Patients and samples
Forty-six participants were enrolled in Shanghai First Maternity and Infant Hospital from April 2019 to September 2020. The collection of the samples was approved by the Ethics Committee and obtained informed consent from all patients. The experimental group included 23 cases of ectopic cyst wall of the ovary endometrioma confirmed by pathology. The endometrium was obtained from 23 donors who received hysteroscopy for benign gynecological diseases, excluding endometriosis in the control group. None of the patients had other immune diseases, acute inflammation, or estrogen-dependent diseases, and no hormone medications were used within three months before surgery. Specimens from both the experimental and control groups were collected during surgery. The specimen was achieved and immediately placed into the specimen preservation solution and then transferred to the laboratory.
Primary cells isolation and culture
Endometrial stromal cells were isolated and cultured according to the methods described previously (44, 45) and with slight modification. Specimen tissues were washed with phosphate buffered saline (PBS) and cut into fragments. 1 mg/mL collagenase type IV (Sigma, USA) was added and digested for 20-40 min in a shaker at 37°C. Tissue homogenate was filtered through a 40 μm cell strainer (Falcon, USA), and the cell suspension was collected and centrifuged for 10 min to collect cells. The isolated primary cells were resuspended with complete medium DMEM/F12 (Hyclone, USA) containing 10% fetal bovine serum (FBS, Sciencell, USA) and cultured in an incubator at 37°C in a humidified atmosphere with 5% CO2.
Cell transfection
HESCs were purchased from American Type Culture Collection and cultured under the same conditions as primary cells. Three designed HK2-short hairpin RNAs (shRNAs) cloned into pGMLV-SC5-puro vector plasmids and scrambled control vector plasmids were transfected respectively with the lentiviral packaging mix into 293T cells to produce the virus for 48 h. The supernatant containing the virus was filtered through a 0.45 μm filter and used to infect HESCs. Seventy-two hours later, the infected HESCs were selected with 30 μg/mL puromycin (Invitrogen, USA), and the knockdown efficiency was detected. The sequences of shRNAs were listed in Supplementary Table 1.
RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
According to the reagent instructions, total RNA was extracted from tissues and cells using RNAiso Plus and transcribed into cDNA (Takara, Japan). Then qRT-PCR was carried out in triplicate on a 384-well plate using a QuantStudio 5 Flex Real-time PCR system (Applied Biosystems, USA). The expression of the target gene relative to ACTB was determined using the 2-ΔΔCT method. The sequences of primers were listed in Supplementary Table 1.
Western Blotting
Tissue and cellular proteins were extracted with RIPA lysate mixed with phosphatase inhibitors and quantified by the BCA method. Following the manufacturer's instructions (Beyotime Biotechnology, China), cytoplasm and nucleus proteins were extracted. The exact amount of protein was electrophoresis with SDS-polyacrylamide gel and then transferred onto a piece of PVDF membrane (Millipore, USA). After blocking, the membrane was incubated with the primary antibody against the target protein on a shaking table at 4℃ overnight. Then the membrane was incubated with a secondary antibody, and the protein bands were quantitated by electrochemiluminescence assay. The antibody details were listed in Supplementary Table 2.
Immunohistochemistry, immunocytochemistry and hematoxylin-eosin (HE) staining
Tissue sections were deparaffinized, rehydrated, retrieved antigen and removed endogenous peroxidase (Biotech Well, China). After being blocked and washed, these sections were incubated with the primary antibody at 4°C overnight. The next day, secondary antibody incubation and chromogenic detection were performed. The sections were then counterstained with hematoxylin, dehydrated, mounted, and examined.
As for immunocytochemistry, cells were seeded in each compartment of slides (Millipore, USA). After fixing with 4% paraformaldehyde and incubating with PBS containing 0.5% Triton X-100, endogenous peroxidase removal and subsequent steps were the same as immunohistochemistry. The antibody details were listed in Supplementary Table 2.
Tissue sections for HE staining were deparaffinized and rehydrated. They were treated with hematoxylin for 5 min, ethanol containing 1% hydrochloric for 45 s, and then immersed in eosin for 5 min. Dehydration and subsequent steps were similar to immunohistochemistry.
Wound healing assay
Cells were seeded in a 6-well plate cultured to 80-90% confluence and then scratched with a pipette tip vertical to the six-well plate. Cell debris was gently washed off with PBS and added FBS-free medium. Images of cells were recorded at 0 h, 24 h, and 48 h after scraping, and scratch areas were measured.
Cell invasion and migration assay
For the invasion assay, 50 μL diluted Matrigel (BD Bioscience, USA) was added to each transwell chamber (Millipore, USA) and solidified at 37℃ before cells were seeded. For both invasion and migration assay, 5×104 cells resuspended in 200 μL of FBS-free medium were seeded in the upper compartment of the chamber, and 600 μL complete medium was added to the lower compartment. After 48 h-incubation, cells on the upper compartment were wiped with a cotton swab. The invaded cells at the lower compartment were fixed in 4% paraformaldehyde and stained with crystal violet. Then invaded cells were photographed under a microscope.
Cell proliferation assay
A total of 100 μL suspension containing 3 000 cells were seeded in each well of a 96-well plate. After different treatments for an appropriate time, 10μL cell counting kit-8 reagent (CCK8, Dojindo, Japan) was added to the culture medium at specific time points of 0 h, 24 h, 48 h, 72 h. After incubating for 4 h at 37°C in 5% CO2, the optical density was measured at 450 nm using a microplate reader.
Detection of glucose consumption, lactic acid production and the activity of hexokinase
Cultured primary endometrial stromal cells were trypsinized and counted and then seeded into a 96-well plate. After 24 h, the experiments of glucose consumption, lactic acid production, the activity of hexokinase detection were performed following the manufacturer’s instructions (Solarbio, China).
Extracellular acidification rate (ECAR) detection
1×105 cells were seeded in each well of a 96-well plate. The XF96 extracellular flux kits were pretreated, and the essential medium was prepared. The next day, 175 μL essential medium was added to each well of the 96-well plate which would be incubated in a CO2-free incubator at 37 ℃ for 1 h. In addition, 25 μL D-glucose (10 mM), Oligomycin (1 μM) and 2-DG (50 mM) per well were added into the upper part of XF96 extracellular flux kits, which were put into a seahorse XF96 cellular flux analyzer. After 30 min, the lower layer of the XF96 extracellular flux kits was replaced with the cell plate, and detection was proceeded according to the standard procedures.
Mice model of endometriosis
The intraperitoneal endometriosis model was constructed according to a previously described method (46) with minor alterations. The uteruses of donor mice were removed and minced, and then injected into recipient mice intraperitoneally. The recipient mice were divided into experimental group and control group randomly. Drug solutions and solvents were intraperitoneally injected into the mice of the experimental group and control group, respectively. All recipient mice were sacrificed 14 days after implantation, and the endometriosis lesions were collected.
Statistical analysis
The statistical analysis was carried out using GraphPad Prism version 6.02. Results are reported as the mean ± SEM from triplicate experiments. Student's t-test and one-way ANOVA were used to analyzed statistics following correction. There was a statistical difference when P < 0.05.