A murine model of EMs
Animal experiments were approved by the Experimental Animal Committee of Shanghai municipal Hospital of Traditional Chinese Medicine, and carried out according to Shanghai municipal Hospital of Traditional Chinese Medicine guidelines for the use of animals. All animal experiments were performed in accordance with the ARRIVE guidelines as described previously (26) to minimize the animals number used in the study and suffering of the animal. The female BALB/c mice (4-6 weeks old, 18-20 g) were obtained from the Shanghai Regan Biotechnology Co., Ltd (Shanghai, China) and were reared in a specific, pathogen-free facility. (27-29). After 1-week acclimatization, mice were randomly divided into two groups: the donor group (n=10) and recipient groups (n=10). Ovariosteresis and estradiol valerat-injection (0.5 µg/mouse/week; Aladdin, Shanghai, China) was carried out to avoid differences of oestrous cycle (28). Mice were anesthetized by 2% isoflurane and then ovaries on both sides were exposed through flank incisions and removed. Donor mice were sacrificed under isoflurane anesthesia and each uterine horn of donor mice was concentrated and peeled in warm PBS to remove uterine muscle. Endometrium tissues were weight and cut into small fragments with scissors and resuspended in sterile PBS with 1× ampicillin (Beyotime, Shanghai, China). After that, endometrium preparation was intraperitoneally injected into two recipient mice (50 mg/mice). Two weeks after EM transplant, endometriosis lesions and eutopic endometrium tissues were removed from the peritoneal cavities and uteri.
Human ectopic and eutopic endometrium tissues (n=17) were provided by Shanghai municipal Hospital of Traditional Chinese Medicine with written informed consent signed by the patients according to Declaration of Helsinki Principles. All of the protocols in this study is approved by Shanghai municipal Hospital of Traditional Chinese Medicine.
Primary murine ESCs were isolated from eutopic and ectopic endometrium tissues of recipient mice. Briefly, endometrium tissues were wash with PBS and minced into small fragments, then treat with 0.3% collagenase I and 0.01% trypsin (Sigma-Aldrich, MO, USA) in DMEM/12F medium (Sigma-Aldrich) for 50 min at 37 °C, followed by filtration through 100 μm and 70 μm nylon cell strainers. All cells were maintained in DMEM/F-12 with 10% fetal bovine serum (Thermo fisher Scientific, DE, USA) and 1× Penicillin-Streptomycin (Beyotime, Shanghai, China) at 37°C under humidified 5% CO2.
Total RNA extraction and quantitative RT-PCR (qPCR) assay
Total RNA was extracted from endometrial tissues or ESCs with RNAiso Plus (TaKaRa Bio Inc., Otsu, Shiga, Japan) according to the manufacturer’s instructions, quantitated by Multiskan SkyHigh (Thermofisher Scientific). Prime Script RT-PCR Kit (TaKaRa) was applied to carry out reverse transcription PCR (RT-PCR). The RT-PCR protocol was 65˚C for 5 min, 30 ˚C for 10 min, and 42˚C for 30 min. qPCR was performed using Accurate 96 (DLAB, China) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The program setting was based on the manufacturer’s recommendations, thermocycling conditions were as follows: initial denaturation at 95 °C for 10 s, denaturation at 95 °C for 10 s, annealing and extension at 65 °C for 10 s; the reaction was performed for 30 cycles. Each reaction was carried out at least in triplicate. ADAMTS9-AS1 and GPX4 RNA transcriptions were quantified by the 2-ΔΔCt method and normalized by endogenous β-actin mRNA. The primer sequences were listed in Supporting Table S1.
Overexpression and RNA interference (RNAi)
The recombinant construct (pcDNA-ADAMTS9-AS1) containing full-length ADAMTS9-AS1 sequence was constructed by inserting their open reading frame into the pcDNA vector. miR-6516-5p mimics were used to mimic the overexpression of miR-6516-5p. siRNA-ADAMTS9-AS1 (siADAMTS9-AS1) was used to knockdown ADAMTS9-AS1 in ESCs and siRNA-control (N.C) was used as a control. The ESCs were seeded into culture dishes before transfection, until the cells adhered on the second day. Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA) was applied to prepare the mixture of siADAMTS9-AS1, N.C, miR-6516-5p or pcDNA-ADAMTS9-AS1 with Lipofectamine™ 3000 according to the instructions.
Cell viability assay
The cell viability of NESCs and EESCs was determined with CCK-8 Cell Proliferation and Cytotoxicity Assay Kit (CCK-8; Solar Bio, Beijing, China) following the manufacturer’s instructions. Before transfection or drug treatment, ESCs were seeded onto 96-well-plates at a density of 6 ´ 103 cells/well to incubate for 24 h. To explore the effect of ADAMTS9-AS1 on regulating ESCs viability and the pattern of ADAMTS9-AS1-induced ESCs cells death, ADAMTS9-AS1 in ESCs was knocked down or overexpressed in the present or absent of cell death inhibitors (Fer-1, 1 μM; ZVAD-FMK, 10 μM; necrostatin-1, 10 μM; 3-MA, 10 mM; and disulfiram, 5 μM) or inducer (Erastin, 10 μM; Sorafenib, 5 μM) purchased from Sigma-Aldrich (Shanghai, China). Then the cells were treated with 15 µL CCK-8 reagent and incubated for another 3 h at 37°C. The absorbance was measured at 450 nm with Multiskan SkyHigh (Thermofisher Scientific, Wilmington, DE, USA).
FDA staining was also performed to determine whether ADAMTS9-AS1 knockdown regulated cell death of EESCs. After being transfected with si-ADAMTS9-AS1 or siRNA-control for 48 hours, EESCs were treated with 100 μg/mL FDA (Thermofisher Scientific, Wilmington, DE, USA) and incubated in dark for 20 min, then observed by microscopy.
Transwell migration assay
To explore the effect of ADAMTS9 AS1 and miR-6516-5p on regulating ESCs migration, the 24-well transwell chambers (Corning, NY, USA) with 8-μm pores were used to assess the migration of EESCs. In brief, the transfected EESCs and NESCs were seeded into the upper chamber at 200 μL 2.5×104 cells density with DMEM/12F medium without serum, 700 μL DMEM/12F medium supplemented with 10 % FBS were added into lower chamber. After incubating for 48 h at 37 ℃ with 5 % CO2, the cells on the lower surface of membranes were fixed by 4% paraformaldehyde (Beyotime, Shanghai, China), stained by 0.1 % crystal violet (Beyotime, Shanghai, China) for 10 min. The images were obtained using BZ-X700 (Keyenth, Osaka city, Osaka Prefecture, Japan), and the number of cells migrated to independent areas was counted using Fiji software.
Measure of iron concentration
Intracellular iron concentrations were measured using an iron assay kit (ab83366, Abcam) according to manufacturer’s instructions. Briefly, ESCs were lysed by Iron Assay Buffer (3×103 cells/well) in a final volume of 100 μL and treated with 5 μL Iron Reducer, plated in 96-well-plate and incubated at room temperature for 30 min. 100 μL iron probe was added to mark ferrous ion. The absorbance was measured at 593 nm with Multiskan SkyHigh (Thermofisher Scientific, Wilmington, DE, USA).
GSH and MDA content
Malondialdehyde (MDA) content in ESCs was assayed using a lipid peroxidation assay kit (ab118970, Abcam) after ADAMTS9-AS1 knockdown or overexpression in the presence or absence of miR-6516-5p or siGpx4. Glutathione (GSH) content in ESCs was assayed using a Glutathione Assay Kit (ab65322, Abcam).
Lipid ROS level
Lipid ROS level was tested using a fluorescent-labelled oxidation sensitive probe (C11-BODIPY, Thermo Fisher Scientific) as previously described (30). In brief, ESCs (5×105) were plated in 24-well plates and treated with the indicated reagents. After 48 h, ESCs were treated with 1.5 μM of C11-BODIPY for 30 min at 37℃. Lipid ROS level was assessed using a flow cytometer (CytoFLEX, Beckman Coulter, FL, USA).
The protein extracted from ESCs lysate was quantified using the BCA Protein Assay Kit (Beyotime). Equal amounts of protein (approximate 50 μg) was loaded and separated on 12% SDS-PAGE for each experiment, then transferred to PVDF membranes (0.45 μm pore size; Millipore Corp., MA, USA). After blocking in 3% BSA at room temperature for 1 h, the membranes were treated with primary antibodies against GPX4 (1:4000, ab125066, Abcam, MA, USA) and β-actin (1:5000, ab8226, Abcam) for 18 h at 4°C, followed by incubation with HRP-conjured secondary antibody (HRP-labeled Goat Anti-Rabbit IgG(H+L); 1:5000; Beyotime) for 1.5 h. The bands were visualized using an ECL reagent (Beyotime) and quantified using Fiji software.
Fluorescence in situ hybridization (FISH)
The subcellular localization of ADAMTS9-AS1 was assessed with FISH Tag™ RNA Multicolor Kit (Carlsbad, CA, USA, USA). ESCs (3×104/well) were loaded on cover glasses, fixed with 4% paraformaldehyde (pH=7.4) for 5 min. After fixation, ESCs were digested by protease K (3 μg/mL) with glycine and acetic anhydride, followed by dehydrated in 75, 95, and 100% ethanol for 3 min each and air-dried at room temperature, and hybridization at 37 °C with probes (150 μL, 450 ng/mL) against ADAMTS9-AS1 for 48 h. Lastly, ESCs were stained by DAPI (Thermofisher Scientific) and observed under a fluorescence scanning microscope (BZ-X700; Keyenth, Osaka city, Osaka Prefecture, Japan).
The recombinant plasmids of pGL3-ADAMTS9-AS1 and pGL3-GPX4-3’UTR or their mutant were constructed in our laboratory by cloning approximate 350 bp cDNA into pGL3 vector (Vector Builder, Guangzhou, China). 4.5×104 ESCs were plated into 48-well plate and co-transfected with 70 nM of miR-6516-5p or miR-cont (used as control) with Lipofectamine™ 3000 (Invitrogen, Carlsbad, CA, USA). Luciferase activity was detected using the Renilla-Lumi™ Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) as the manufacturer’s protocol.
Biotin-labeled miR-6516-5p and miR-cont were transcribed in vitro using the Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermofisher Scientific, Wilmington, DE, USA). About 2 × 107 cells were dissolved in the standard lysis buffer (Thermofisher Scientific, Wilmington, DE, USA) plus 10 U/mL RNase Inhibitor (Beyotime, Shanghai, China). Next, each binding reaction was added streptavidin-labeled beads and incubated for 90 min. Lastly, ADAMTS9 AS1 in the eluate was quantified by q-PCR and GAPDH mRNA was used as a reference.
Data were analyzed using GraphPad 7.0 (GraphPad Software, La Jolla, CA, USA) and presented as mean ± SEM from at least three independent experiments. The significance between different groups was assessed using student t test or one-way ANOVA followed by Tukey-Kramer multiple comparisons test. P values lower than 0.05 was considered to indicate a statistically significant difference.