2.1. Cell Culture
The human endometrial carcinoma cell line Ishikawa and KLE were grown in DMEM-F12 complete medium (HyClone, China) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% antibiotics (penicillin and streptomycin) (Boster, China) at a final concentration of 100 µg/ml. All cells were grown at 37 ℃ in a humidified atmosphere of a 5% CO2 incubator. All the cells were free of mycoplasma contamination.
2.2. Clinical Specimens
A total of seven paired EC and adjacent tissues of patients who underwent surgery or biopsy in the Department of Gynecology, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China) from September 2019 to March 2021 were collected for qRT-PCR, IHC and western blotting. All patients had complete clinical data and did not receive immunotherapy, chemotherapy, or radiotherapy. This study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (No. 2022-S017). All patients have signed the written informed consent forms before surgical resection.
2.3. Immunohistochemistry (IHC) Staining and Scoring
Samples were embedded in paraffin and sliced into sections at a thickness of 4 µm. The sections were then incubated with GPX4 antibody (Proteintech, China) which was diluted at 1: 3000 at 4 ℃ overnights. The following day, the stained sections were incubated with secondary antibody at room temperature for 30 min in darkness and visualized with DAB solution. Finally, the nucleus was counterstained by haematoxylin. A Motic microscope (Motic, China) was used to visualize and photograph the sections. The IHC staining scores were evaluated by two independent observers blinded to the corresponding patients based on the staining intensity (SI) and the percentage of immunoreactive cells (PR). The SI score was calculated from 0 to 3: 0 = no staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The PR was scored from 1 to 4: 1 = 0–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 75–100%. The PR and the SI were multiplied to produce a weighted score for each patient. A score of 8–12 was defined as a high expression level and a score of 0–7 was defined as low expression.
2.4. Total RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The adherent cells were harvested with the RNAiso reagent (Takara, Japan). The extraction of RNA was performed according to the manufacturer's manual. The cDNA was obtained from RNA using the reverse transcription kit (Vazyme, China). The cDNA was used as templates, and the SYBR Green Fast qPCR Mix (Abclonal, China) was used for real-time quantitative PCR. The fold changes of RNA transcripts were calculated by the 2 − ΔΔCt method and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as the internal control. The primer sequences used were shown as follows: GPX4-F: 5’-GAGGCAAGACCGAAGTAAACTAC-3’; GPX4-R: 5’-CCGAACTGGTTACACGGGAA-3’; GAPDH-F: 5’-GAGTCAACGGATTTGGTCGT-3’; GAPDH-R: 5’-GACAAGCTTCCCGTTCTCAG-3’.
2.5. Western Blotting
Fresh tissues or adherent cells were first washed twice with PBS and then lysed on ice with RIPA lysis buffer (Servicebio, China) containing Cocktail and PMSF for 1 hour. Cell lysates were centrifuged and quantified using BCA Protein Assay kits (Biosharp, China). A total of 30 µg of protein was loaded for electrophoretic separation on sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After incubation with 5% skimmed milk at room temperature for 1 hour and washing, the membranes were further incubated with primary antibody at 4 ℃ overnight. Membranes were blotted with the following antibodies: anti-GPX4 (1: 1000; Proteintech, China), anti-ELK1 (1: 1000; Proteintech, China), and anti-GAPDH (1: 10000; Abclonal, China). Horseradish peroxidase-conjugated secondary antibody was added and incubated at room temperature for 60 minutes, followed by capture with enhanced chemiluminescence (ECL) (Biosharp, China).
2.6. Cell Proliferation Assay
Cell Counting Kit-8 (CCK8) assay (Targetmol, China) was used to detect cell viability. A total of 100 µl cell suspension (3×103 cells) was first plated in a 96-well plate, and then 10 µl of CCK8 solution was added to each well at 24, 48, 72, 96, 120 h. After another 2 h of incubation, the absorbance was measured at 450 nm by using a microplate reader (Thermo Fisher Scientific, USA). Clone formation assay was used to detect the number of colonies. Cells were seeded into well plates (300 cells per well) and cultured for 14 days. The above-mentioned cells were then fixed for 30 min with 2 ml of methanol and then stained cells with crystal violet solution for about 30 min.
2.7. Cell Migration Assays
Transwell chambers of 8 µm pore size (Corning, USA) were used to assess migration. 100 µl cell suspension without serum was used in the upper chamber with Ishikawa and KLE (1.0 × 105/well). The lower chamber contained a chemo-attractant (medium with 20% FBS). After 24-hour incubation at 37 ℃, the cells were then fixed with 4% paraformaldehyde (Servicebio, China) for 30 min and stained with 0.1% violet crystal (Servicebio, China) for 10 min at room temperature. After washing with PBS solution, cells that passed the membrane were counted and imaged in 3 different randomly selected views under 100 × magnification using CX23 Olympus light microscopy (Olympus, Japan).
2.8. Flow Cytometry (FCM) Analysis
Use the Annexin V-FITC Apoptosis Detection Kit (Beyotime, China) to evaluate cell apoptosis. Use PI/RNase Staining Buffer (BD Biosciences, USA) to analyze the cell cycle. The flow cytometer used was ID7000 Spectral Cell Analyzer (Sony, Japan). The data were analyzed by using FlowJo.
2.9. Lipid Reactive Oxygen Species (ROS) Assay
Cells were collected and then centrifuged. After that, DCFH-DA (Beyotime, China) was prepared to pretreat the cells at 37 ℃ for 20 min. Following incubation, the cells were collected and washed twice in a free-serum medium. Subsequently, the cells were resuspended in PBS and were analyzed by flow cytometry to detect the levels of ROS within the cells.
2.10. Fe2 + and Malondialdehyde (MDA) Assay
Firstly utilized a BCA protein assay kit (Biosharp, China) to detect the protein concentrations in cell supernatants. After that, Fe2+, as well as the MDA levels, were both estimated by the iron assay kit (NJJCBIO, China) and the MDA detection kit (Beyotime, China), respectively.
2.11. Evaluation of Mitochondrial Membrane Potential (MMP)
The MMP was measured by a mitochondrial membrane potential assay kit (Beyotime, China). JC-1 monomers (green) can form aggregates (red fluorescence) in the mitochondria with high △Ψm, which cannot form aggregates in the mitochondria with low △Ψm. Results were detected by using a confocal laser microscope (Olympus, Japan).
2.12. Dual-Luciferase Reporter Assay
Ishikawa cells were seeded in 6-well plates at a 70% concentration and transfected with relevant plasmid and the luciferase vector. After 48 h, cells were lysed and the luciferase activities were measured using the dual-luciferase reporter assay kit (Beyotime, China) according to the manufacturer’s protocol. The luminescence intensities of firefly and Renilla luciferases were recorded by a microplate reader. For data analysis, the luciferase activity was normalized by comparing it with Renilla luciferase activity.
2.13. Chromatin Immunoprecipitation (ChIP) Assay
Chromatin immunoprecipitation was performed using Ishikawa cells, which were treated with 37% formaldehyde (Sigma, USA) at 1% final concentration for 10 min at room temperature to cross-link proteins to DNA. After that, the remaining steps were carried out according to the manufacturer’s instructions for the ChIP assay kit (Beyotime, China). The antibodies used were anti-ELK1 (Proteintech, China) and normal rabbit IgG (Proteintech, China). The enriched DNA was analyzed utilizing the primers by real-time PCR.
2.14. Tumor Xenograft Assay
Female BALB/c-nu nude mice (age, 4–5 weeks)were purchased from China Charles River and housed in a standard pathogen-free environment laboratory. The mice were randomized into two groups. Ishikawa (1x106) were suspended in 100 µl serum-free DMEM and subsequently injected into the right flanks of the mice. After 25 days, the mice were cervical dislocated under anesthesia, and the weight, length, and width of xenografts were measured. Tumor volume = (length × width2)/2. Tumor samples were partially embedded in paraffin for histopathological analysis. Animal experiments were performed according to the protocols approved by Tongji Medical College's Animal Care and Use Committee.
2.15. Statistical Analysis
GraphPad Prism 8 (GraphPad Software, USA) was used for statistical analysis. The experimental data of every 3 independent replicates were presented as the mean ± standard error of the mean (SEM). The Student's t-test was used for comparisons between two independent sample groups. One-way ANOVA was used for one-way comparisons between multiple groups, while two-way ANOVA tests were used for two-way comparisons between multiple groups. P < 0.05 was considered to indicate a statistically significant difference. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.