All tissue samples were obtained with full and informed patient consent, and all experiments were followed the ethical principles outlined by the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. This study was approved by institutional ethics review board of the Second Xiangya Hospital, Central South University.
Clinical sample collection
In this study, snap-frozen cyst walls of ovarian endometriomas and matched eutopic endometrium of the uterus, from the same patient who underwent laparoscopy, were simultaneously collected (20 to 40 years of age, proliferative phase, n = 30, revised classification of American Fertility Society, r-AFS stage III/IV). Paired specimens, selected randomly, and twenty normal endometrium from women free of endometriosis (control group) (20 to 40 years of age, proliferative phase, n = 20) were as controls. All the patients had regular menstrual cycles and did not receive hormonotherapy or immunosuppressor for at 6 months prior to the specimen collection. Furthermore, patients in the control group had no evidence of tumor in the endometrium and did not have histological diagnosis of adenomyosis. The specimen diagnosis was determined by histopathology and the menstrual cycle was confirmed by both last menstrual period and histological ascertain. Some of the tissue specimens were fixed in formalin and some were stored at − 80 °C.
Samples of eutopic and ectopic endometrial tissues were taken from three EMS patients. Total RNA was isolated using Trizol reagent (15,596,018, Invitrogen, Calsbad, CA, USA) according to the manufacturer’s protocol. RNA sample concentrations were determined by OD260/280 using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity was checked by agarose gel electrophoresis at a 28S:18S ratio of ≥1.5. miRNA sequencing libraries were An Illumina TruSeq® Small RNA Library Prep Kit. A total of 5 μg of RNA was used in per sample, which was quantified and checked using the KAPA library quantification kit (KAPA Biosystems, Wilmington, MA, USA). Paired-end sequencing was performed on an Illumina NextSeqCN500 sequencer. High-throughput sequencing of EMS eutopic and ectopic endometrial tissue samples was performed to screen for differentially expressed miRNAs in EMS patients.
GSE7846 microarray data were obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds), a dataset consisting of endometrial cells from five non-EMS patients and five EMS patients. Differentially expressed genes (DEGs) between the EMS and non-EMS samples were analyzed using the R language “limma” package (http://www.bioconductor.org/packages/release/bioc/html/limma.html), with p value < 0.05 as the threshold value.
Isolation and cultivation of ESCs
EcESCs, and normal human ESCs (hESCs) were isolated from the above collected tissues. The tissue samples were added with 3 times the volume of 0.25% type IV collagenase-trypsin ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich Corporation, St. Louis, MO, USA) solution and pre-warmed to 37 °C. After digestion, the tissue solutions were filtered through 150 μm and 74 μm stainless steel cell filters (GongLu; Hangzhou, China) to obtain cells, respectively. These cells were finally cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (A4192001, Gibco, Waltham, CA, USA) containing 10% fetal bovine serum (FBS, 16140071, Gibco) and 1% antibiotic-antifungal solution (100 ×, 15,240,112, Gibco). After purification, the morphology was observed by immunofluorescence staining and the expression of vimentin in the cells was identified by immunofluorescence and immunohistochemistry (IHC).
For Transwell co-culture system, EcESCs, dimethyl sulfoxide (DMSO) and the sphingomyelin inhibitor GW4869 (10 μM, Sigma-Aldrich) were added to the apical chamber, and EcESCs were added to the basolateral chamber for 24 h. Cells were differently treated by DMSO or GW4869.
Primary and passaged ESCs were seeded onto sterile slides, and the adherent cells were removed after incubation, washed in phosphate buffer saline (PBS), fixed in 4% paraformaldehyde for 20 min, and blocked for 2 h in blocking solution. Cells slides were incubated for 3–5 h at room temperature with Vimentin primary antibody (1/200, 5741, Cell Signaling Technology, Hercules, CA, USA), and then with fluorescence secondary antibody immunoglobulin G (IgG) H&L (ab6785, 1:100, Abcam, Cambridge, UK) in the dark room. Cell nuclei were stained with 4′,6-Diamidine-2-phenylindole dihydrochloride (DAPI, C1025, Beyotime, Nantong, China) for 15 min, and cell morphology and Vimentin expression were observed under fluorescence microscope.
Cells were transfected with the plasmids of short hairpin RNA against AFAP1-AS1 (sh-AFAP1-AS1), BCL9 overexpression vector (oe-BCL9), or their corresponding negative control (sh-NC or oe-NC). Expression vectors were transfected into 293 T cells separately using the Lipofectamine 2000 (Invitrogen), and the supernatant was collected to obtain the viral solution. For transfection, EcESCs (1 × 106) were seeded in 6-well plates with 2 mL of medium per well, and infection was performed when cell confluence reached 50%. Separately, 800 μL of fresh viral solution was mixed with 800 μL of complete medium, while Polybrene (6 μg/mL, TR-1003-G, Sigma-Aldrich) was added. At 12 h after transfection, fresh complete medium was added and cells were further cultured at 37 °C in 5% CO2. Cells were placed in medium containing puromycin (1 μg/mL, A1113803, Thermo Fisher Scientific) 48 h after infection to screen stably transfected cell lines. Cells were collected when they were mortal in the puromycin-containing medium and overexpression or silencing efficiency was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) or Western blot analysis.
Cells were transfected with plasmids of miR-15a-5p mimic, miR-15a-5p inhibitor or the matched NC. Cells were seeded into six-well plates and transfected with the above plasmids (50 nM; GenePharma, Shanghai, China) according to the Lipofectamine 2000 reagent instructions. Cells were collected 48 h after transfection.
Isolation and identification of Exo
Exo was obtained from the supernatant of EcESCs medium using the Exo Extraction Kit (ExoQuick; SBI, CA, USA) according to the instructions. When EcESCs confluence reached 90% in the culture dish, the medium was replaced with Exo-free 1640 medium containing FBS. After 24 h of incubation, the cell culture medium supernatant was collected from 20 mL of medium (1 × 107 cells) and centrifuged at 3000×g for 15 min. Then 1000 μL of ExoQuick Exosome precipitation solution was added to 1000 μL of supernatant and the mixture was frozen at 4 °C for 30 min. After centrifugation at 1500 g for 30 min, the Exo precipitate was resuspended with 100 μL of sterile 1× PBS and its supernatant was used as a control without Exo. Exo morphology was subsequently observed by transmission electron microscopy (TEM) and the Exo protein markers CD9, CD81, CD63, and Tsg101 were identified by Western blot analysis.
Transmission electron microscopy (TEM)
After the separation of Exo, images of Exo were taken using TEM. Exo was resuspended and fixed in 30 μL of 2% paraformaldehyde and then adsorbed onto a discharged copper grid. Exo was stained after addition of 4% uranyl acetate. Exo images were acquired by a Hitachi H7650 TEM (Japan).
Nanoparticle tracking analysis (NTA)
A total of 20 μg Exo was dissolved in 1 mL PBS and vortexed for 1 min to maintain a uniform distribution of Exo, followed by measurement of Exo size distribution using a NTA (Malvern Instruments Ltd., Malvern, UK).
Internalization of Exo
Exo was labeled with the membrane labeling dye PKH67 green fluorescence (HR8569, BjBalb, Beijing, China). Exo secreted by control cells was labeled using PKH67 dye, and the labeled Exo was co-cultured with EcESCs for 24 h, after which the cells were fixed with 4% paraformaldehyde and the nuclei were stained with 10 μg/mL of DAPI staining solution (C1025, Beyotime) for 10 min. The uptake of labelled Exo by recipient cells was observed using a Nikon Eclipse fluorescence microscope (Nikon, Tokyo, Japan).
Total RNA was extracted from cells, Exo or tissues using TRIzol® reagents (15,596,018, Invitrogen), while lncRNA and mRNA levels were detected by reverse transcription using the PrimeScript™ RT-qPCR kit (RR047A, Takara, Japan). Reverse transcription was performed to detect miRNA levels using the PrimeScript™ miRNA RT-qPCR kit (B532451, Sangon Biotech, Shanghai, China). The SYBR® Premix Ex Taq™ II kit (RR820A, Takara) was used for sample configuration and samples were subjected to real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal reference for lncRNA and mRNA while U6 was used as an internal reference for miRNA. The primers used for amplification were provided by General Biotechnology (Shanghai, China). Primer sequences are listed in Supplementary Table 1. The relative transcript levels of the target genes were calculated using the relative quantification method (2-ΔΔCT method), in which mRNA relative transcript levels of the target genes = 2-ΔΔCt. Three replicate wells were set up for each sample and each set of experiments was repeated 3 times.
Western blot analysis
Cells were digested with trypsin and collected. Cells were lysed with enhanced radio immunoprecipitation analysis (RIPA) lysis buffer containing protease inhibitors (AR0102, Boster, Wuhan, China) and protein concentrations were determined using the bicinchoninic acid (BCA) protein quantification kit (AR0146, Boster). The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophores (SDS-PAGE), which were electrotransferred to polyvinylidene fluoride (PVDF) membranes. The membrane was sealed with 5% BSA for 1 h at room temperature and then incubated with diluted primary antibodies of CD9 (1/1000, ab195422, Abcam), CD81 (1/1000, ab109201, Abcam), CD63 (1/1000, ab134045, Abcam), Tsg101 (1/1000, ab83, Abcam), Calnexin (1/1000, ab22595, Abcam), BCL9 (1/1000, 22,947–1-AP, Proteintech, VA, USA), and α-tubulin (1/2000, ab52866, Abcam) overnight at 4 °C. The membranes were added with horseradish peroxide (HRP)-labelled goat anti-mouse secondary antibody (1:2000, ab6808, Abcam) for 1 h at room temperature. An appropriate amount of enhanced chemiluminescence (ECL) working solution (AR1174, Boster) was added. The transfer film was incubated at room temperature for 1 min, and X-ray film was placed in the dark box for 5–10 min before development and fixation. The bands in the Western blot images were quantified in grey scale using Image J analysis software, and α-tubulin was used as an internal reference.
Cell counting kit-8 (CCK-8)
Cell proliferation capacity was assessed using the CCK-8 (K1018, Apexbio, USA) kit. Groups of EcESCs at logarithmic growth phase were taken and cells were seeded into 96-well culture plates at a density of 1 × 103 cells/well, with 100 μL of medium containing 10% FBS added to each well. Cells were incubated for the indicated time, then 10 μL of CCK-8 solution was added to each well of the plate and incubated for an additional 4 h at 37 °C. After incubation, optical density (OD) values were measured at 450 nm, and absorbance values were recorded on day 1, day 2 and day 3. Cell growth curves were plotted with five parallel wells set up for each experiment.
For cell migration capacity analysis, 2 × 105 transfected cells were suspended in serum-free DMEM (200 μL) and added to the apical chamber of a Transwell uncoated with Matrigel (356,234, BD Bioscience, San Jose, CA, USA).
For cell invasion analysis, Matrigel (356,234, BD Bioscience) was diluted (1:10) in serum-free DMEM and diluted Matrigel (100 μL) was added to the apical chamber of the Transwell and incubated for more than 30 min. Then 2 × 105 transfected cells were then seeded into the apical chamber coated with Matrigel.
The basolateral chambers were both spiked with DMEM containing 10% FBS (600 μL) and then incubated at 37 °C in an incubator for 24 h. Cells were then fixed with 4% paraformaldehyde for 15 min and stained with crystal violet (0.1%) for 15 min. The stained positive cells were then observed using an inverted light microscope (CarlZeiss, Germany) and photographed for imaging, with ImageJ software used for positive cell counting.
Dual luciferase reporter gene assay
The target binding sequence of AFAP1-AS1 to miR-15a-5p 3’untranslated region (UTR) was inserted into the psh-Check2 plasmid, wild type (WT-AFAP1-AS1), followed by the construction of a AFAP1-AS1 sequence mutation vector using a point mutation kit (Takara), as MUT-AFAP1-AS1. The target binding site of BCL9 to miR-15a-5p was inserted into the pGL3 promoter vector (WT-BCL9), followed by the construction of a BCL9 sequence mutagenesis vector (MUT-BCL9) using a point mutation kit (Takara).
The constructed vector was then co-transfected with miR-15a-5p mimic or miR-NC (50 nM; GenePharma) in ESC using Lipofectamine 2000 reagent (11,668,019, Thermo Fisher Scientific), respectively. At 48 h post-transfection, relative luciferase activity was measured using the Dual Luciferase Reporter Assay Kit (E1910; Promega, Madison, WI, USA), and firefly luciferase activity was measured in the Dual Luciferase Reporter Assay System (Promega) with renilla luciferase activity used as an internal reference.
RNA-pull down assay
Cells were transfected with biotin-labeled NC-Bio, AFAP1-AS1(MUT)-Bio, and AFAP1-AS1(WT)-Bio (50 nM each). After 48 h of transfection, cells were collected and washed with PBS. Cells were then incubated with specific cell lysis buffer (Ambion, Austin, TX, USA) for 10 min, after which 50 mL of sample cell lysis buffer was dispensed. The residual lysate was incubated with M-280 streptavidin magnetic beads (Sigma-Aldrich) pre-coated with RNase-free and yeast tRNA (Sigma-Aldrich) for 3 h at 4 °C. RNA was extracted and detected by RT-qPCR.
RNA immunoprecipitation (RIP)
Cells were lysed in an ice bath for 5 min with lysis buffer and enzyme inhibitor (1,111,111, Roche, Germany), centrifuged at 14,000 rpm for 10 min at 4 °C to remove the supernatant. The binding of AFAP1-AS1 to miR-15a-5p protein was then detected using a RIP kit (Millipore, Billerica, MA, USA). One third of the cell extracts was removed as input and the remaining two thirds were incubated with antibodies for co-precipitation, respectively. Specifically, cells were mixed with 5 μg of antibody AGO2 (ab32381, 1/50, Abcam) at room temperature for 30 min, while IgG (ab109489, 1/100, Abcam) and magnetic beads were resuspended in 100 μL of RIP Wash Buffer. The magnetic bead-antibody complex was washed and resuspended in 900 μL of RIP Wash Buffer and incubated overnight at 4 °C with 100 μL of cell extract. Samples of the co-precipitation reaction system were placed on magnetic base to collect the bead-protein complexes. The co-precipitation reaction system samples and the Input were digested with proteinase K and RNA was extracted.
Paraffin sections of nude mouse endometriotic tissue were placed in a thermostat and stored at 60 °C for 2 h. Sections were dewaxed with xylene and hydrated with graded ethanol (100, 95, 85, and 70%). Then the sections were soaked in citrate buffer (0.01 mol/L, pH = 6.0) and heated at 95–100 °C for 30 min for antigen repair. Sections were washed with PBS and incubated with 0.5% TritonX100 for 30 min, followed by incubation with BCL9 primary antibody (1/100, 22,947–1-AP, Proteintech) overnight at 4 °C. Subsequently, sections were incubated with HRP-labelled goat anti-rabbit IgG secondary antibody (1:500; Life Technologies, Carlsbad, CA, USA) for 1 h at room temperature, followed by treatment with diaminobenzidine solution for 3–5 min. Sections were counterstained with hematoxylin for 1–3 min, dehydrated, and mounted with neutral balsam. A brown chromogen on the membrane indicates a positive immunoreaction. Images were visualized using a Nikon ECLIPSE Ti microscope (Fukasawa, Japan) system, and five high magnification fields were randomly selected during analysis to calculate the rate of positive cells out of 100 cells per field.
Establishment of EMS nude mice model
Twenty Balb/c female nude mice (6–8 weeks, Kaixue Biotech, Shanghai, China) were housed in a sterile environment for 2 weeks in adaptive housing, with a light/dark cycle (12 h/12 h) at 23–25 °C. Ectopic endometrial tissues were obtained from 10 EMS patients, and endometrial fragments were resuspended in PBS and injected intraperitoneally into anesthetized nude mice through a 19-gauge needle (all patients’ tissue fragments were mixed well and each nude mouse was injected with tissues containing approximately 30 mg/0.2 mL of PBS). After 7 days of endometrial tissue implantation (day 7 of moulding), the nude mice were randomly treated with 10 mice in each treatment.
After EcESCs were transfected with plasmids of sh-NC and sh-AFAP1-AS1, the Exo was extracted from the culture medium supernatant separately and resuspended in PBS to a concentration of 30 μg/200 μL. Ten nude mice were injected intraperitoneally with sh-AFAP1-AS1-enriched Exo (Exo-sh-AFAP1-AS1) every 2 days; the other ten nude mice were injected intraperitoneally with sh-NC-enriched Exo (Exo-sh-NC) as control. After 24 h of the last injection (day 14 of modeling), the nude mice were euthanized and endometriotic tissues were collected for subsequent experimental analysis. The animal experiment protocol was approved by the Animal Ethics Committee of Second Xiangya Hospital, Central South University.
All data were processed using SPSS 21.0 statistical software (SPSS, Inc., Armonk, NY, USA) and GraphPad Prism7. Measurement data derived from three times of experiments were expressed as mean ± standard deviation. Comparisons between two groups were made using independent samples t-test, and comparisons among multiple groups were performed by one-way analysis of variance (ANOVA). Cell proliferation at different times was performed by two-way ANOVA. A p < 0.05 indicates that the difference is statistically significant.
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