2.1 Ethics Approval and Consent to Participate
The written informed consent of each patient participating in the study was obtained. The study protocol and informed consent were approved by the ethics committee of the Affiliated Hospital of Medical School of Ningbo University. All of the methods were carried out in accordance with the Declaration of Helsinki.
2.2 Sample collection
All samples (normal/ectopic endometrial tissues and leucorrhea) were collected in the Affiliated Hospital of Medical School of Ningbo University from March 2020 to March 2021. A total of 51 females were enrolled in our study. Among all patients, 26 patients who were diagnosed with EMS through laparoscopy and histopathological examination served as the control group. The remaining 25 patients with non-endometriosis who were admitted to the hospital during the same period included as the control group. Inclusion criteria: 1. No history of treatment with hormones or antibiotics within three months before laparoscopic surgery; 2. No hepatitis, tuberculosis, tumor and other diseases. Exclusion criteria: 1.Treated with hormones and antibiotics recently; 2. With serious organic diseases; 3. Combined with other gynecological diseases such as inflammation of the reproductive system and tumors. (The general information of the enrolled patients was shown in Supplementary table 1). All subjects who had regular menstrual cycles were women of childbearing age who were in non-menstrual period three days before the sample collection. Mast cell line-Luva was generous gift from a laboratory at Zhejiang University.
2.3 Exosomes isolation from tissues and leucorrhea
We used differential centrifugation to extract exosomes from tissues and leucorrhea.Briefly: ectopic tissue was disaggregated into a single cell suspension with type IV collagenase (Solarbio, China). Leucorrhea was diluted with PBS to make a mixed solution. The supernatant and leucorrhea solution were centrifuged at 4°C with a high-speed centrifuge (Thermo, USA) at 500g for 10mins to remove living cells, 2000g for 10mins to remove dead cells, and 10,000g for 20mins to eliminate the cell debris. Every step was repeated twice. The supernatant was then centrifuged at 100,000g twice with ultracentrifuge (Beckman, USA) for 70 minutes each time. The exosomes were resuspended or lysed with different reagents for subsequent experiments.
2.4 Exosomal size identification
Transmission electron microscopy (TEM) was used to identify the size of exosomes. Briefly, the exosome was dropped on the copper net for 5 mins at room temperature. 3% phosphotungstic acid solution stained the nanoparticles. Then, 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)
2.5 Immunoblotting for exosomal markers
Exosomes were lysed with a RIPA buffer, resuspended in the loading buffer, boiled at 95°C for 5 minutes, and then electrophoresed on SDS-PAGE. Proteins were transferred to polyvinylidene fluoride membrane, which was blocked with 5% non-fat dry milk 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 a dilution of 1:1000 followed by incubation at 4°C overnight. The next day protein was incubated with 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) to analyze.
2.6 Library construction and small RNA sequencing
ExoRNA was extracted with Trizol reagent (Invitrogen,USA), and purified RNA was sent to Aksomics Biological Engineering Co., Ltd. (Shanghai, China) for performing tRFs & tiRNAs sequencing analysis. The brief steps were as follows: agarose gel electrophoresis was used to detect the integrity of the total RNA sample, and NanoDrop ND-1000 quantitative analyzer (thermos, USA) quantified RNA concentration. TRF&tiRNA-seq library preparation includes: 1).3'-adapter ligation; 2). 5'-adapter ligation; 3). cDNA synthesis; 4). PCR amplification; 5). size selection of 134-160bp PCR amplified fragments (corresponding to ~14-40nt small RNA). The library was quantitatively analyzed with Agilent 2100 bioanalyzer. According to the quantitative results, the library was mixed in equal amounts. The DNA fragments in the mixed library were denatured with 0.1M NaOH to generate single-stranded DNA molecules, which were loaded onto the kit at a concentration of 1.8 pM. According to the manufacturer's instructions, the NextSeq 500/550 V2 kit (#FC-404-2005, Illumina) was used for sequencing with the NextSeq system. R package edgeR software was used to screen the differentially expressed TRFs and tiRNAs based on the count value.
2.7 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℃. The cDNAs were synthesized by using a reverse transcription kit, according to 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 using RT-PCR quantitation kit (CWBio, Beijing, China). Briefly, after an initial denaturation step at 95 °C for 10 min, the amplifications were carried out with 40 cycles at a melting temperature of 95 °C for 15 s, and an annealing temperature of 60 °C for 30 s. The relative expression levels of mRNAs were calculated with 2–ΔCt method. PCR productions of tRF-Leu-AAG-001, tRF-Leu-TAG-015 were tested by 3% agarose. The sequences of the specific primers were presented in table 1.
2.8 3D cell culture
Ectopic tissues were digested into the single-cell suspension with type IV collagenase (Solarbio, China). After centrifugation to pellet the cells, NanoShuttle (50μl, Greiner bio-one Co., Germany) was added to the cell suspension, and incubated the cell-nano mix suspension was incubated at 37 ℃ for 1hour. After centrifugation to remove the supernatant, the number of cells was adjusted to 8*104/150ul with the medium mix. The cells were inoculated into a 96 well microplate (cell-repellent surface, Greiner bio-one Co., Germany). Then we hold the microplate on a magnetic driver (Greiner bio-one Co., Germany). The cell balls were placed in a 37°C, 5% cell incubator and incubated for 15 minutes, and then the magnetic driver was removed.
2.9 Fluorescence positioning
We used immunofluorescence and RNA fluorescence probes for 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 96-well plate for 1h, Cy3-labeled tRF-Leu-AAG-001 fluorescence probe was added and incubated overnight at 37°C. The next day, cell balls were washed with PBS for 5 minutes, protected from light, three times, then added anti-CD117-FITC antibody and incubating at 37°C for 1 hour. Aspirated the secondary antibody and washed with PBS in the dark. Finally, added DAPI solution at room temperature for 5 minutes, photograph the fluorescence with Olympus confocal microscope.
2.10 knockdown of tRF-Leu-AAG-001 by Small interfering RNA
tRF-Leu-AAG-001 siRNA and negative control (NC) were designed and compounded by Sangon Biotech. Luva was seeded into 6-well plates, and then they were transfected of siRNA by using Lipofectamine 2000(Invitrogen, USA). After 24h, cells were digested and transferred to T75 culture flask, and we collected the cell supernatant for exosomes isolation at 24h and 48h.
2.11 Tube formation assay
The 96-well plate was pre-coated by Matrigel. Before the test, human umbilical vein endothelial cells (HUVECs) were cultured with ECM medium containing 100x growth factor and 5%FBS for 24h. HUVECs were co-cultured with four groups for 24 hours, including luva group, luva treated with tRF-Leu-AAG-001 siRNA group, exosomes derived from luva group, and exosomes treated with tRF-Leu-AAG-001 siRNA group. After treatment, HUVECs were added to 96 wells with 2.5*104 cells per well. The vascularization phenomenon was observed under the Olympus microscope. ImageJ software was used to measure blood vessel nodes and capillary length.
This assay was performed to identify the internalization of the exosomes from mast cells into HUVECs. Briefly, isolated exosomes were re-suspended in 200 ul of 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 Techology,China) and incubated at 37℃ for 1hour without shaking. Labeled exosomes were centrifuged at 10000g for 70 min, and the supernatant was carefully filtered with a 0.22-μm filter. PHK67-labeled 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 subsequently observed under a Confocal laser scanning microscope(LEICA TCS SP8,Germany).
2.13 Statistical Analysis
The experimental data were 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 performed using a Two-tailed Student's t-test, and multiple comparisons were performed using a One-Way Analysis of Variance (ANOVA). P value<0.05 indicates statistical significance.