Clinical samples
We recruited twenty-four patients with and without PCOS who were undergoing in vitro fertilization-embryo transfer/intracytoplasmic sperm injection (IVF-ET/ICSI) at Shanghai Jiai Genetics and IVF Institute, in the period from January to June 2019. The Human Research Ethics Committee of the Obstetrics and Gynecology Hospital of Fudan University approved the study. Eleven patients were diagnosed as PCOS according to the Rotterdam revised criteria (32) (2 out of 3): oligo- or anovulation; clinical and/or biochemical signs of hyperandrogenism; and polycystic ovaries with exclusion of other etiologies (congenital adrenal hyperplasia, androgen-secreting tumors, Cushing’s syndrome). Thirteen participants, forming the non-PCOS group, were recruited because tubal factor infertility or male factor, who regular menstrual cycles (26-35 days), normal ovarian morphology and no clinical or biochemical evidence of hyperandrogenism. Patients with genetic diseases, thyroid diseases, a family history of type 2 diabetes mellitus within 3 months before the recruitment were excluded. Follicular fluid and granulosa cells from such patients were usually collected as clinical samples for the study of PCOS disease (33, 34). The clinical characteristics of PCOS and non-PCOS group women are shown in the Table 1.
Isolation of human GCs
Human GCs were collected from PCOS and non-PCOS patients undergoing IVF/ICSI. Oocyte retrieval was performed under the guidance of the transvaginal ultrasonography after human chorionic gonadotropin administration. After oocyte retrieval, follicular fluid was centrifuged at 2000 rpm for 10min, the precipitates were digested with hyaluronidase (80 IU/ml) (Sigma, St. Louis, Mo., USA) for 30 min at 37℃, then transferred into lymphocyte separation medium (LTS1077-1; TBD, Tianjin, China) and centrifuged at 1500 rpm for 10 min (35). GCs were isolated from the interface layer, then washed and resuspended in phosphate buffer saline (PBS) (GNM10010; Genom, Hangzhou, China). Owing to the difficulties in obtaining human sufficient quantities of GCs and in maintain primary cultures, fresh GCs cells were used for RNA or protein extraction immediately after pufification.
Cell culture
We used an immortalized human GCs line KGN for functional studies. The KGN cell line is a steroidogenic human granulosa-like tumor cell line. KGN cells were obtained from Shandong University and were cultured with phenol red–free DMEM/F12 (GNM11039; Genom) supplemented with 10% fetal bovine serum (10270-106; Gibco, NY) and 1% penicillin-streptomycin (C125C5; NCM, Suzhou, China) (36). KGN cells were cultured in a humidified atmosphere (5% CO2) at 37℃ and digested with 0.25% trypsin (containing EDTA) (GNM25200; Genom) when passaged.
Bioinformatics analysis
Potential target genes of let-7i (let-7i-5p) were predicted by three databases: Starbase (http://starbase.sysu.edu.cn/), Mirwalk (http://zmf.umm.uni-heidelberg.de/apps/zmf/mirwalk2/), and TargetScan (http://www.targetscan.org). There are 116 genes overlapping in all three databases. We evaluated the binding site strength. IMP1-3 were unverified targets with high binding scores.
Vector construction and transfection
Lentiviruses expressing IMP2 (lenti-IMP2) (31592-1; GeneChem, Shanghai, China) and the control lentiviruses (lenti-ctrl) (CON335; GeneChem) were transfected respectively into KGN cells at 70-80% confluency of cells using HiTransG P reagent (GeneChem). Puromycin (REVG1001; GeneChem) was used to select stably transfected clones according to the manufacturer’s protocol. Small interfering RNA (si-RNA) targeting IMP2 (si-IMP2) and siRNA control (si-ctrl) were purchased from GenePharma (Shanghai, China). Si-IMP2-1 (si-1) sequence (5’-3’), GCGAAAGGAUGGUCAUCAUTT; si-IMP-2 (si-2) sequence, ACAGGACUGUCCGUGCUAUTT; si-IMP2-3 (si-3) sequence, GCUGUUAACCAACAAGCCATT. let-7i mimics (miR10000415, micrONTM hsa-let-7i-5p mimic), mimics-negative control (mi-ctrl) (miR1N0000001, micrON mimic NC #22), let-7i inhibitors (miR20000415, micrOFFTM hsa-let-7i-5p inhibitor), inhibitors-negative control (in-ctrl) (miR2N0000001, micrOFF inhibitor NC #22) were purchased from RiboBio Co., Ltd (Guangzhou, China). SiRNA (100nM), miRNA mimics (150nM), mi-ctrl (150nM), miRNA inhibitors (150nM) and in-ctrl (150nM) were transfected respectively into KGN cells at 40-50% confluency of cells using Lipofectamine 3000 (l3000015; Invitrogen, CA) according to the manufacturer’s protocol. After transfection for 48h, the cells were collected for further investigations.
RNA extraction and quantitative real-time polymerase chain reaction
Total RNA from human GCs or KGN cells was extracted using the TRIzol reagent (9109; TaKaRa, Dalian, China). 1mL of TRIzol reagent was added gently to each well of 6-well plate. The concentration and quality of all the RNA samples were evaluated using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific), and the 260/280 values for all of samples were above 1.8 and 1.9. To measure mRNA or miRNA expression, RNA (1 μg) was then reverse transcribed using the PrimerScriptTM RT reagent Kit (RR037A, TaKaRa) in a 10μl reaction volume. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TB Green Premix Ex TaqTM II (RR820B, TaKaRa). Let-7i levels were measured by qRT-PCR using Bulge-Loop hsa-let-7i-5p Primer Set (MQPS0000414; Ribobio) with U6 small nuclear RNA (MQPS0000002; Ribobio) as an internal control. Other genes’ expression was normalized to GAPDH. Each sample was run in triplicate. Data were analyzed according to 2−ΔΔCt method. The primer list is included in the Table 2.
Protein extraction and western blotting analysis
Human GCs and KGN cells were lysed on ice in radioimmunoprecipitation assay lysis buffer (P0013B; Beyotime, Haimen, China), supplemented with the protease inhibitor cocktail (HY-K0010, HY-K0021, HY-K0022, MedChemExpress, Monmouth Junction, NJ, USA) of 1mM each. After centrifugation at 12,000g for 30min, protein concentrations were quantified using a bicinchoninic acid protein assay kit (P0010; Beyotime). Equal amount of protein was separated by sodium dodecyl sulfonate polyacrylamide gel and electro-transferred to polyvinylidene difluoride membrane (IPVH00010; Millipore, Billerica, USA). After blocking with 5% non-fat milk for 2 h at room temperature, membranes were incubated with anti-IMP2 (1:2000, ab124930; Abcam, Cambridge, UK), anti -Aromatase (1:1000, ab18995; Abcam) and anti-GAPDH antibodies (1:10000, ab181602; Abcam) respectively overnight at 4℃ and were washed by 0.1% TBST. Then, the membrane was incubated with horseradish peroxidase–conjugated anti-rabbit secondary antibodies (1:5000, SA00001-2, Proteintech) for 1 h at room temperature. Protein expression was determined using an enhanced chemiluminescence detection system (General Electric Company, Fairfield, USA).
5-ethynyl-2′-deoxyuridine incorporation assay
According to the manufacturer’s instructions, Cell-LightTM EdU Apollo567 In Vitro Kit (C10310-1; Ribobio) was used for cell proliferation assay. For 5-ethynyl-2′-deoxyuridine (EdU) assay, KGN cells were cultured in 96-well plates at a density of 3×103 per well. At 48h after transfection with let-7i mimics or let-7i inhibitors or corresponding controls, 50 μM EdU labeling medium was added to the cell culture and incubated for 2 h at 37°C with 5% CO2. Then, the cells were fixed with 4% paraformaldehyde for 30 min, incubated with 2mg/ml glycine (50 μl per well) for 5 min. After being washed with PBS, the cells were permeabilized with 0.5 % Triton X and treated with 1×Apollo solution for 30 min at room temperature in the dark. The cells were incubated with 100 μl 1×Hoechst 33342 solution for 30 min at room temperature in the dark and then washed with PBS. Finally, the images were captured with a fluorescence microscopy (Olympus, Tokyo, Japan) and the ratio of EdU-positive cells to Hoechst-positive cells was calculated from five random fields in three wells.
Cell cycle assay
PI/RNase staining buffer solution (550825, BD Pharmingen) was performed to detect nuclear DNA from cell suspensions. KGN cells transfected with let-7i mimics or let-7i inhibitors or corresponding controls were plated in 12-well plates and incubated at 37°C for 48h. Then, the cells were collected and washed with buffer solution three times, and the cell cycle distribution was analyzed using propidium iodide (PI) staining and flow cytometry (BD FACS Calibur, USA). Data were analyzed by Flow Jo v.10.0.7 software.
Cell counting Kit-8 assay
Cell viability was determined using Cell-Counting Kit-8 (CCK-8) (ck04, DOJINDO, Japan). KGN cells transfected with let-7i mimics or let-7i inhibitors or corresponding controls were reseeded in 96-well plates at 1.5×103 cells/well in 100 μl cell suspension. 10 μl CCK-8 reagent was added to each well quickly and then the cells were cultured for 1.5 h at 37°C at indicated time points (0, 24, 48, and 72 hours). Optical density was measured at 450 nm using a microplate reader. Each experiment was carried out in triplicate at least.
Apoptosis assay
We used the Annexin V/ PI staining for the analysis of apoptotic cells. 1×106 KGN cells transfected with let-7i mimics or corresponding controls were digested with 0.25% trypsin (without EDTA) (GNM15050; Genom) and washed twice with ice-cold PBS. The cells were then stained and treated with the Annexin V PE apoptosis detection Kit (559763, BD Pharmingen) according to the manufacturer’s guidelines. Experiments were performed by flow cytometry (BD FACS Calibur, USA) and data were analyzed by Flow Jo v.10.0.7 software.
Measurement of estradiol levels
KGN cells were seeded in 24-well plates in serum-free and treated with 100 nM testosterone (T) and 1 μM Forskolin (FSK) for 24 h after transfection with let-7i mimics or let-7i inhibitors. T and FSK were purchased from Sigma-Aldrich Co. Ltd. T, as a substrate for aromatase, is converted to E2 under the action of aromatase in GCs (37). Since the expression level of functional FSH receptor is very low in KGN cells, FSK, as a substitute for FSH, was utilized to activate FSH receptor in GCs and assess the effect of let-7i on steroidogenesis (38). The culture medium was assayed immediately; The concentration of estradiol (E2) in the conditioned medium was determined by an ELISA kit (CSB-E05108h; CUSABIO), as per the manufacturer’s instructions. The assay was validated by the parallelism between the pooled and serial-diluted samples and the standard curve.
Dual-luciferase reporter assay
The luciferase reporter plasmids contain either wildtype 3’UTR segment of IMP2 (55335-1; GeneChem) or mutant 3’UTR segment of IMP2 (3’UTR-M, 55336-1; GeneChem). KGN cells grown in 24-well plates were co-transfected with 0.1 μg of the report plasmid and 0.02 μg of the Renilla luciferase plasmid, as well as 0.4 μg hsa-let-7i plasmid (55333-2; GeneChem) or miRNA empty vector plasmid (miRNA-NC) using X-tremegene HP (06366236001, Roche, Germany) transfection reagent. 48 h after transfection, the firefly and Renilla luciferase activities in the cell lysates were detected with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions.
Statistical analysis
All data were analyzed by SPSS version 23.0 (IBM, Armonk, NY, USA) and GraphPad Prism version 7 (GraphPad, La Jolla, USA). The Shapiro-Wilk’s test was used to assess whether the data had a Gaussian distribution. Comparisons between two groups were performed with Student’s t test for quantitative data with a Gaussian distribution, or Mann–Whitney U-test for data with a non-Gaussian distribution. For data with more than two groups were analyzed by one-way analysis of variance (ANOVA) or the Kruskal-Wallis test. Correlation between let-7i expression and IMP2 expression groups was analyzed by Pearson’s rank correlation test. QRT-PCR data were analyzed according to 2−ΔΔCt method. Data are shown as the mean ± standard deviation (SD) from at least three independent experiments. A P value of <0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.