The human granulosa-like tumor cell line (KGN) used in this study was obtained from Ji he Biotechnology Co., LTD (Shanghai, China). The KGN was cultured in medium (C11995500BT, Gibco, USA) with 10% fetal bovine serum (FBS) (10270–106, Gibco, USA) and 1% penicillin–streptomycin (SV30010, Hyclone, USA) and grown in 5% CO2 atmosphere at 37 °C. The Plasmid YB-1 RNAi were obtained from Genechem (Shanghai, China). Si-MALAT1, miR-211-5p inhibitor, si-FOXO3 and their relative NC were obtained from GenePharma (Shanghai, China). Cells were plated into 6-well plates (TCP001006, Jet Bio-Fil, China) the day before transfection and then transfected with plasmids or siRNA using Lipofectamine 2000 reagent (11668019, Thermo Fisher Scientific, USA) at 70% or 50% cell fusion After 48 h, cells were used for further experiments.
The establishment of the POF model in vitroKGN were plated into 6-well plates 12 h prior to treatment. When approximately 30% cell confluence was reached, 100 μm of H2O2 was added directly for 24 h for induction of cellular senescence.
Patient group and GCs acquisitionThe serum samples and GCs utilized in this research were acquired from healthy volunteers and POF patients at the Fourth Affiliated Hospital of Jiangsu University. All patients and healthy volunteers were informed, and written consent was signed. Each group had 10 patient samples. The research protocol was approved by Jiangsu University Ethics Committee.
Western blot analysisAfter total proteins were harvested, a western blot was performed according to standard methods to detect protein expression. Briefly, total proteins were separated by SDS-PAGE (P1200, Solarbio, China) and then transferred to polyvinylidene difluoride membranes (1620184, Bio-Rad, USA). After blocking, the membranes were first imprinted with YB-1 (ab12148, Abcam, UK), P53(WL01919, Wanleibio, China), P21(WL0362, Wanleibio, China), FOXO3 (WL02891, Wanleibio, China) or GAPDH (ab9485, Abcam, UK) primary antibodies for 12 h at 4 °C followed by membrane wash and secondary antibodies (#14708, Cell Signaling Technology, USA) incubation for 1.5 h. The protein band signals were detected by ECL chemiluminescent substrate (E412-01, Vazyme, China) in ChemiDoc™ XRS + with Image Lab™ software (Bio-Rad, USA).
Isolation and culture of BMSCsThe harvesting of BMSCs consisted of the following steps. First, bone marrow was harvested by flushing the tibia and femur of 5-week-old SD rats. Then, suspensions containing BMSCs were placed in α‐MEM (C12571500BT, Gibco, USA) containing 20% FBS and 1% penicillin–streptomycin cultured in a cell culture incubator. In the next step, the suspension containing unaffixed BMSCs was discarded after 73 h. Subsequently, the cells were passaged when they were 80–90% fused. Finally, BMSCs that were passaged for 3–7 generations were used for experiments or extraction of sEVs.
SEVs isolation, quantification, and characterizationSEVs isolation was performed using an exosome isolation kit (UR52121, Umibio, China). After removing cell fragments, the supernatant was added exosome concentration solution (ESC) in the proportion of 4:1. After blending, the mixed liquid was placed at 4 °C for 2 h. Then, sEVs precipitation is separated out by centrifugation at 10000 × g for 1 h. Then, the PBS-washed sEVs were purified with an exosome purification filter at 3000 × g for 10 min at 4 °C. Lastly, the sEVs were stored at − 80 °C for further investigation. BCA protein assay kit (CW0014, CWBIO, China) was used to determine the protein concentration of sEVs. The marker protein CD9 (ab236630, Abcam, UK) was detected by western blot analysis. Transmission electron microscopy (TEM; H-7800, Hitachi, Japan) and nanosight tracking analysis (220-Twin, Particle Metrix, GER) were used to measure the appearance and size of sEVs.
Measurement of intracellular ROS levelsThe Kit (S0033M, Beyotime Biotechnology, China) for measuring intracellular ROS levels was purchased from Beyotime Biotechnolog. Cells and ovary tissues of different groups were treated with DCFH-DA diluted according to the instructions at 37 °C for at least 30 min and then rinsed gently with PBS for three times. Finally, the images were captured immediately under Leica DM4 B & DM6 B Upright Microscope (Leica, GER) and the image capture software was LAS X.
Assay of cellular senescence (S-A-β-gal staining)The Cell Senescence S-A-β-galactosidase Staining Kit (C0602, Beyotime, China) was employed to assess S-A-β-gal activity. The S-A-β-gal staining efficiency was determined as the proportion of positively stained cells to the sum of cells in a single field of view. The images were captured immediately under Leica DM4 B & DM6 B Upright Microscope and the image capture software was LAS Core.
Real-time quantitative PCR (qPCR)Treatment of cells with TRIZOL reagent (15596–026, Thermo Fisher Scientific, USA) is the method of total RNA isolation. cDNA was obtained with a reverse transcription kit (KR118, TIANGEN, China and B532451, Sangon, China) in S1000 Thermal Cycler (1852196, Bio-Rad, USA). The qPCR primers were customized at Sangon Biotech (Shanghai, China). qPCR was used to analyze targeted RNA expression by utilizing SYBR PCR kit (B639271 and B532461, Sangon, China) and was performed in 7500 fast real-time qPCR system (4351106, Thermo Fisher Scientific, USA). The relative expression of RNA was performed using the 2-△△Ct method. The primer sequences were displayed in Supplementary Information: Table S1.
Data acquisition and analysis of LncRNA-MiRNA-mRNA networkYB-1-associated RIP-seq (GSE130781) and CLIP-seq (GSE150925) were collected from Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/), and ceRNA network was predicted in the online website of Starbase (https://rnasysu.com/encori/).
RNA immunoprecipitation (RIP) assayThe reagents used for the RIP experiments were EZ-Magna RIP kit (Millipore, Billerica, USA). The experimental steps were succinctly described as follows. First, RIP lysis buffer was applied to lysed HEK-293 cells. Second, PBS-washed magnetic beads were washed by RIP wash buffer, RNAs magnetic beads were conjugated with an anti-YB-1 antibody or negative control anti-IgG. Next, 100 μl cell lysate, 900 μl RIP buffer, and magnetic beads mixed continuously at 4 °C until overnight. Next, the magnetic beads combining YB-1-RNA were digested by proteinase K for 0.5 h at 55 °C. Then, the magnetic beads combining YB-1-RNA were treated with phenol and chloroform after a RIP buffer wash. Next, salt solution I, II, precipitate enhancer and anhydrous ethanol were added, and the RNA-containing liquid was placed at − 80 °C overnight. Finally, the RNA was obtained by centrifugation of the above liquid and dissolved in Dnase/Rnase-free ddH2O for qPCR analysis.
Pull-down assay with biotinylated miRNAHEK-293 cells were transfected with biotinylated miR-211-5p probe and the negative control probe customized at Genepharm (Shanghai, China). Then, cells were lysed with 550 μl lysis buffer containing protease inhibitor (78,430, Thermo Fisher Scientific, USA) and Rnase inhibitor (R0102, Beyotime, China). Cell lysates and Streptavidin-Dyna beads (65801D, Thermo Fisher Scientific, USA) were mixed overnight at 4 °C with constant rotation. Finally, RNA was extracted by adding 750 μl Trizol LS per sample and 250 μl water and then subjected to qPCR as explained above.
Luciferase report assayThe Phusion Mutagenesis kit (F541, Thermo Fisher, USA) was used to mutate the binding sites. cDNAs that contained the wild-type sequences WT-FOXO3 or mutated binding sequencesor MUT-FOXO3 with miR-211-5p in FOXO3 3′ UTR were cloned into the pGL4 luciferase reporter vector (Promega, USA). HEK-293 cells were co-transfected with the recombinant plasmid together with miR-211-5p mimics or mimics NC (synthesized from Genepharma, China). Then, the co-transfected cells were obtained in the Reporter Lysis Buffer after transfection for 24 h. The luciferase activity was assayed using the Dual-Luciferase Reporter Assay System (Promega, USA).
The establishment and treatment of the POF model in vivoSD female rats (five-week-old) purchased from The Experimental Animal Center of Jiangsu University were used for subsequent experiments after 7 days of acclimatization. Seventy-two rats were confirmed to have normal estrus cycles and randomly divided into four groups: WT, POF + PBS, POF + sEVs, and POF + si-YB-1 sEVs group. The establishment of POF rat model was accomplished by injection of CTX. The specific method was 50 mg/kg/d CTX injection for one day followed by 8 mg/kg/d CTX injection for 13 consecutive days (Yang et al. 2020a). Detailed animal information is provided in Supplementary Information: Animal protocol.
Location of BMSCs-sEVs in vivo or vitroIn vivo, BMSCs-sEVs were stained with fluorochrome Dil (C1036, Beyotime, China) and injected into normal rats 6, 12, 24, 48, and 72 h later the lung, heart, liver, kidney, spleen, ovary, uterus of the rats and the location of BMSCs-sEVs in vivo detected via IVIS® Lumina LT Series III (PerkinElmer, USA) (wavelength = 550 nm). Repeat each experiment three times. In vitro, sEVs were incubated with 1 μM PKH26 (mini26, Sigma-Aldrich, USA) for 10 min at room temperature before unbound dye was removed by centrifugation. Subsequently, the labeled sEVs were added to the culture medium of KGN cells. Cells were fixed with 4% paraformaldehyde (P0099-100 ml, Beyotime, China) after 24 h of treatment. Following DAPI staining (C1006, Beyotime, China) Leica TCS SP5 II laser scanning confocal microscopy (Leica, GER) was used to detect the uptake of PKH26-labeled sEVs by KGN cells.
Enzyme-linked immunosorbent assaySerum was extracted by centrifugation from blood samples of rats collected from the heart. The ELISA kits (BPE30597, BPE30608, BPE30083, BPE30623, Lengton, China) are used to measure levels of some hormones in the serum, including FSH, E2, AMH, and LH.
Immunohistochemistry (IHC)The rat ovary tissues were fixed, embedded, and sectioned. the slides were incubated with the anti-YB-1 or anti-FOXO3 at 4 °C overnight. Next, slides were submerged in enhancers and secondary antibody for 1 h. Then DAB (P0203, Beyotime, China) was utilized for the chromogenic reaction. Counterstain slides with hematoxylin (ST2067-20 g, Beyotime, China). Then dehydrated, cleared, and evaluated. Finally, differential fields were randomly selected under the light microscope to capture images. The images were captured immediately under Leica DM4 B & DM6 B Upright Microscope and the image capture software was LAS Core.
Assessment of reproductive potentialAfter two weeks of sEVs treatment, reproductive tests were performed. 6 rats in each group were mated with 3 sexually mature SD male rats. Mating success was determined by the presence or absence of sperm plugs. Each female rat was mated again after 3 weeks postpartum. Fertility levels, including pregnancy rate and number of offspring, were recorded for each group of rats. Female rats that had three consecutive positive sperm plugs but did not reproduce were ruled infertile.
Statistical analysisThe data were based on three independent experiments and were displayed with mean ± standard deviation. GraphPad Prism software 8.0 for Windows (GraphPad Software, USA) was implicated in data analysis. Student’s t test was applied to evaluate the statistical significance between the two groups. Differences between two or more groups were analyzed using one-way ANOVA. P < 0.05 was defined as a significant difference (*P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001) in this study.
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