2.1. Cell culture
Five types of human EC cell lines (Ishikawa, RL95-2, HEC-1A, HEC-1B, and KLE) were obtained from the American Type Culture Collection (ATCC, USA). The normal endometrial cells we used were human primary endometrial epithelial cells. Cells were grown in DMEM/F12 complete medium (HyClone, China) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and penicillin and streptomycin (Boster, China) at final concentrations of 100 µg/mL. All cells were free from Mycoplasma contamination and were grown at 37°C in a humidified atmosphere of 5% CO2.
2.2. Clinical specimens
A total of twelve paired EC and adjacent normal tissues of patients who underwent surgery or biopsy at 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 immunohistochemistry (IHC) and western blot. 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).
2.3. Bioinformatical analysis
The Cancer Genome Atlas (TCGA, http://www.cancergenome.nih.gov/) was used for gene set enrichment analysis (GSEA) to analyze the expression and prognosis of USP39 in patients with EC. The ggplot2 package in R was applied to perform a volcano plot of all the differentially expressed genes based on the RNA-seq results.
2.4. 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 anti-L-lactyl lysine rabbit mAb (pan-anti-Kla; PTM Bio, China), anti-lactyl-histone H3 (Lys18) rabbit mAb (anti-H3K18la; PTM Bio, China), and anti-USP39 (Proteintech, China) at 4°C overnight. The slides were then rinsed three times with PBS, incubated with a secondary antibody at room temperature for 30 min in the darkness, and visualized after being stained with a DAB solution. Three randomly selected fields were observed under a microscope (Motic, China). The IHC staining scores, based on the staining intensity (SI) and the percentage of immunoreactive cells (PR), were evaluated by two independent observers who were blinded to the patient identity. The SI scores were assigned from 0 to 3 as follows: 0 = no staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The PR was scored from 1 to 4 as follows: 1 = 0–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 76–100%. The PR and SI scores 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 a low expression.
2.5. Total RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR)
Adherent cells were harvested with the RNAiso reagent (Takara, Japan), and RNA was extracted according to the manufacturer's manual. The concentration of RNA was measured using a Tecan Infinite M200 Pro (Thermo Fisher Scientific, USA). cDNA was obtained from the RNA using a reverse transcription kit (Vazyme, China), and was used as a template for qRT-PCR with the SYBR Green Fast qPCR mix (ABclonal, China). The fold changes of RNA transcripts were calculated using the 2−ΔΔCt method, and β-Actin was used as an endogenous control. All qRT-PCR runs were conducted in triplicate. The primer sequences used were as follows: USP39-F: 5’-GGTTTGAAGTCTCACGCCTAC-3’; USP39-R: 5’-GGCAGTAAAACTTGAGGGTGT-3’; β-Actin-F: 5’-GGATTCCTATGTGGGCGACG-3’; and β-Actin-R: 5’-GATAGCACAGCCTGGATAGCA-3’.
2.6. Western blot
Fresh tissues or adherent cells were washed twice with PBS and then lysed on ice for 1 hour with RIPA lysis buffer (Servicebio, China) containing a cocktail and PMSF. After centrifugation, total protein concentrations were determined using a BCA protein assay kit (Biosharp, China). Individual samples (20 µg/lane) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10–12% gels and transferred onto polyvinylidene difluoride (PVDF) membranes. After incubation with 5% dry milk in TBST at room temperature for 1 hour, the membranes were washed and incubated with pan-anti-Kla (PTM Bio, China), anti-H3K18la (PTM Bio, China), anti-USP39 (Proteintech, China), anti-PGK1 (Proteintech, China), anti-PI3K (Abmart, China), anti-p-PI3K (Abmart, China), anti-AKT (Abmart, China), anti-p-AKT (Abmart, China), anti-HIF-1α (Abmart, China), anti-β-Actin (ABclonal, China), and anti-H3 (Proteintech, China) at 4 ℃ overnight. After washing the membranes, the bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence (ECL) kit (Biosharp, China). The relative expression levels of the targets to those of β-Actin or H3 were determined by densitometric analysis using the ImageJ software.
2.7. Glucose uptake, lactate accumulation, and ATP content assays
EC cells were seeded into 6-well plates and treated with 2-deoxy-D-glucose (2-DG), oxamate, or a plasmid for 24 hours. Afterward, the cells were washed twice with PBS and lysed with a lysis buffer. The concentrations of glucose, lactate, and ATP were determined using commercial assay kits (NJJCBIO, China), according to the manufacturer’s instructions.
2.8. Cell viability assay
Cell Counting Kit-8 (CCK-8; Targetmol, China) was used to measure cell viability. Cells were seeded into 96-well plates at a density of 3×103 cells per well in a 100-µL suspension. Every 24 h for 5 days, 10 µL of the CCK-8 solution was added to each well in 100 µL of a serum-free medium. After incubation for 2 h at 37°C, the absorbance of each well was measured at 450 nm using a microplate reader (Thermo Fisher Scientific, USA). Finally, the numbers of living cells over 5 days were plotted in a graph to reflect the cell proliferation rate.
2.9. Colony formation assay
For the colony formation assay, cells were seeded into 6-well plates at a density of 1×103 cells per well and cultured for 14 days. Subsequently, the cells were washed with PBS, fixed with 1 mL of methanol for 30 min, and stained with a crystal violet solution for approximately 30 min. After being washed and air dried, the colonies formed were counted manually.
2.10. 5-Ethynyl-2′-deoxyuridine (EdU) assay
The EdU assay was performed with Ishikawa and KLE cells using an EdU-555 cell proliferation kit according to the manufacturer’s protocol (Beyotime, China). Cells were seeded into 6-well plates, and 24 h later, a 10 µM EdU solution was added to the cells for 2 h. Thereafter, the cells were fixed with 4% paraformaldehyde (PFA) and rinsed three times with PBS and once with 0.5% Triton X-100. Next, a click additive reaction system was added to label the proliferated cells, and Hoechst 33342 was added for cell counting. Finally, cells were visualized using a fluorescence microscope (Olympus, Japan).
2.11. Cell migration assay
Transwell 24-well plates with an 8-µm pore size (Corning, USA) were used to assess cell migration. Serum-free culture medium (100 µL) was added to the upper chamber with Ishikawa or KLE cells (1.0 × 105/well), and 700 µL of a medium with 20% FBS as a chemo-attractant was placed into the lower compartment of the chamber. After 24-h incubation at 37°C, cells that crossed the membrane were fixed with 4% paraformaldehyde (Servicebio, China) for 30 min and stained with 0.1% crystal violet (Servicebio, China) for 10 min at room temperature. Migrated cells were imaged and counted in three randomly selected fields of view using a CX23 light microscope (Olympus, Japan) with a 100× magnification.
2.12. Flow cytometry (FCM) analysis
An annexin V-FITC apoptosis detection kit (Beyotime, China) was used to evaluate cell apoptosis, and a PI/RNase staining buffer (BD Biosciences, USA) was used to analyze the cell cycle. The flow cytometer used was an ID7000 spectral cell analyzer (Sony, Japan), and the data were analyzed using the FlowJo software.
2.13. Chromatin immunoprecipitation (ChIP)-qPCR
Ishikawa and KLE cells (3 × 106) were seeded into 100-mm dishes, and after treatment with 1% formaldehyde, the cells were lysed with 1 ml of RIPA lysis buffer. Genomic DNA was isolated and sheared into 200–400-bp fragments using a sonicator. After centrifugation, the supernatants were collected, and the chromatin was precipitated with anti-H3K18la (1:50; PTM Bio, China) or IgG (Proteintech, China) at 4°C overnight. The following steps were conducted according to the manufacturer's instructions for a ChIP assay kit (Beyotime, China). The primers designed for specific promoter regions of USP39 are listed in Supplementary Table 1.
2.14. Co-immunoprecipitation (Co-IP) assay and mass spectrometry (MS)
For immunoprecipitation (IP), cells were harvested and lysed with NP40 buffer (Servicebio, China), followed by the addition of PMSF and a protease inhibitor cocktail. After centrifugation, the supernatant was collected in fresh tubes. Approximately 1 mg of the cell lysate was incubated with 1.0 µg of primary antibodies (anti-IgG and anti-USP39) with a rotation overnight at 4°C. Then, 25 µL of washed protein A agarose beads (MCE, USA) were added to the lysate, and the incubation continued for 2 h at 4°C with a rotation. Subsequently, the beads were collected by centrifugation and washed three times with NP40 buffer. The immune complex was released by boiling the beads with an SDS loading dye and analyzed by western blot. Purified peptides were pickup by autosampler and transferred into a C18 analytical column (Thermo Fisher Scientific, USA) for separation. A Q Exactive Plus mass spectrometer coupled with an EASY-nLC 1200 system (Thermo Fisher Scientific, USA) was used to acquire LC-MS/MS data and the MS raw data were analyzed with MaxQuant (V1.6.6) using the Andromeda database search algorithm, filtered with 1% FDR at both protein and peptide levels.
2.15. In vivo tumor formation
After random grouping into an experimental and control group, SPF BALB/c-nu female nude mice were subcutaneously injected with treated Ishikawa and normal Ishikawa cells (1 × 106), respectively. The tumor size was measured every 5 days, and then the mice were killed by cervical dislocation on day 25. The xenograft volume was calculated using the following formula: tumor volume (mm3) = (length × width2)/2. The tumors were weighed and embedded in paraffin for an IHC assay. Animal experiments were performed according to the protocols approved by the Animal Care and Use Committee of the Tongji Medical College.
2.16. Mouse model of EC lung metastasis
After random grouping, SPF BALB/c-nu female nude mice were injected via the tail vein with Ishikawa cells (1 × 106 in 0.1 ml of PBS). After 6 weeks of treatment, the mice were killed by cervical dislocation. The lungs were excised, fixed, and embedded in paraffin to observe probable lung metastasis.
2.17. Statistical analysis
GraphPad Prism 8 (GraphPad Software, USA) was used for statistical analysis. Experimental data from three independent replicates are presented as the mean ± standard error of the mean (SEM). A Student's t-test was used for comparisons between two independent sample groups with a normal distribution of data. One-way ANOVA was used for one-way comparisons, and two-way ANOVA was used for two-way comparisons among 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.