Construction of POF mouse model

Female C57BL/6 mice (n = 31) aged 10 weeks were purchased from Sbev (Beijing) Biotechnology Co., LTD. Mice were kept at 24–26 °C, 55–60% humidity, and 12 h/12 h alternating light and dark conditions. Mice were fed with irradiated mice/rat chow (Double Lion, Suzhou, China) and water. After one week of adaptive feeding, when the mice were in good condition, formal experiments were performed. All mice experimental operations were approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University.

For experiment 1: To the screen differentially expressed tsRNA in POF model. The 16 mice were randomly divided into two groups (8 mice/group): control group and POF group. In the control group, mice were given a normal diet. In the POF group, mice were high-fat (8 g/kg) diet (Formula: 83.5% base feed + 15% lard + 1.5% cholesterol) which was purchased from Spiff (Beijing) Biotechnology Co., LTD, and mice were administered 400 µL 30% D-glucose once a day via gavage for 30 days. The feed for the high fat diet of the POF group Finally, mice were euthanized with a CO2 overdose. The serum and ovarian tissue were collected.

For experiment 2: To verify the participation of tsRNA-3043a in POF development in vivo. The 15 mice were randomly divided into three groups (5 mice/group): POF group, POF + tsRNA-3043a agomir group, and POF + agomir Negative control (NC) group. In the POF group, mice were treated as described in Experiment 1 above. In the POF + tsRNA-3043a agomir or NC group, mice were treated as in the POF group, and injected with 0.2 mL tsRNA-3043a agomir or NC (4 mg/kg) in the tail vein. Finally, mice were euthanized with a CO2 overdose. The serum and ovarian tissue were collected.

Hematoxylin-eosin (HE) staining

To detect ovarian tissue morphology, HE staining was performed. Firstly, ovarian tissues were fixed in 4% polyformaldehyde for 24 h and then embedded in paraffin followed by being cut into 4 μm sections. Secondly, the slices were dewaxed by xylene and gradient ethanol to water. Thirdly, the slices were stained with Harris hematoxylin for 5–10 min and then stained with eosin for 1–3 min. Finally, the slices were dehydrated in gradient ethanol, transparentized by xylene, and sealed with neutral gum. A light microscope (OL YMPUS CK31: Olympus, Japan) equipped with TVO.63XC-MO imaging system (MSHOT) was used to image acquisition and analysis. To avoid counting the same follicles repeatedly, only those with visible oocyte nuclei are counted.

ELISA assays

The content of luteinizing hormone (LH), estradiol (E2), and follicle-stimulating hormone (FSH) in serum was detected by mice E2 ELISA kit (m1001962, mlbio, China), mice LH ELISA kit (ml063366, mlbio, China), mice FSH ELISA kit (m1001910, mlbio, China) according to manufacturer’s instructions. The absorbance value of each well was tested at 450 nm using a Microplate Reader (Thermo Scientific, USA). The standard substances were used and the reverse pipetting and peak recovery tests was conducted to reduce the variance between assays, The mean intra and inter-assay coefficient of variation for all ELISA assays (i.e. precision) was ≤ 10%.

Detection of SOD activity

The SOD of ovarian tissue was determined using the SOD activity test kit (BC0170, Solarbio, USA). About 0.1 g of ovarian tissue sample was homogenized in extraction buffer followed by centrifuging 8000 g at 4 °C for 10 min to retain supernatant. Before determination, the reagents 1, 3, and 5 were bathed in water at 37 °C for 8 min and then mixed with samples. After bathing at 37 °C for 30 min, the absorption value of each tube was determined at 560 nm.

RNA sequencing

Total RNA was extracted from the ovarian tissues or KGN cells using TRizol reagent (Invitrogen) following the manufacturer’s instructions. Oligo (dT) magnetic beads were used to purify mRNA from total RNA. RNA concentration and purity were displayed by a microspectrophotometer, and RNA integrity was determined using agarose gel electrophoresis.

For small RNA sequencing, total RNA was ligated with a 3’ adaptor and hybridized to the reverse primers, and then ligated with a 5’ adaptor. Next, the product was subjected to synthesis of the first-strand of cDNA followed by PCR amplification. The cDNA libraries were sorted by 8% SDS-PAGE to obtain 134–160 bp long fragments. The prepared cDNA libraries were qualified and absolutely quantified using Agilent BioAnalyzer 2100 followed by RNA sequencing on an Illumina NextSeq 500 system with single-end 50 strategy.

For mRNA sequencing, total RNA of each sample was fragmented and reverse transcribed into first-strand of cDNA, followed by double-stranded cDNA synthesis and amplification of cDNA libraries by PCR using Illumina TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). After quality inspection, cDNA libraries were sequenced on Illumina HiSeq 2500 platform with pair-end 150 strategy.

Raw data from RNA sequencing were filtered by FastQC to obtain clean reads. The differential expression level of tsRNAs and mRNA were calculated by R package edgeR with thresholds |Log2FC| > 0.5 and P value < 0.05. RNAhybrid and Miranda software was applied to predict the target gene of tsRNAs. The target genes of tsRNAs and mRNA were analyzed by GO and KEGG enrichment. Raw data were deposited to NCBI (PRJNA1020648 with SUB13861060 and SUB13860953).

qRT-PCR analysis

Total RNA of each sample was reverse transcribed into cDNA using a cDNA Synthesis Kit (K1622, Thermo, USA) on the PCR Amplifier (Life ECO-96, Bioer Technology, China). For tsRNAs, primers were the specific primers. For mRNAs, primers were the random primers. Afterward, the cDNA product was subjected to quantitative Real-Time PCR using 2 × Master Mix kit (Roche) on an ABI Q6 Real-Time PCR system (Applied Biosystems Inc., USA). The procedure of PCR amplification was as follows: 95 °C for 10 min; 95 °C for 15 s and 60 °C for 60 s (40 cycles); 95 °C for 10 s; 60 °C for 60 s; slowly heated from 60 °C to 99 °C. U6 and GAPDH was used to normalize the expression of tsRNA and mRNA, respectively. The relative expression of genes was obtained by the 2−ΔΔCt method. All reactions were repeated three times. The primers used in this study are shown in Supplemental Table 1.

Cell culture and transfection

The human ovarian granulosa tumor cell KGN cell line was purchased from iCell Bioscience Inc (iCell-h298, China) and cultured in DMEM/F12 + 10% FBS + 1% P/S medium under 95% air and 5% CO2 condition with 37 °C.

Transient transfection KGN of tsRNA-3043a inhibitor, mimics or NC using Lipofectamine 2000 reagent (Invitrogen). Briefly, cells were seeded on a six-plate with 3 × 105 cells/well. RNA and Lipofectamine 2000 were diluted with OPTI-MEM at the ratio of 5: 45 and mixed for 20 min. After that, when reaching about 80–90% confluence, the mixture was added into cell samples. The sequences of tsRNA-3043a inhibitor/mimics/NC were synthesized by GenePharma and shown in Supplemental Table 1.

In vivo, tsRNA-3043a agomir was used to overexpression of tsRNA-3043a. tsRNA-3043a agomir is a double strand with modifications on the anti-sense strand synthesized by GenePharma. To ensure stability and inhibition, modifications were made on the antisense strand with two thioskeletal modifications at the 5’ end, a cholesterol modification and four thioskeletal modifications at the 3’ end and full chain methoxylation.

For overexpression of FLT1, the full-length of FLT1 was inserted into pcDNA3.1 plasmid, and then recombinant pcDNA3.1-FLT1 vector or blank vector was transfected into KGN cells. The siRNA was utilized to knock down FLT1 expression and was shown in Supplemental Table 1.

CCK8 assays

KGN cell proliferation was detected by CCK8 kit (Beyotime). KGN cells were prepared to single cell suspension with 1 × 104 cells/mL. Cells at the logarithmic growth stage were inoculated into 96-well plates (#3599, Corning). Each well was spread with 100 µL. Each sample was set with six replicated wells. After culture for hours 0, 24, 48, 72, and 96, 10 µL CCK8 reagent was added. The absorbance (OD value) was determined using a microplate reader (Infinite M1000, Tecan) at 450 nm.

Flow cytometry

Flow cytometry was used for the apoptosis of KGN cells. Cells were collected, washed with PBS, and re-suspended with 195 µL 1 × Binding Buffer to make a single cell suspension (5 × 105 cells/mL) followed by added 5 µL of Annexin V-FITC. Afterward, cells were incubated for 15 min at room temperature under dark and centrifuged at 1000 rpm for 3 min to discard the supernatant. Cells were re-suspended in 190 µL of 1X Binding Buffer and mixed with 10 µL Propidium lodide. Finally, KGN cell apoptosis was detected by flow cytometer (CytoFLEX LX Flow Cytometer, Beckman).

Oil red O staining and β-galactosidase staining

The accumulation of lipid droplets in KGN cells was detected by oil red O staining. Appropriately 600 µL of oil red O staining solution was mixed with 400 µL of water and then filtered with a 0.45 μm filter membrane. KGN cells were fixed in 4% paraformaldehyde at 25 °C for 30 min and washed with 1×PBS followed by stained with freshly prepared oil red O solution for 60 min at 25 °C. The images were taken under a microscope.

The senescence of KGN cells was measured by a β-galactosidase Assay Kit (G1580, Solarbio, China) based on senescence-associated β-galactosidase (SA-β-Gal) activity, according to the product instruction. Cellular senescence was observed under a light microscope (NIKON).

Dual luciferase gene reporter assay

The physical binding between tsRNA-3043a and FLT1 was assessed by dual luciferase gene reporter assay. The full-length FLT1 sequence (wild-type) and binding site point mutations (mut) were cloned into psiCHECK plastid. Next, psiCHECK- FLT1 and psiCHECK- FLT1-mut cotransfected into 293T cells with tsRNA-3043a mimics and tsRNA-3043a mimics NC, respectively. After incubation for 48 h, cells were lysed and mixed with Firefly luciferase substrate and measured Firefly luciferase activity. Finally, Renilla luciferase buffer and substrate were added followed by detecting Renilla luciferase activity. After normalizing for Firefly luciferase activity, relative luciferase activity was achieved by comparing activity from firefly luciferase with renilla luciferase.

Western blot

RIPA lysis buffer (Thermo) was applied to prepare protein samples and then total protein concentration was determined by BCA kit. Protein (20 µg) was separated by 10% SDS-PAGE and transferred onto PVDF membranes. Next, PVDF membranes were blocked with TBST solution containing 5% skim milk at 4 °C for 12 h and incubated with GAPDH (1:2000, 60004-1-Lg; Proteintech, China) and VEGFR-1/FLT-1 (1: 1000, 13687-1-AP; Proteintech, China) for 3 h at 25 °C. Membranes were washed 3 times with 1% TBST for 15 min each time. Membranes were incubated with Goat Anti-Mouse IgG H&L(HRP) (1:1000, ab205719, Abcam, USA) and Goat Anti-Rabbit IgG H&L(HRP) (1/20000, ab6721, Abcam, USA) for 2 h. Protein bands were stained with ECL luminescence reagent (Thermo) and visualized by a Chemiluminescence (QINXIANG SCIENTIFIC Instrument Co. LTD, Shanghai, China). Finally, the gray value of the bands was obtained by image J software.

Immunohistochemistry (IHC)

Ovarian tissues were fixed in 4% paraformaldehyde for 24 h and then dehydrated by gradient ethanol and xylene followed by being embedded in paraffin and cut into 4 µm slices. Next, slices were deparaffinized with xylene and hydrated with gradient ethanol followed by incubation with antigen retrieval solution for 30 min. The sections were blocked with 5% BSA and incubated with the anti-FLT1 antibody (1:200, 13687-1-AP, Proteintech) overnight at 4°C. Afterward, the sections were incubated with the corresponding secondary antibody (G1213-100UL, Servicebio) for 50 min at 25°C followed by 3,3’-diaminobenzidine and hematoxylin staining. Finally, sections were sealed with neutral balsam and photographed using an Olympus CK31 microscope (Tokyo, Japan).

Statistical analysis

GraphPad Prism Version 9.0.0 Software (La Jolla, CA, USA) was applied for data statistical analysis. All data were represented as mean ± standard deviation (SD). The difference between the two groups was determined by t test, and between multiple groups was compared by one-way ANOVA followed by Tukey’s post-hoc test. The p < 0.05 was defined as statistically significant.