Preparation of peritoneal fluid samples
Peritoneal fluid samples were collected from patients with regular menstrual cycles who underwent a salpingo-oophorectomy or evisceration for the treatment of ovarian endometriotic cysts without any hormonotherapy for at least 3 months prior to operation (EMS group, n = 20) and premenopausal patients who underwent hysterectomies for subserousal leiomyoma without evidence of endometriosis (control group, n = 16) under sterile condition after the laparoscopy in order to minimize blood contamination. The protocols were approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University (2019-KY-197). All patients signed informed consent.
Preparation and identification of PFMCs
PFMCs were prepared and identified according to previous report [18]. Peritoneal fluid samples collected from EMS and control groups as described above were centrifuged at 200g for 5 min, and the supernatant was removed. Cell pellets were re-suspended in phosphate buffered saline (PBS), and isolated by Histopaque-1077 (Sigma, USA) according to the manufacturer’s instructions. Cells were centrifuged at 150g for 30 min, and collected at the interface. For the identification of PFMCs (purity > 97%), indirect immunofluorescence (IIF) was conducted. Anti-CD3 (Abcam, USA), anti-B19 (Abcam, USA), anti-CD56 (Invitrogen, USA), and anti-CD14 (Abcam, USA) monoclonal antibodies were used to identify T lymphocytes, B lymphocytes, natural killer lymphocytes, and macrophages.
Isolation and purification of CD4+ T cells
Peripheral blood mononuclear cells (PBMCs) were collected from healthy fertile women and isolated by Histopaque-1077 (Sigma, USA) according to the manufacturer’s instructions, washed twice with RPMI-1640 medium (Gibco, USA), counted by a Neubauer hemocytometer, and re-suspended at 1 × 106 cells/mL. MagniSort™ Human CD4 T cell Enrichment Kit (Invitrogen, USA) was used to isolate CD4+ T cells according to the manufacturer’s instructions (purity > 95%). Naïve CD4+ T cells were isolated using MagniSort Human CD4 Naive T cell Enrichment Kit (eBioscience, USA). The protocols were approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University. All patients signed informed consent.
For CD4+ T cell transfection, lentivirus-mediated H19 over-expression (lenti-H19), lentivirus-mediated miR-342-3p mimic, lentivirus-mediated miR-342-3p inhibitor lentiviral vectors and scramble sequence was set as negative control.
Th17 polarization induction
CD4+ T cells differentiation into Th17 cells were performed according to previous report [19]. CD4+ T cells (5 × 105) were incubated for 48 h with anti-CD3 (1 μg/mL) (Abcam, USA), anti-CD28 antibody (1 μg/mL) (Abcam, USA), IL-1β (20 ng/mL)(Gibco, USA), IL-6 (20 ng/mL) (Gibco, USA), IL-23 (20 ng/mL) (Invitrogen, USA), IFN-γ-neutralizing antibody (2 μg/mL) (Cell Signaling Technology, USA), and IL-4-neutralizing antibody (2 μg/mL) (Cell Signaling Technology, USA).
Quantitative real-time RCR (qRT-PCR)
Total RNAs from PFMCs and CD4+ T cells were extracted by Trizol (Invitrogen, USA), and inversely transcribed into cDNA using the High-Capacity cDNA archive kit (Invitrogen, USA). qRT-PCR was conducted to measure H19 and miR-342-3p expression using PowerUp™ SYBR™ Green Master Mix (Invitrogen, USA). The relative expressions of H19 and miR-342-3p were expressed as a function of threshold cycle (Ct) and analyzed by 2−ΔΔCt method. Specific primers for H19 and miR-342-3p were as follows: H19, F: 5′-GCTCCACTGACCTTCTAAAC-3′; miR-342-3p, F: 5′-UCUCACACAGAAAUCGCACCCGU-3′.
Western blot
PFMCs and CD4+ T cells were lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, China). Protein samples (50 ng) was separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Invitrogen, USA). The membrane was incubated with primary antibodies against IER3 (Invitrogen, USA), β-actin (Abcam, USA) and horseradish peroxidase-conjugated secondary antibody (Abcam, USA). Blots were detected by enhanced chemiluminescence, and band intensities were quantified using image software Image Lab (Bio-Rad, USA). β-actin was used as an internal control.
Flow cytometry
PFMCs or CD4+ T cells were collected and re-suspended at 2 × 106 cells/mL. Cells were detected by BD FACSCanto II flow cytometry (BD, USA) and analyzed using CELLQuest software. Cells positive for both CD4 and intercellular IL-17A were considered as Th17. Cells were collected and incubated with APC-conjugated anti-CD4 antibody (Invitrogen, USA), anti-IL-17A antibody (Invitrogen, USA) and anti-IFN-γ (Invitrogen, USA) for the observation of Th17 cells.
Enzyme-linked immuno sorbent assay (ELISA)
The cytokine IL-17 level from CD4+ T cell culture supernatant was detected by the IL-17A Human ELISA Kit (Invitrogen, USA).
Luciferase reporter assay
The sequence of IER3 3′UTR or the mutated sequence was predicted to interact with miR-342-3p and inserted into pGL3 vector (Promega, USA) which were noted as IER3 3′UTR wild-type (WT) or IER3 3′UTR mutant (MUT) that constructed by Sangon Biotech (Shanghai, China). The reporter plasmid (IER3 3′UTR WT or IER3 3′UTR MUT, 500 ng) and microRNAs (miR-342-3p mimic, miR-342-3p inhibitor, or negative control, 1000 ng) were transfected into HEK293 cells for 48 h for Luciferase reporter assay. Luciferase activity was measured by dual Glo™ Luciferase Assay System (Promega).
Isolation and culture of ESC
ESCs were isolated and cultured according to previous report [5]. Endometrial tissues were collected from patients with regular menstrual cycles but without endometriosis and/or adenomyosis who underwent a hysterectomy for the treatment of uterine leiomyoma under sterile conditions and none of the included patients had experienced hormonotherapy. Tissues were digested with 0.1% collagenase type IV (Sigma, USA) at 37 °C for 30 min with constant agitation. Then, sterile gauzes (pore diameter size: 200 mesh) was used to filtrate the tissue pieces to remove debris. The supernatant was discarded during gentle centrifugation, and the cells were re-suspended in DMEM/F-12 medium (Gibco, USA). For removal of epithelial cells, ESCs were passed through sterile gauzes (pore diameter size: 400 mesh). For removal of leukocytes and erythrocytes, the filtrated suspension was layered over Ficoll, and centrifuged at 800×g for 20 min. The middle layer was collected and washed with D-Hanks solution. Then, ESCs were placed in a culture flask, and allowed to adhere for 20 min. The adherent stromal cells were cultured as monolayer in flasks with DMEM/F-12 (Gibco, USA) containing 10% Fetal bovine serum (Gibco, USA), and incubated in 5% CO2 incubator at 37 °C. The protocols were approved by the Ethics Committee of The First Affiliated Hospital of Zhengzhou University. All patients signed informed consent.
Co-culture of CD4+ T cells and ESC
To establish ESC and CD4+ T cells co-culture unit, ESCs were cultured in 48-well plates at a concentration of 2 × 105 cells/well until they adhered to the plastic. Then, the media were removed, and CD4+ T cells with different treatment were applied over ESCs at the same concentration for 48 h.
MTT assay
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect ESC proliferation. ESCs (2 × 104) were seeded into a 96-well plate and incubated overnight. 20 μL MTT (5 mg/mL; Invitrogen, USA) was added to each well and cultured for 4 h. Then, cells were lysed using dimethylsulfoxide (150 μL/well; Sinopharm Chemical Reagent, China). The optical density was read at 570 nm.
Resazurin assay
Resazurin assay was also used to detect ESC proliferation according to previous report [20]. ESCs (2 × 104) were seeded into a 96-well plate and incubated overnight. 20 μL resazurin solution (0.1 mg/mL; Invitrogen, USA) was added to each well and cultured at 37 °C for 4 h. Fluorescence intensity was monitored, the excitation at 530 nm and emission at 590 nm was measured using a microplate reader (Thermo Scientific, USA).
Intraperitoneal endometriosis model
The nude mouse endometriosis model was established according to previous report [5]. The nude mice (8 week-old) and female C57BL/6 mice were purchased from Laboratory animal center of Zhengzhou University. The animal experiments were approved by Ethics Committee of The First Affiliated Hospital of Zhengzhou University. Nude mice were used to construct an allotransplantation of intraperitoneal endometriosis model. At 0th day, the uterus of female C57BL/6 mice was minced, and the tissue debris was intraperitoneally injected into the nude mice. At 5th day, CD4+ T cells from female C57BL/6 mice were transfected with lenti-NC or lenti-H19, induced for Th17 polarization and transferred to the abdominal cavity in endometriosis nude mice. So the nude mice were divided into EMS, EMS + Th17, EMS + lenti-NC + Th17, and EMS + lenti-H19 + Th17 groups, with six mice in each group. At 14th day, the nude mice were sacrificed, and the endometriosis-like lesions and PF were collected for the detection of lesion weight, and LncRNA H19, miR-342-3p and IER3 expressions.
Statistical analysis
All data were presented as mean ± standard deviation and analyzed by SPSS software (Version 15.0, USA). Each experiment was repeated for three times. The differences between groups were assessed by t-test or one-way analysis of variance (ANOVA), with p < 0.05 considered statistically significant.
