Introduction

Premature ovarian failure (POF), also well-known for primary ovarian insufficiency, is a gynecological disease that causes amenorrhea, infertility, menopause, and genitourinary symptoms (Lin et al. 2017). POF is characterized by premature loss of ovarian follicles before the age of 40 years (Persani et al. 2010). POF occurrence is attributed to premature follicular depletion due to accelerated atresia of the primordial follicle or altered maturation/supplementation, resulting in inadequate folliculogenesis (Persani et al. 2010). Currently, treatment of POF has been aimed at maintaining ovarian reserve (Kalich-Philosoph et al. 2013), activating dormant follicles (Kawamura et al. 2016) and mitigating follicular loss through potentially protective agents such as melatonin (Ma et al. 2017). Hormone replacement therapy (HRT) is another common treatment for POF, but HRT has contraindications, such as unexplained vaginal bleeding, acute liver damage, and breast cancer. However, these seemingly viable symptomatic treatments have not provided sufficient benefit to patients in clinical trials, and these treatments rarely promote the development of remaining follicles in patients. Therefore, there is an urgent need for a new targeted therapeutic strategy to activate dormant primordial follicles and stimulate follicular growth in patients with POF.

Although understanding of the pathogenesis of POF is limited, it has been found that ovarian granulosa cell damage and apoptosis play a key role in the process of POF (Qu et al. 2022). Oxidative stress is the key to granule cell damage and is involved in the pathogenesis of POF (Rani et al. 2016). The imbalance of oxidative stress between intracellular reactive oxygen species (ROS) production and antioxidant capacity (Superoxide dismutase, SOD; glutathione, r-glutamyl cysteingl + glycine, GSH) leads to oxidative stress and induces oocyte aging (Rani et al. 2016). For example, X-ray irradiation reduces the cell viability of ovarian granulosa cells, induces intracellular ROS accumulation, increases oxidative stress, and leads to ovarian damage (Zhao et al. 2023). Oxidative stress induced by CTX is one of the main causes of ovarian dysfunction, which can destroy follicles by inducing granulosa cell apoptosis and reducing glutathione (GSH) levels in the ovary, leading to impaired ovarian function (Codacci-Pisanelli et al. 2017). Therefore, in this study, a decrease in SOD levels was used as a marker of oxidative damage in granulosa cells.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) are among the oldest small RNAs in all domains of life and are produced by tRNA cleavage (Chen et al. 2021), but their function is still largely unknown. Recent studies have demonstrated that tsRNA plays an important role in various diseases, including reproductive, neurological, tumor and other fields (Yu et al. 2020). For example, tsRNAs were served as a paternal epigenetic factor in sperm to contribute to intergenerational inheritance (Chen et al. 2016). In adult ovaries of Onychostoma macrolepis, 235 tsRNAs were abnormally expressed between the periods of breeding and late overwintering, such as tRFi-Lys-CTT-1 and tRFi-Gly-GCC-1(Peng et al. 2022). In mature spermatozoa, tRNAGln−TTG-derived tsRNAs controlled the early cleavage of preimplantation embryos (Chen et al. 2020). These studies suggest that tsRNA plays an important role in reproduction-related life activities. However, it has not been reported whether tsRNA can be involved in POF pathology by regulating oxidative damage. In contrast, the remaining types of non-coding RNAs, such as miRNAs (Qu et al. 2022; Liu et al. 2021), lncRNAs (Zheng et al. 2021; Zhang et al. 2022), and circRNAs (Xing et al. 2023) have been reported in POF. Therefore, we focused only on the role of tsRNAs in POF.

In this study, we aimed to clarify the specific expression profile of tsRNA in ovarian tissues of POF mice by high-throughput sequencing and to explore the role and mechanism of the key molecule tsRNA-3043a in oxidative damage in POF. This study will enrich our understanding of POF and provide new directions for the targeted treatment of POF.

Materials and methods

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.

Results

Ovarian pathological in POF mice

The animal experimental flowchart is shown in Fig. 1A. To observe the pathological characteristics of POF, HE staining of mice was performed. Compared to controls, POF mice had abnormal ovarian tissue morphology and increased inflammatory cell infiltration (Fig. 1B). The number of normal follicles in the ovarian of POF mice was significantly lower than that of the NC mice (Fig. 1B, p < 0.001). The ovarian weight of POF mice also significantly decreased compared with the NC group (Fig. 1C, p < 0.001). The levels of serum indicators such as E2 (p < 0.001), LH (p < 0.05), and FSH (p < 0.001) in peripheral blood of POF mice exhibited significant descent, compared with the control mice (Fig. 1D). In addition, SOD activity in ovarian tissue, an indicator of oxidative stress, was also significantly decreased in POF mice, suggesting an oxidative stress imbalance (Fig. 1E, p < 0.05). These data indicate successful construction of a mouse model of POF with normal follicle loss and concomitant oxidative stress imbalance.

Fig. 1
figure 1

Ovarian pathological in POF mice. (A) The animal experimental flowchart. (B) HE staining was performed on ovarian tissues (n = 3) and the number of normal follicles was counted. Scale bar: 50 μm. (C) Ovarian weight (n = 8) determination. (D) Concentration of LH, E2, and FSH in serum of mice were detected by ELISA (n = 8). (E) SOD activity in ovarian tissue (n = 8) was detected by SOD activity detection kit. Scale bar: 100 μm

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Dysregulation of tsRNA expression profile in ovarian of POF mice

To investigate the role and molecular mechanisms of tsRNA in POF progression, ovarian tissues collected from 3 POF mice and 3 control mice were subjected to RNA sequencing. Compared with control mice, a total of 81 tsRNAs were significantly differentially expressed between POF mice and control mice, including 79 up-regulated tsRNAs and 2 down-regulated tsRNAs in POF mice (Fig. 2A). These differentially expressed tsRNAs predicted to have 13,764 potential target genes (Fig. 2B). GO analysis reveals transcriptional regulation as a biological process for the preferential enrichment of these target genes, such as “negative regulation of transcription, DNA − templated”, “regulation of transcription, DNA − templated”, “transcription, DNA − templated”, “positive regulation of transcription, DNA − templated”, “negative regulation of transcription from RNA polymerase II promoter”, “regulation of transcription from RNA polymerase II promoter”, “transcription from RNA polymerase II promoter”, and “positive regulation of transcription from RNA polymerase II promoter”(Fig. 2C). KEGG analysis showed that these target genes of differentially expressed tsRNAs were mainly enriched in signaling pathways related to oxidative stress, such as “MAPK signaling pathway”, “FoxO signaling pathway”, “Wnt signaling pathway”, and “Rap1 signaling pathway” (Fig. 2D). Therefore, the aberrant differentially expressed tsRNAs may be involved in oxidative stress in granulosa cells and thus participates in the POF process through transcriptional regulation.

Fig. 2
figure 2

Dysregulation of tsRNA expression profile in ovarian of POF mice. (A) Volcano plot of differentially expressed tsRNAs of mice between POF vs. NC. (B) The predicted target gene of differentially expressed tsRNAs (C) GO analysis of target gene of differentially expressed tsRNAs (D) KEGG pathway analysis of target gene of differentially expressed tsRNAs

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tsRNA-3043a promotes POF progression

To screen the specific tsRNA involved in the pathophysiology of POF, the top 3 tsRNAs with abundant expression, large fold change, and conserved in humans were screened for qRT-PCR validation. The results showed that compared with the control group, only tsRNA-3043a expression was significantly up-regulated in the POF group, whereas the expression of tsRNA-3010b and tsRNA-3025a did not change significantly (Fig. 3A, p < 0.05). Therefore, tsRNA-3043a was selected for subsequent study.

Fig. 3
figure 3

tsRNA-3043a promotes POF progression. (A) Detection of candidate tsRNA expression by qRT-PCR. (B) The overexpression of tsRNA-3043a in KGN cells was detected by qRT-PCR. (C) CCK8 assay was used to detect the effect of tsRNA-3043a mimics on the viability of POF model KGN cells. (D) Flow cytometry was utilized to assess the effect of tsRNA-3043a mimics on apoptosis of POF model KGN cells. (E) Oil red O and β-galactosidase staining was used to detect the effect of tsRNA-3043a on lipid accumulation and cell senescence in POF model KGN cells, respectively. Scale bar: 100 μm

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tsRNA-3043a (TTCCCGGCCCATGCACCA) is derived from tRNA-Gly-GCC, also named as mmu_tsr016393 in tsRBase database (http://www.tsrbase.org/entry.php?acc=mmu_tsr016393) and tDR-57:74-GlyGCC-1-M3 according to the tDRname rules (Holmes et al. 2023).

To investigate the effect of tsRNA-3043a on POF in vitro, human ovarian granulosa cells (KGN cell line) were transfected with tsRNA mimics/NC. The overexpression efficiency of tsRNA-3043a in KGN cells was confirmed by qRT-PCR (Fig. 3B, p < 0.05). CCK8 assay was used to detect the effect of tsRNA-3043a on the viability of KGN cells. Compared with the control group, the proliferation of KGN cells in POF model group was significantly inhibited, and this effect was further aggravated by tsRNA-3043a mimics (Fig. 3C, p < 0.001). On the contrary, POF modelling resulted in a significant increase in KGN cell apoptosis, which was further exacerbated by tsRNA-3043a overexpression (Fig. 3D, p < 0.001). Moreover, compared to the control group, boosted lipid accumulation and cellular senescence was observed in the POF group by oil red O and SA-β-Gal staining, respectively. Overexpression of tsRNA-3043a made lipid accumulation and cellular senescence more pronounced (Fig. 3E). In addition, tsRNA-3043a knockdown cells were analyzed in parallel. In contrast to overexpression of tsRNA-3043a, tsRNA-3043a inhibitor significantly enhanced cell proliferation and inhibited apoptosis, lipid accumulation, and cellular senescence (Fig. 4). Taken together, tsRNA-3043a promotes POF progression through inducing apoptosis, lipid accumulation, and senescence of granule cell.

Fig. 4
figure 4

tsRNA-3043a inhibitor suppresses POF progression. (A) The knockdown of tsRNA-3043a in KGN cells by using tsRNA-3043a inhibitor was detected by qRT-PCR. (B) CCK8 assay was used to detect the effect of tsRNA-3043a inhibitor on the viability of POF model KGN cells. (C) Flow cytometry was utilized to assess the effect of tsRNA-3043a inhibitor on apoptosis of POF model KGN cells. (D) Oil red O and β-galactosidase staining was used to detect the effect of tsRNA-3043a inhibitor on lipid accumulation and cell senescence in POF model KGN cells, respectively. Scale bar: 100 μm

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tsRNA-3043a overexpression alters transcriptome of KGN cells

The KGN cell line has all the conserved features of natural granulosa cells and is considered an easily accessible system, we selected KGN to characterize the signaling pathway in human folliculogenesis through mechanistic experiments. Total RNA was extracted from the POF cell model induced by KGN cell transfected with tsRNA-3043a mimics or NC and RNA sequencing was performed. As shown in volcano map, compared with the NC group, 100 mRNAs were up-regulated and 198 mRNAs were down-regulated in the tsRNA-3043a mimics group (Fig. 5A). Heat map that differentially expressed genes (DEGs) were clearly separated between the two groups (Fig. 5B). GO analysis displayed that DEGs were mainly involved in biological processes related to phosphorylation regulation, such as “peptidyl − tyrosine phosphorylation”, “protein phosphorylation”, and “protein autophosphorylation” (Fig. 5C). KEGG pathway analysis showed that these DEGs were enriched into lipid associated pathways, including “ovarian steroidogenesis”, “Ras signaling pathway”, “steroid hormone synthesis”, “Rap1 signaling pathway”, “MAPK signaling pathway” and “PI3K-Akt signaling pathway” (Fig. 5D). Interestingly, we found that FGF family and FLT1, which were significantly downregulated in the tsRNA-3043a mimic group, were enriched to these pathways (Fig. 5E). In short, tsRNA-3043a overexpression significantly altered the transcriptome profile of KGN cells, and this alteration was associated with phosphorylation regulation and lipid metabolism.

Fig. 5
figure 5

tsRNA-3043a overexpression alters transcriptome of KGN cells. (A) Volcano map of DEGs in POF model KGN cells between tsRNA-3043a mimics and mimics NC groups. (B) Cluster heatmap of DEGs. (C) Bubble plot of GO enrichment of DEGs. (D) Bubble plot of KEGG enrichment of DEGs. (E) The network of tsRNA-3043a – target gene – pathways

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FLT1 inhibits POF progression

To verify the reliability of RNA sequencing, we screened six target genes (FGF23, FGF6, FGF3, FGFR4, FLT1, and FGFR3) that were significantly down-regulated and highly abundant in the tsRNA-3043a mimics group for qRT-PCR validation. The results showed that six DEGs were downregulated in the tsRNA-3043a mimics group compared with the control group, which was consistent with the transcriptome data (Fig. 6A, p < 0.05). FLT1 had the largest fold change, thus, FLT1 was used as the target gene for subsequent experiments. Notably, RNAhybrid 2.2 predicted that FLT1 and tsRNA-3043a had the complementary pairing of 14 bases (Fig. 6B). The results of double-luciferase reporter experiment indicated that compared with the NC group, tsRNA-3043a mimics significantly inhibited the luciferase activity in the presence of psiCHECK-FLT1, whereas did not alter the luciferase activity of psiCHECK-FLT1-mut (Fig. 6B, p < 0.001). These data indicated that tsRNA-3043a could bind to the target gene FLT1.

Fig. 6
figure 6

FLT1 inhibits POF progression. (A) qRT-PCR was used to validate the expression of DEGs. (B) Dual-luciferase reporter assay verified tsRNA-3043a binding to target gene FLT1. (C) The mRNA expression of FLT1 was detected by qRT-PCR. (D) The protein expression of FLT1 was detected by western blot. (E) Cell proliferation of KGN cells was detected by CCK8. (F) Apoptosis of KGN cells was detected by flow cytometry. (G) Oil red O staining was used to detect cellular lipid deposition. Cell senescence was detected by SA-β-Gal staining. Scale bar: 100 μm

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To explore the effect of FLT1 on the POF in vitro, FLT1 was overexpressed in KGN cells and POF modelling was performed. The overexpression efficiency of FLT1 at mRNA (Fig. 6C, p < 0.001) and protein (Fig. 6D, p < 0.001) level were confirmed. CCK8 results showed FLT1 overexpression significantly increased proliferation compared with the control group (Fig. 6E, p < 0.001). Flow cytometry proved that the number of apoptosis was significantly reduced in the FLT1 overexpression group compared with the control group (Fig. 6F, p < 0.001). Oil red O staining revealed that lipid deposition in the FLT1 overexpression group was lower than those in the control group (Fig. 6G). SA-β-Gal staining demonstrated that FLT1 overexpression led to a clear decrease in the SA-β-Gal expression, suggesting that FLT1 overexpression inhibited cell senescence (Fig. 6G). In summary, FLT1 suppresses POF progression of KGN cells.

tsRNA-3043a induces POF by targeting FLT1 in KGN cells

To investigate whether the promotion of tsRNA-3043a in POF is dependent on the regulation of FLT1, we performed rescue experiments, the tsRNA-3043a mimics and pcDNA3.1-FLT1 vector were transfected into KGN cells at the same time. As shown in Fig. 7A (p < 0.05)and 7B, qRT-PCR and western blot results proved that FLT1 expression was significantly down-regulated in POF cells at mRNA and protein levels after transfected with tsRNA-3043a mimics compared with the mimics NC group, while pcDNA3.1-FLT1 vector reversed this inhibition effect. Moreover, proliferation of POF cells was significantly reduced by the tsRNA-3043a mimics, but co-transfected with pcDNA3.1-FLT1 vector significantly restored the proliferation of POF cells (Fig. 7C, p < 0.001). Overexpression of tsRNA-3043a also significantly provoked the apoptosis, lipid deposition, and cell senescence of POF cells, whereas simultaneous overexpression of FLT1 significantly reversed these effects (Fig. 7D and E, p < 0.001). Furthermore, the rescue effect of FLT1 knockdown on tsRNA-3043a inhibitor was also investigated. As shown in Fig. 8, concurrent FLT1 knockdown abolished the elevation of FLT1 expression and cell proliferation seen with tsRNA-3043a inhibitor; siFLT1 also reversed the suppression of apoptosis, lipid deposition, and cell senescence induced by tsRNA-3043a inhibitor. Therefore, these data suggested that tsRNA-3043a promoted POF through inhibition of FLT1.

Fig. 7
figure 7

tsRNA-3043a induces POF by targeting FLT1 in KGN cells. (A) The mRNA expression of FLT1 in KGN cells was detected by qRT-PCR. (B) The protein expression of FLT1 in KGN cells was detected by western blot. (C) CCK8 was used to detect proliferation of KGN cells. (D) Apoptosis of KGN cells was detected by flow cytometry. (E) Oil red O staining was used to detect cellular lipid deposition. Cell senescence was detected by SA-β-Gal staining. Scale bar: 100 μm

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Fig. 8
figure 8

Rescue experiments using FLT1 knockdown in cell POF model upon tsRNA-3043a inhibitor. The mRNA (A) and protein (B) expression of FLT1 in KGN cells was detected by qRT-PCR and western blot, respectively. (C) CCK8 was used to detect proliferation of KGN cells. (D) Apoptosis of KGN cells was detected by flow cytometry. (E) Oil red O staining was used to detect cellular lipid deposition. Cell senescence was detected by SA-β-Gal staining. Scale bar: 100 μm

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tsRNA-3043a facilitates POF by inhibiting FLT1 in mice

To test the effect of tsRNA-3043a, we conducted an in vivo experiment with mice (Fig. 9A). We constructed POF mouse models while stimulating the mice by tail vein injection of tsRNA-3043a agomir. HE staining results showed that compared with the agomir NC group, the number of normal follicles of POF mice in the tsRNA-3043a overexpression group was significantly reduced (Fig. 9B). Administration of tsRNA-3043a agomir resulted in a significant decrease in ovarian weight (Fig. 9C, p < 0.05) as well as SOD activity in ovarian tissue (Fig. 9D, p < 0.0001). ELISA results proved that compared with the agomir NC group, the levels of E2, FSH, and LH were significantly reduced in the serum of POF mice in the tsRNA-3043a overexpression group (Fig. 9E, p < 0.001). In addition, expression of FLT1 in ovarian tissue was measured by immunohistochemistry. As expected, tsRNA-3043a overexpression significantly suppressed the FLT1 expression compared with the agomir NC group in POF mice (Fig. 9F). These data indicated that tsRNA-3043a facilitates ovarian failure by inhibiting FLT1 in POF mice.

Fig. 9
figure 9

tsRNA-3043a facilitates POF by inhibiting FLT1 in mice. (A) The animal experimental flowchart. (B) HE staining (n = 3) was used to observe ovarian tissue morphology and follicles. (C) Weighing of ovarian tissue (n = 5). (D) The level of SOD in mouse ovarian tissue (n = 5) was detected. (E) The levels of E2, FSH, and LH in serum of mice (n = 5) were detected by ELISA. (F) The expression of FLT1 in mouse ovarian tissue was detected by immunohistochemistry. Scale bar = 20 μm

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Discussion

POF is an important cause of female infertility, and it is still incurable. HRT is currently the most common treatment for POF patients to get rid of menopausal syndrome (Lin et al. 2017). The method of IVA (in-vitro activation) can treat infertility in POF patients with residual follicles (Kawamura et al. 2016). Vitamin D inhibits the formation of neutrophil extracellular traps and interferes with the development of POF (Chen et al. 2023). However, these treatments cannot effectively restore ovarian function and fertility, and may also bring adverse reactions and strong immune resistance. The paucity of treatment is attributed to the ambiguity of the molecular mechanisms of POF. In this study, we revealed a novel molecular mechanism by which tsRNA-3043a promotes POF via inhibiting FLT1 (Fig. 10). Targeting tsRNA-3043a may be a novel strategy for pharmacological treatment of POF.

Fig. 10
figure 10

Diagram of the hypothesis mechanism of tsRNA-3043a facilitates POF by inhibiting FLT1

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tsRNAs are small regulatory non-code RNAs that cleavage from mature tRNA or precursor tRNA, which exert biological roles through multiple mechanisms, including interaction with proteins or mRNA, repression of translation, regulation of gene expression, chromatin and epigenetic modifications (Xie et al. 2020). However, only three studies on “tsRNA ovarian” were retrieved from the PubMed. One of them was a comprehensive analysis of non-coding RNAs in the ovarian of Onychostoma macrolepis in the late overwintering and breeding period, and identified 235 differentially expressed tsRNAs, including tRFi-Lys-CTT-1 and tRFi-Gly-GCC-1(Peng et al. 2022). Secondly, Panoutsopoulou et al. found that i-tRF-GlyGCC expression was abundant in ovarian tumors and can be used for prognostic assessment of patients with epithelial ovarian cancer (Panoutsopoulou et al. 2021). Thirdly, Panoutsopoulou et al. demonstrated that the 3’U-tRFValCAC promotes a malignant phenotype in ovarian cancer (Panoutsopoulou et al. 2023). Although these three studies are different from the mechanism of POF, they may strongly confirm that tsRNAs play an important role in the pathology of ovarian. In addition, tsRNAs are often thought to play similar roles to miRNAs in a manner of “miRNA-like” (Krishna et al. 2021). Studies have shown that miRNAs play a key role in the process of POF. For example, inhibition of miR-146b increased apoptosis of POF granule cells (Yu et al. 2022). Overexpression of miR-21 in stem cells inhibits granulosa cell apoptosis by targeting PDCD4 and PTEN to improve ovarian structure and function with chemotherapeutic POF in rats (Fu et al. 2017). The tsRNAs are similar in size and function to miRNAs. Therefore, these studies support our results that tsRNA-3043a promotes POF.

FLT1, also known as VEGFR1, is the receptor of VEGF/PIGF and a vascular endothelial growth factor. In this study, we reveal that FLT1 was down-regulated in POF mice and FLT1 overexpression inhibited cellular senescence of POF KGN cells. Some studies have confirmed that FLT1 is essential for the normal development of granulosa cells. For example, Munakata demonstrated that low FLT1 expression is the causal factor of the suboptimal development of oocyte-and-granulosa cell complexes in vitro culture (Munakata et al. 2016). Granulosa cells induce ovarian vessel growth by down-regulating the soluble antagonist FLT1 (Gruemmer et al. 2005). FLT1 is localized in the granulosa and thecal cells of preovulatory follicles and is associated with the development of follicles through controlling angiogenesis (Otani et al. 1999). Therefore, these data implicated that targeted regulation of FLT1 can modulate ovarian granulosa cell function. In our research, tsRNA-3043a promoted the apoptosis of granulosa cells and caused cell senescence by targeting FLT1. Therefore, new drugs targeting tsRNA-3043a/FLT1 axis may be an effective therapeutic strategy to repair damaged ovarian function and improve POF.

Conclusions

In conclusion, 81 tsRNAs were aberrantly expressed in the ovarian tissues of POF mice, with upregulated tsRNA-3043a promoting POF by promoting oxidative damage, apoptosis, and senescence of ovarian granulosa cells. Overexpression of tsRNA-3043a significantly altered the transcriptome profile of POF model cells and promoted POF progression by targeting and inhibiting FLT1 expression in vitro and in vivo. This study firstly elucidated the role and mechanism of tsRNA-3043a in the regulation of POF, and provided a theoretical basis for the development of targeted drugs for the clinical treatment of POF.