomy and Reproductive Medicine, Hengyang Medical College, University of South China. The KGN cells were cultured with high glucose Dulbecco’s modified Eagle’s medium (DMEM, #11965084, Gibco, Waltham, USA) with 10% foetal bovine serum (FBS, #10099141C, Gibco) in a humidified atmosphere with 5% CO2 at 37 °C. For the in vitro cellular model of PCOS, KGN cells were cultured with high glucose DMEM with 10% foetal bovine serum (FBS) and 100 nM testosterone (#M6105, AbMole BioScience, Houston, USA) in a humidified atmosphere with 5% CO2 at 37 °C.
The miR-143-3p mimics, miR-155-5p mimics, miR-143-3p inhibitor and miR-155-5p inhibitor were purchased from RiboBio (Guangzhou, China). Cell transfection was performed using the riboFECT CP Transfection Kit (#C10511-05, RiboBio) when the cells reached 50–60% confluence. The transfection efficiency was evaluated by a riboMONITOR Transfection Indicator Kit (#C10410-5, RiboBio) at 12 h after transfection.
Luciferase reporter assay
A wild-type (WT) or mutant (MUT) mRNA fragment was constructed and inserted downstream of the luciferase reporter gene of psiCheck2 (#C8021, Promega, Madison, USA). When KGN cells reached 50–60% confluence, miRNA mimics (100 nM) or the mimic control and psiCheck2-mRNA-3′ untranslated region (3′ UTR)-MUT (15 mg/L) or psiCheck2-mRNA-3′ UTR-MUT (15 mg/L) were cotransfected into KGN cells by ExFect Transfection Reagent (#T101-01, Vazyme, Nanjing, China). At 36 h after transfection, the activities of Renilla luciferase and firefly luciferase were determined by the Dual-luciferase Reporter Assay System (#E1910, Promega). Renilla luciferase activity was normalized to firefly luciferase activity, and relative luciferase activity was calculated.
Quantitative real-time PCR (qPCR)
RNA extraction was performed by using the TRIzol (#15596026, TRIzol™, Thermo Fisher, Waltham, USA) method according to the manufacturer’s protocol, and the stem-loop method was used to reverse transcribe miRNA into cDNA. Total RNA was reverse transcribed into cDNA according to HiScript III-RT SuperMix for qPCR (#R323-01, Vazyme) instructions. qPCR analysis was performed on an Applied Biosystems QuantStudio (#A34321, Thermo Fisher Scientific) by using ChamQ Universal SYBR qPCR Master Mix (#Q711-02, Vazyme). β-actin was used as an internal reference, and the gene expression level was calculated by the comparative 2−△△Ct method. The primer sequences are shown in Table 1.
Western blot analysis
The protein samples were extracted using ice-cold radioimmunoprecipitation assay buffer (#89901, Thermo Fisher Scientific). The protein concentration was determined by a bicinchoninic acid assay kit (#PC0020, Solarbio, Beijing, China). Denatured proteins were separated by 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (#1610173, TGX FastCast Acrylamide Kit, Bio-Rad, Hercules, USA) and then transferred to polyvinylidene membranes. The membrane was blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% skim milk powder for 2 h. After the membrane was washed with TBST, it was incubated with primary antibodies against hexokinase (HK) 2 (#A20829, 1:1000 dilution, ABclonal, Woburn, USA), pyruvate kinase muscle isozyme M2 (PKM2, #4053, 1:1000 dilution, Cell Signaling Technology, Danvers, USA), lactate dehydrogenase A (LDHA, #3582, 1:1000 dilution, Cell Signaling Technology) and β-actin (#3700, 1:1000 dilution, Cell Signaling Technology) overnight at 4 °C. Then, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H + L) (#AS003, 1:5000 dilution, ABclonal) or HRP-conjugated goat anti-mouse IgG (H + L) (#AS014, 1:5000 dilution, ABclonal) at room temperature for 2 h. Finally, the protein chromogenic bands were detected by the Tanon 5500 chemiluminescence imaging system by using an enhanced chemiluminescence kit (#CW0049, CWBIO, Boston, USA) and analysed by ImageJ (https://imagej.nih.gov/ij/).
Determination of pyruvate and lactate levels
At 36 h after different treatments, pyruvate and lactate concentrations in culture medium were determined by a pyruvate determination kit (#A081-1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and lactate test kit (#A019-2-1, Nanjing Jiancheng Bioengineering Institute), respectively. The culture media of the different processed cells were collected in a 1.5 ml centrifuge tube and photographed (Table 2).
At 36 h after different treatments, cells were stained with a Cell-Light EdU Apollo567 In Vitro Kit (#C10310-1, RiboBio) according to the manufacturer’s instructions. EdU-positive signals were detected by the EVOS (#AMF7000, Thermo Fisher Scientific) imaging system (40X) and were analysed by ImageJ (https://imagej.nih.gov/ij/).
Data were analysed using GraphPad Prism 8.0 and IBM SPSS Statistics 26. The data are presented as the mean ± standard deviation (SD). Significant differences between/among different treatment groups were analysed by using an unpaired t test or one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison tests. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.
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