Patients and sample collection
Fresh endometrial eutopic tissues from 11 patients with endometriosis were collected during the late proliferative phase of the menstrual cycle (Day11 ~ 13) and from 11 women of reproductive age who underwent oviduct obstruction as controls. The diagnosis was confirmed by laparoscopy. Samples were collected from October 2018 to December 2019 at the Reproductive Hospital Affiliated to Shandong University. Women undergoing hysteroscope-laparoscopic surgery for suspected endometriosis were confirmed by histological examination, no comorbidities such as PCOS, POI or IBD, so did the controls. Informed consent was obtained from patients before surgery. All surgical patients received no hormonal treatments 3 months prior to surgery. Patients data were collected, including age, body mass index, and hormone levels. The study was approved by the Institutional Review Board (IRB) of the Reproductive Hospital Affiliated to Shandong University.
Cell culture and transfection assay
Human endometrial stromal cells (HESC)were cultured with 90% non-phenol red DMEM/F12 (Gibco) supplemented with 10% FBS and 1% P/S in a 5% CO2 atmosphere at 37°C. si-STING were purchased from Genepharma (GenePharma, Co., Ltd., Shanghai, China). When HESC reached 70–80% confluence, transfection was conducted with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cells were harvested after 48 h for other assays.
Protein extraction and western blotting
Radioimmunoprecipitation assay buffer (RIPA, Beyotime, Shanghai, China) was used to lyse cells after transfection for 48 h, followed by the determination of the protein concentration using bicinchoninic acid (BCA; Beyotime). The protein was separated by 10% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer to a polyvinylidene difluoride (PVDF) membrane (Immobilon-P). After blocking in 5% non-fat milk at room temperature for 1 h, the primary antibody was added and incubated in a refrigerator at 4°C overnight. Tris-buffered saline-Tween (TBST) was used to wash the membrane for 15 min 3 times, and the corresponding secondary antibody was added and incubated for 1 h at room temperature (about 25℃). After washing each sample for 15 min 3 times with TBST, the protein was detected using the ECL detection system (Millipore, Billerica, MA, USA). Primary antibodies against rabbit anti-human STING (1:1000 dilution, CST13647, USA), rabbit anti-human TBK1(1:1000 dilution, CST38066, USA), rabbit anti-human p-TBK1(1:1000 dilution, CST5483, USA), rabbit anti-human IRF3(1:1000 dilution, CST4302, USA), rabbit anti-human p-IRF3(1:1000 dilution, CST29047, USA), and mouse anti-human Tublin (1:10000 dilution; Proteintech 11224-1-Ig, Rosemont, IL, USA) were used. Goat anti-rabbit and Goat anti-mouse HRP-conjugated secondary antibodies were obtained from ZSGB-BIO (1:10000 dilution, Beijing, China).
5-Ethyl-2′-Deoxyuridine Incorporation (EdU) assay
For the EdU assay, HESC were incubated with 50 µmol/L EdU (RiboBio, Guangzhou, China) for 6 h at 37°C and then fixed with 4% paraformaldehyde for 30 min at room temperature. After incubation with Apollo reaction reagent for 30 min, the cells were stained with Hoechst 33342. The Olympus 1X51 (Tokyo, Japan) was used to capture images of HESC, and cells in five random fields per group were counted. The EdU incorporation rate was estimated as the ratio of EdU-positive cells to Hoechst-positive cells.
Transwell assays
Transwell assays were performed using 24-well plates with 8-µm pore size inserts (Corning Life Sciences, Corning, NY, USA) according to the manufacturer’s protocols. HESC were plated into Transwell chambers after 48 h of transfection. The essential difference between the migration and invasion assays is that, for migration assays cells can migrate through the inserts directly, but for invasion assays they have to invade the matrix to reach the inserts. Details are shown as follows.
In the migration assay, the HESC(105cells/well) were seeded into the upper chamber in 200 µL of serum-free phenol red-free DMEM/F12 and allowed to migrate for 24 h to the lower chamber, which contained phenol red-free DMEM/F12 with 10% FBS.
In the invasion assay, Matrigel (1 mg/mL; BD Biosciences, Franklin Lakes, NJ, USA), prepared in serum-free phenol red-free DMEM/F12 medium, was placed in the upper chamber and incubated for 8 h at 37°C. Similar to the migration assay, HESC (105 cells/well) were seeded in the upper chamber with 200 µL of serum-free medium and allowed to invade the lower chamber, which contained medium with 10% FBS, for 36 h.
Transwell filters were fixed with 4% paraformaldehyde for 30 min, stained with hematoxylin for 30 min, and fixed on a glass slide. Cells that did not migrate or invade were wiped off with cotton swabs. The results were expressed as the number of cells that migrated or invaded per field, as counted using the Olympus 1X51 microscope in five random fields.
Immunohistochemistry analysis
Fresh tissue samples were washed with PBS several times to remove blood. Then, they were fixed in 4%PFA for 24h and embedded into paraffin. The samples were cut into 4um sections and mounted onto glass slides. Deparaffinized, rehydrated sections were incubated with 3%H2O2 for 30min to block endogenous peroxidase activity. Antigen retrieval was performed using a pressure-cooker for 15min in EDTA buffer in boiling water. The sections were rinsed in PBS, blocked with 10% BSA for 30min, and then incubated with primary antibodies rabbit anti-human STING (diluted 1:100, CST13647, USA) overnight in a wet chamber at 4 ℃. HRP-conjugated goat anti-rabbit or mouse IgG was used as the second antibody. HRP activity was detected by measuring the level of the DAB for several seconds. The sections were counterstained with haematoxylin before mounting. The sections were observed under an Olympus 1X51 microscope.
Elisa experiment
For the Elisa assay, protocols were based on Human IFN-beta ELISA Kit (R&D 41410-1). Add 50ul sample diluent, 50ul standard in blank microtiter plate, incubated 1h then aspirated and wash 3 times. Add 100ul diluted HRP solution, incubated 1h then as aspirated and wash 3 times. Add 100ul TMB substrate, incubated 15min in the dark do not seal or wash. Add 100ul Stop Solution and read plate within 5min at 450nm.
Statistical Analysis
Statistical analyses were performed using SPSS 21.0 (Statistical Package for the Social Sciences, SPSS, Inc., Chicago, IL, USA). Values were expressed as means ± SD, and comparisons were performed using Student’s t-tests. P < 0.05 was considered statistically significant. Each experiment was repeated three times.