Ethical approval and consent to participate
Written informed consent was obtained from each patient participating in the study. The study protocol and informed consent were approved by the Ethics Committee of the Affiliated Hospital of Ningbo University Medical School. All methods were performed in accordance with the Declaration of Helsinki.
All samples (normal/ectopic endometrial tissues and leucorrhoea) were collected at Ningbo University Medical School Affiliated Hospital from March 2020 to March 2021. A total of 51 women were enrolled in our study. Among all patients, 26 patients who were diagnosed with SME by laparoscopy and histopathological examination served as a control group. The remaining 25 patients without endometriosis who were admitted to hospital during the same period were included in the control group. Inclusion criteria: 1. No history of hormonal or antibiotic treatment in the three months preceding laparoscopic surgery; 2. No hepatitis, tuberculosis, tumors and other diseases. Exclusion criteria: 1. Recent hormonal and antibiotic treatment; 2. With serious organic diseases; 3. Combined with other gynecological diseases such as reproductive system inflammation and tumors. (General information of enrolled patients was presented in Supplementary File 1: Table S1). All subjects who had regular menstrual cycles were women of childbearing age who were nonmenstrual three days prior to sample collection. The mast cell-Luva line was a generous donation from a laboratory at Zhejiang University.
Isolation of exosomes from tissues and leucorrhoea
We used differential centrifugation to extract exosomes from tissues and leucorrhoea. Briefly: ectopic tissue was disaggregated into a single cell suspension with collagenase type IV (Solarbio, China). The leucorrhoea was diluted with PBS to make a mixed solution. The supernatant and leucorrhoea solution were centrifuged at 4°C with a high-speed centrifuge (Thermo, USA) at 500 g for 10 min to remove live cells, 2000 g for 10 min to remove dead cells and 10,000 g for 20 min to remove cell debris. Each step was repeated twice. The supernatant was then centrifuged at 100,000 g twice with an ultracentrifuge (Beckman, USA) for 70 min each time. The exosomes were resuspended or lysed with different reagents for further experiments.
Identification of exosomal size
Transmission electron microscopy (TEM) was used to identify the size of exosomes. Briefly, the exosome was deposited on the copper net for 5 min at room temperature. A 3% phosphotungstic acid solution colored the nanoparticles. Then, the exosomes were analyzed with a transmission electron microscope (Hitachi H-7650). The diameter distribution of exosomes was examined by nanoparticle tracking analysis (NTA) (Malvern NanoSight NS500).
Immunoblot for exosomal markers
Exosomes were lysed with RIPA buffer, resuspended in loading buffer, boiled at 95°C for 5 min, and then electrophoresed on SDS-PAGE. Proteins were transferred to a polyvinylidene fluoride membrane, which was blocked with 5% skim milk powder in TBST. Immunodetection was performed with anti-HSP70 antibody (1:1000, Proteintech, China), anti-Flotillin-1 antibody (1:1000, Proteintech, China), anti-CD63 antibody (1:1000, Proteintech , China) and anti-calnexin antibody (1:1000, Proteintech, China) at 1:1000 dilution followed by incubation at 4°C overnight. The following day, the protein was incubated with an appropriate HRP-conjugated secondary antibody (1:5,000, Abcam, USA). Bands were revealed using ECL Plus and then imaged on the Electrophoresis Gel Imaging Analysis System (D-Digital, USA) for analysis.
Library construction and small RNA sequencing
The exoRNA was extracted with Trizol reagent (Invitrogen, USA) and the purified RNA was sent to Aksomics Biological Engineering Co., Ltd. (Shanghai, China) to perform sequencing analysis of tRFs and tiRNAs. The brief steps were as follows: agarose gel electrophoresis was used to detect the integrity of the total RNA sample, and the Quantitative Analyzer NanoDrop ND-1000 (thermos, USA) quantified the RNA concentration. Preparation of the TRF&tiRNA-seq library includes: (1) ligation of the 0.3′ adapter; (2) 5′ adapter ligation; (3) cDNA synthesis; (4) PCR amplification; (5) size selection of PCR-amplified fragments of 134-160 bp (corresponding to a small RNA of about 14-40 nt). The library was quantitatively analyzed with the Agilent 2100 Bioanalyzer. According to the quantitative results, the library was mixed in equal amounts. DNA fragments in the mixed library were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, which were loaded onto the kit at a concentration of 1.8 µM. According to the manufacturer’s instructions, the NextSeq 500/550 V2 Kit (#FC-404–2005, Illumina) was used for sequencing with the NextSeq System. EdgeR software from the R package was used to screen for differentially expressed TRFs and tiRNAs based on count value.
Quantitative real-time polymerase chain reaction (qRT-PCR)
According to the manufacturer’s instructions, total RNAs were extracted from purified exosomes and cultured cells using Trizol reagent (Invitrogen, USA). The extracted RNA was stored at -80°C. The cDNAs were synthesized using a reverse transcription kit, according to the manufacturer’s instructions (CWbio, Beijing, China). qRT-PCR for cellular and exosomal RNA including tRF-Leu-AAG-001, tRF-Leu-TAG-015, IL-6, IL-10, IL-1β, TNF-α and GAPDH were performed by RT-PCR quantification kit (CWBio, Beijing, China). Briefly, after an initial denaturation step at 95°C for 10 min, amplifications were performed with 40 cycles at a melting temperature of 95°C for 15 s and an annealing temperature of 60°C for 30 s. Relative mRNA expression levels were calculated with the 2-ΔCt method. The PCR productions of tRF-Leu-AAG-001, tRF-Leu-TAG-015 were tested by 3% agarose. The sequences of the specific primers have been presented in Table 1.
3D cell culture
Ectopic tissues were digested in single cell suspension with type IV collagenase (Solarbio, China). After centrifugation to pellet the cells, NanoShuttle (50 μl, Greiner bio-one Co., Germany) was added to the cell suspension and incubated. The cell-nano mixture suspension was incubated at 37°C for 1 h. After centrifugation to remove the supernatant, the number of cells was adjusted to 8*104/150ul with the medium mix. Cells were inoculated into a 96-well microplate (cell repellent surface, Greiner bio-one Co., Germany). Then we hold the microplate on a magnetic conductor (Greiner bio-one Co., Germany). Cell balls were placed in a 37°C, 5% cell incubator and incubated for 15 min, then the magnetic conductor was removed.
We used immunofluorescence and RNA fluorescence probes for the co-localization of tRF-Leu-AAG-001 and mast cells. We purchased the Cy3-labeled tRF-Leu-AAG-001 fluorescence probe from Ruibo Biotech and purchased the mast cell marker: anti-CD117-FITC antibody from Thermo Fisher. Briefly: 3D primary ectopic cells were inoculated in a 96-well plate for 1 h, a Cy3-labeled tRF-Leu-AAG-001 fluorescence probe was added and incubated overnight at 37°C. The following day , cell balls were washed with PBS for 5 min, protected from light three times, then added anti-CD117-FITC antibody and incubated at 37°C for 1 h. Aspirate the secondary antibody and wash with PBS in the dark. Finally, add DAPI solution at room temperature for 5 min, photograph the fluorescence with an Olympus confocal microscope.
knockdown of tRF-Leu-AAG-001 by small interfering RNAs
tRF-Leu-AAG-001 siRNA and Negative Control (NC) were designed and composed by Sangon Biotech. Luva were seeded in 6-well plates, then they were transfected with siRNA using Lipofectamine 2000 (Invitrogen, USA). After 24 h, the cells were digested and transferred to a T75 culture flask, and we collected cell supernatant for exosome isolation at 24 h and 48 h.
Tube formation test
The 96-well plate was pre-coated with Matrigel. Prior to testing, human umbilical vein endothelial cells (HUVECs) were cultured with ECM medium containing 100× growth factor and 5% FBS for 24 h. HUVECs were co-cultured with four groups for 24 h, including luva group, luva treated with siRNA group tRF-Leu-AAG-001, exosomes derived from luva group, and exosomes treated with siRNA group tRF -Leu-AAG-001. After treatment, HUVECs were added to 96 wells with 2.5*104 cells per well. The phenomenon of vascularization was observed under the Olympus microscope. ImageJ software was used to measure blood vessel nodes and capillary length.
This test was performed to identify the internalization of mast cell exosomes in HUVECs. Briefly, the isolated exosomes were resuspended in 200 µl PBS in a 1.5 ml microcentrifuge tube. Then, mast cell-derived exosomes were labeled according to the instructions using the PKH67 Green Fluorescent Cell Linker Mini Kit (Umibio Science and Technology, China) and incubated at 37°C for 1 h without shaking. The labeled exosomes were centrifuged at 10,000 g for 70 min and the supernatant was carefully filtered with a 0.22 μm filter. PHK67-tagged exosomes were then co-cultured with HUVECs for 24 h in a 6-well plate. The cells were then prepared for immunofluorescence analysis, and the internalization of exosomes was then observed under a confocal laser scanning microscope (LEICA TCS SP8, Germany).
Experimental data was statistically analyzed using GraphPadPrism8.0 (GraphPad Software, USA) and SPSS software (version 21.0; IBM, Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation (SD). Statistical comparisons between the two groups were made using a two-tailed Student’s t-test, and multiple comparisons were made using a one-way analysis of variance (ANOVA). P a value