Cell lines and cell culture
Human KLE endometrial adenocarcinoma cells were obtained from the Shanghai Cell Biological Research Institute (Shanghai, China), and ECC-1 cells were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA). KLE cells are ERα-, while ECC-1 cells are ERα + . KLE and ECC-1 cells were thawed and cultured in DMEM/F12 medium with 0.005 mg/ml insulin, 1% antibiotic–antimycotic solution, and 10% fetal bovine serum (FBS) at 37°C in 5% CO2. Cells treated with XCT790 (Sigma-Aldrich, St. Louis, MO, USA) were incubated in phenol red-free medium (Thermo Fisher) containing 1% serum replacement 2 (Sigma-Aldrich). ECC-1 and KLE ECs were incubated with 10 µM XCT790 (in dimethyl sulfoxide [DMSO]; Sigma-Aldrich) or DMSO (control) for 24 hours. Lentiviral vectors expressing siRNAs targeting TFEB (named TFEB-KD) and ERRα (named ERRα-KD) were constructed. The following siRNA target sequence in the TFEB gene (GenBank accession No. NM_013261) was selected: 5′-GAG ACG AAG GTT CAA CAT CAA-3'. The siERRα sequence was 5'-GAG CGA GAG GAG TAT GTT CTA-3′. The lentiviral vector used to overexpress TFEB (named TFEB-OV, GenBank accession NM_001167827.3) and ERRα (named ERRα-OV, GenBank accession NM_004451.5). Overexpression of ERRα or TFEB was achieved in KLE and ECC-1 cells and named KLETFEB−OV, ECC-1TFEB − OV, KLEERRα−OV and ECC-1ERRα−OV, respectively. In addition, KLE and ECC-1 cells with ERRα or TFEB expression downregulated through lentivirus-mediated siRNA were named KLETFEB−KD, ECC-1TFEB − KD, KLEERRα−KD and ECC-1ERRα−KD, respectively.
Bioinformatics Data Analyze
Gene expression data (575 cases, workflow type: HTSeqCounts) were downloaded from The Cancer Genome Atlas (TCGA) official website for the Uterine Corpus Endometrial Carcinoma (UCEC) projects. The dataset included 23 normal endometrial specimens and 543 EC specimens, with 9 repeated cancerous specimens excluded. Patient clinical information, gene-level copy number variation (CNV) profiles, gistic2 thresholds analyzed by the GISTIC2.0 method and somatic nonsilent mutation (gene-level) data were acquired from the University of California, Santa Cruz (UCSC) Xena website. The Database for Annotation, Visualization and Integrated Discovery (DAVID) (version 6.8) provides a comprehensive set of functional annotation tools that help investigators understand the biological meaning behind a large list of genes. GO functional annotation and KEGG analysis of the DEGs were performed, and the results were visualized with the cluster Profiler R package.
Rna Extraction, Rt-qpcr And Western Blotting (Wb)
RNA extraction, RT-qPCR and Western blotting (WB)
Samples were collected from an equal number of intact cells in TRI Reagent® (#TR118; Molecular Research Center). After reverse transcription on 500 ng of total RNA with RevertAid Reverse Transcriptase, oligo-dT and random hexamers (Thermo Fisher Scientific), quantitative PCR amplification was performed on a ViiA™ 7 real-time PCR system (Applied Biosystems) using Takyon Low Rox SYBR® MasterMix dTTP Blue (#UFLSMT-B0701; Eurogentec). Relative gene expression was calculated using the ddCt method, with GAPDH as the reference gene. Standard techniques were used for protein quantification, separation, transfer, and blotting. Primary antibodies against the following targets were used: TFEB (1:1000; Cell Signaling Technology, China), ERRα (1:500; Abcam, UK), LPCAT1(1:1000; Proteintech, China), LPCAT3 (1:1000; Proteintech, China), MMP2 (1:1000; Beyotime Biotechnology, China), Cortactin (1:1000; Beyotime Biotechnology, China), E-cadherin (1:1000; Cell Signaling Technology, China), vimentin (1:1000; Cell Signaling Technology, China), MRPS2 (1:1000; Proteintech, China) and GAPDH (1:2000; Proteintech, China).
Cells were grown to confluence in 6-well plates, and a 200-µL tip was used to introduce a scratch in the monolayer. The scratch areas in the wells were washed with PBS and 1 mmol/L R-flurbiprofen until the cells in those areas were removed thoroughly and imaged at 0 and 24 hours postscratching. The horizontal migration rate was calculated using the following formula: (width 0 h − width 24 h)/width 0 h × 100%.
Chromatin-immunoprecipitation (Chip) Assay
Cells were harvested followed by cross-linking for 10 min with 1% (vol/vol) formaldehyde. Afterwards, cells were lysed by sonication. The cell lysates were immunoprecipitated with anti-TFEB (ab2636, Abcam) overnight at 4°C. After washing and elution, the crosslinks were reversed for 4 hours at 65°C. The eluted DNA was purified and analyzed by qPCR using a Bio-Rad SYBR Green intercalating fluorophore system with the following ERRα primers: 5’-AGT TTA TGT GGC TGT GGG CA-3’ (forward) and 5’-GGA TAT TTG CTG TCT TTA TAT TC-3’ (reverse). The Ct value of each sample was normalized to the corresponding input value.
Luciferase Reporter Assays
Bioinformatics methods were used to analyze and predict the potential transcription factor binding sites in the ERRα promoter region. The ERRα promoter sequence (64303524 bp to 64305524 bp) relative to the transcription start site was amplified by PCR and inserted into the pGL3-basic vector (E1751, Promega). KLE cells were cotransfected with empty pcDNA3.1 vector or TFEB-S211A pcDNA3.1 plasmid in 24-well plates with Lipofectamine 2000. After 48 hours, the firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit (E1901, Promega) and a microplate reader (Synergy H1, Bio-Tek), and the ratio of firefly/Renilla luciferase activity was determined.
Lipid And Metabolite Profiling
Liquid Chromatography Mass Spectrometry (LC/MS) analyses were performed using a high-performance liquid chromatography system (1260 series; Agilent Technologies) and mass spectrometer (Agilent 6460; Agilent Technologies). Briefly, 107/ml EC cells or 20 mg EC tissue was homogenized in 1.5 mL of chloroform/methanol (2:1, v/v), vortexed for 1 minute, centrifuged at 3,000 rpm for 10 minutes, added to 800 µL organic phase in a clean tube, and dried with nitrogen. Sample preparation processes were performed in accordance with the above method of parallel preparation of quality control samples. Mass spectrometric analysis was conducted by adding 200 µL isopropanol/methanol solution (1:1, v:v), and the supernatant was used for analysis. For targeted metabolomic analyses, multiple reaction monitoring transitions representing the metabolites were simultaneously monitored, and positive/negative polarity switching was used. Data analyses were performed according to the instructions of Shanghai Applied Protein Technology20.
Tandem Mass Tag (Tmt) Labeling Proteomics
The total protein in KLE cells and KLE-XCT790 cell samples was extracted and evaluated by SDS-PAGE and staining. The qualified protein samples were labeled with trypsin and TMT. The labeled polypeptides were mixed into one component in equal quantities. After desalination, high-pH RP-HPLC was used for grading. Eight different polypeptide components were obtained, and each component was separated by nano-HPLC and detected by mass spectrometry. Then, maxQuant search software was used for protein identification and quantitative analysis. After the quantitative results were standardized, statistical analysis was conducted to screen out the differentially expressed proteins.
Mitochondrial Stress Detection
Mitochondrial stress detection was conducted by using the Seahorse XF Cell Mitochondrial Stress Test Kit. First, prepare the test solution (Seahorse XF DMEM medium with additives including 1 mmol/L sodium pyruvate, 2 mmol/L glutamine, and 10 mmol/L glucose). The test solution was heated in a water bath to 37°C and prepared for use. Next, prepare the compound storage and working fluid. Using a Seahorse XF cell mitochondrial stress test kit, oligomycin (blue cap), 0.5 µM FCCP (yellow cap), and rotenone/antimycin A (red cap) were properly prepared into a working solution and added to the dole on the probe plate. Seahorse XF cell culture microplates were removed from a 37°C CO2 incubator, and the cells were examined under a microscope to confirm the degree of confluence. Remove the test solution from the water bath. The cell growth medium in the cell culture microplates was replaced with preheated detection solution using a multichannel pipette, and the cell culture microplates were placed in a CO2-free incubator at 37°C for 1 hour. Then, run the experiment on the computer and analyze the data.
All tissues were assembled into a tissue microarray. Immunostaining for ERRα and TFEB was performed according to standard procedures. Rabbit polyclonal anti-ERRα (dilution 1:100; Abcam, UK) and rabbit polyclonal anti-TFEB (dilution 1:100; Cell Signaling Technology, China) antibodies were used. The percentage of positive cells was scored as 0 (cells < 5%), 1 (5–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The positive staining intensity was scored as 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The expression levels of ERRα and TFEB were assessed to determine their immunoreactive scores (IRSs) using the algorithm IRS = Si × Pi (where Si and Pi represent the intensity and percentage of positively stained cells, respectively). Samples were divided into four groups based on their IRS: 0, negative (-); 1–4, weakly positive (+); 5–8, positive (++); and 9–12, strongly positive (+++).
Participants And Specimens
EC tissue and normal endometrial tissue samples and blood samples with relevant clinical data were obtained from surgical patients in Fujian Provincial Maternity and Children’s Health Hospital of Fujian Medical University, China from 2013 to 2018. None of the patients received any preoperative radiation, chemotherapy or hormone therapy. Finally, we collected 111 tissue specimens, including 79 EC specimens and 32 normal endometrium specimens. The samples were embedded in paraffin, and all diagnoses of the pathological sections were made by experienced pathologists. In addition, according to ERRα immunoreactive scores, 35 cases with the highest score and 20 cases with the lowest score were selected for lipidomic analysis. Finally, because of 1 EC tissue sample missed (IRS = 7), a total of 54 patient tissues from 35 EC patients and 19 patients with normal endometrium were also collected for lipidomics analysis (Supplement Fig. 1). All patients were informed of the experiments and signed informed consent forms. This research protocol was approved by the Ethics Committee of Fujian Provincial Maternity and Children’s Hospital (No. FMCH-2018-14).
Statistical analysis was performed using GraphPad Prism 8.0 software and IBM SPSS (version 22). Statistical significance was determined by Student’s t test or by ANOVA, and related parameters were analyzed using Pearson’s correlation. Correlation coefficients for graded data were obtained using Pearson correlation analysis. Receiver operating characteristic (ROC) curves and the Youden Index were used to determine the cut-off point of continuous variable. The univariate binary logistic regression analyses was used to analyse indicators associated with EC. Differences with p-values less than 0.05 were considered significant.