Reagents and antibodies
Equol (CAS: 94105-90-5) was purchased from Aladdin Reagent (Shanghai, China). The Caspase-3 Activity Assay Kit (C1116), the Cell Cycle Analysis Kit (C1052) and the Mitochondrial Membrane Potential Assay Kit (JC-1, C2006) were obtained from Beyotime Biotechnology (Shanghai, China). The Annexin V-FITC/PI Apoptosis Kit (KGA106) and the cell counting kit-8 (CCK-8; KGA317) were purchased from KeyGen Biotech Co., Ltd. (Nanjing, China). The Diaminobenzidine Kit (DAB-1031) was purchased from Maixin Inc. (Fujian, China). The MagicOmics Micro Proteomics Sample Preparation Kit (MagicOmics-MMB) was purchased from QLBIO (Beijing, China). Antibodies against ubiquitin (A19686) were acquired from ABclonal Technology (Wuhan, China). Anti-cleaved caspase-3 (AF7022) antibodies were purchased from Affinity Biosciences (Changzhou, China). Antibodies against cleaved caspase-7 (#8438), cleaved caspase-9 (#20750), and cleaved poly (ADP-ribose) polymerase (cleaved PARP, #5625) were obtained from CST Company (Danvers, MA, USA). Anti-cytochrome C (Cyt C, 10993-1-AP), Anti-TRIM21 (12108-1-AP), and Anti-GAPDH (60004-1-Ig) antibodies were acquired from Proteintech Group, Inc. (Wuhan, China). HRP-conjugated Goat Anti-Rabbit IgG (SE134) and Goat Anti-Mouse IgG (SE131) antibodies used for western blotting were purchased from Solarbio (Beijing, China). HRP-conjugated Goat Anti-Rabbit IgG (#31460) and Goat Anti-Mouse IgG (#31430) antibodies used for immunohistochemistry were purchased from Solarbio (Pittsburgh, PA, USA). CY3-conjugated Goat Anti-Rabbit IgG (ab6939) and FITC-conjugated Goat Anti-Mouse IgG (ab6785) antibodies were purchased from Abcam (Cambridge, UK). In addition, Anti-Annexin A2 (ANXA2, sc-28385) antibodies were obtained from Santa Cruz Biotechnology (CA, USA).
Clinical sample collection and untargeted metabolomic analysis
Serum samples were collected from 29 GTN (20 invasive mole and 9 choriocarcinoma) patients and 30 normal healthy individuals attending the Shengjing Hospital of China Medical University after the written informed consent to participate was obtained. All experiments were conducted with the approval of the Ethics Committee of Shengjing Hospital of China Medical University for Ethical Review.
We conducted the untargeted metabolomics using ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry. Fifty milligrams of samples were weighted to an EP tube. After the addition of 1000 μL of extract solvent (acetonitrile:methanol:water, 2:2:1, containing 1 μg/mL of internal standard 2-Chloro-l-phenylalanine), the samples were vortexed for 30 s, homogenized at 45 Hz for 4 min, and sonicated for 5 min in ice-water bath. The homogenate and sonicate circle were repeated for 3 times, followed by incubation at − 20 °C for 1 h and centrifugation at 12,000 rpm and 4 °C for 15 min. The resulting supernatants were transferred to LC–MS vials and stored at − 80 °C for UHPLC-QE Orbitrap/MS analysis. LC–MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Q Exactive (Orbitrap MS, ThermoFisher Scientific, Pittsburgh, PA, USA). The injection volume was 2 μL. The mobile phase A was 0.1% formic acid in water for positive, and 5 mmol/L ammonium acetate in water for negative, and the mobile phase B was acetonitrile. The preprocessing results generated a data matrix that consisted of the retention time (RT), mass-to-charge ratio (m/z) values, and peak intensity. OSI-SMMS (version 1.0, Dalian Chem Data Solution Information Technology Co. Ltd.) was used for peak annotation after XCMS data processing with in-house MS/MS database. Based on the orthogonal projection to latent structures-discriminant analysis (OPLS-DA) models, the variable importance in projection (VIP) scores were obtained. The metabolites with Log2fold changes (Log2FC) > 1.5, P-value < 0.05, and VIP ≥ 1 were considered to be differential metabolites (DMs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted to uncover the biological pathways associated with DMs.
Choriocarcinoma cell lines and cell transfection
Human choriocarcinoma cell lines JEG-3 and Bewo were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China), which were cultured in MEM (Solarbio) and Ham’s F-12 K (Servicebio Biotechnology, Wuhan, China) mediums supplemented with 10% fetal bovine serum, respectively. Cells were maintained in a 37 °C cell incubator with an atmosphere of 5% CO2. Both JEG-3 and Bewo cells were transfected with the small interference RNA (siRNA) targeting TRIM21 (siTRIM21) or co-transfected with siTRIM21 and the siRNA targeting ANXA2 (siANXA2) using Lipofectamine 3000 (Invitrogen, CA, USA), according to the manufacturer’s instructions.
Cell viability assay
Cells were treated with desired concentrations of Equol (0–512 μM) for 24 h, transfected with siTRIM21, or co-transfected with siTRIM21 and siANXA2 for 48 h. The transfected cells were then treated with 40 μM Equol for 24 h. Cell viability was detected by CCK-8 assay, according to the manufacturer’s protocol. The optical density at 450 nm was recorded by an 800™ TS microplate reader (BioTek, CA, USA).
Cell cycle assay
JEG-3 and Bewo cells were seeded into 6-well plates at a density of 4 × 105 cells per well and then treated with vehicle or Equol (20 μM and 40 μM) for 24 h. At the end of incubation, cells were harvested and fixed with 70% pre-cooled ethanol for 12 h, centrifuged, and then stained with propidium iodide utilizing a Cell Cycle Analysis Kit according to the manufacturer’s introductions. Cell cycle was analyzed by a NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA).
Annexin V-FITC/PI apoptosis assay
JEG-3 and Bewo cells were seeded into 6-well plates (4 × 105 cells per well) and then treated under different conditions. Cells were directly treated with vehicle or Equol (20 μM and 40 μM) for 24 h, transfected with siTRIM21, or co-transfected with siTRIM21 and siANXA2 for 48 h. Then the transfected cells were treated with 40 μM Equol for 24 h. Subsequently, cells were harvested and stained with Annexin-V-FITC and PI, as described by the protocol for the Annexin V-FITC/PI Apoptosis Kit. Cell apoptosis was analyzed by utilizing a NovoCyte flow cytometer (ACEA Biosciences). For analysis, the annexin V-FITC+/PI− cells were identified as early apoptotic cells, the annexin V-FITC+/PI+ cells as late apoptotic cells, and the annexin V-FITC−/PI− cells as viable cells.
Western blot analysis
Cellular lysates were prepared using RIPA lysis buffer (Solarbio) containing Phenylmethylsulfonyl fluoride (PMSF, Solarbio). Proteins were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon®-P PVDF membranes (Millipore, USA). The membranes were blocked with 5% (M/V) skim-milk in TBST buffer for 1 h at room temperature and then incubated with antibodies against cleaved caspase-3, cleaved caspase-7, cleaved caspase-9, cleaved PARP, Cyt C, TRIM21, ANXA2, and GAPDH at 4℃ overnight. After four washes, the membranes were incubated with the HRP-conjugated Goat Anti-Rabbit IgG and Goat Anti-Mouse IgG antibodies for 1 h at 37 °C. The proteins were visualized using the Gel Imaging System (Beijing Liuyi Factory, China) after reaction with ECL reagent (Solarbio). Quantitative data was obtained using the Gel-Pro-Analyzer software.
Caspase-3 activity assay
Caspase-3 activity was determined using a Caspase-3 Activity Assay Kit, according to the manufacturer’s protocol. Briefly, cells were treated with Equol (20 μM and 40 μM) for 24 h, followed by lysing in lysis buffer on ice for 15 min and centrifugation at 16,000×g for 10 min. The protein concentration of the supernatant for each sample was measured by the Bradford’s method. The reaction was started by adding caspase-3 substrate Ac-DEVD-pNA (10 μL). After incubation for 2 h at 37 °C, the cleavage of the substrate was detected at 405 nm using a microplate reader (ELX-800, BioTek). The activity of caspase-3 was expressed as changes in DEVDase activities.
Mitochondrial membrane potential (MMP) assay
The MMP of cells was analyzed using JC-1 staining. Briefly, cells were stained with JC-1 solution for 20 min at 37 °C. After being rinsed with JC-1 buffer for two times, the MMP of cells was assessed by fluorescence microscopy of JC-1-stained cells under an Olympus IX53 inverted microscope (OLYMPUS, Japan).
Label-free quantitative (LFQ) proteomics
Cells were lysed in lysis buffer containing protease inhibitor. After centrifuging at 14,100×g for 20 min, the supernatant was collected. The protein concentration was measured using the Bradford’s method. The proteins from each sample were subjected to alkylation and digestion. LC–MS/MS analysis of tryptic peptides was conducted on a RIGOL L-3000 HPLC System (RIGOL, Beijing, China) coupled to a Thermo Scientific™ Orbitrap Eclipse™ (ThermoFisher Scientific) via a Nanospray Flex™ (NSI) ion source (ThermoFisher Scientific). Peptides were separated using a gradient from 6 to 12% B in 13 min, then 12–30% B in 33 min and stepped up to 40% B in 7 min followed by a 10 min wash where solvent A was 0.1% formic acid in water and solvent B was 80% acetonitrile and 0.1% formic acid in water. MS spectra was collected in the Orbitrap mass analyzer (120,000 resolution, 350–1500 m/z range) with an automatic gain control target of 4 × 105 and a maximum ion injection time of 50 ms. All raw files were analyzed using the Proteome Discoverer suite (version 2.4, Thermo Fisher Scientific). The proteins with Log2FC > 1.0 and P-value < 0.05 were considered to be differentially expressed proteins (DEPs). Gene Ontology (GO) analysis was conducted to obtain the functions of DEPs.
Molecular docking
The molecular structure of Equol (PubChem CID, 91,469) was retrieved from PubChem Compound (https://pubchem.ncbi.nlm.nih.gov/). The whole protein chain of TRIM21 (AlphaFold ID, AF-P19474-F1) was downloaded from the AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/). Molecular docking was conducted to predict the binding affinities and modes of interaction between Equol and TRIM21 using an online analysis website (https://www.dockeasy.cn/).
RNA extraction and quantitative real-time PCR (qRT-PCR) analysis
Total RNA was extracted using the TriPure reagent (BioTeke, Beijing, China) according to the manufacturer’s instructions. Final concentrations were determined using the NANO 2000 Spectrophotometer (ThermoFisher Scientific). The RNase inhibitor-treated RNA was reverse transcribed to cDNA using the BeyoRT II M-MLV reverse transcriptase (Beyotime). Amplification and detection were performed on the ExicyclerTM 96 Real-Time PCR System (BIONEER, Korea). The mRNA levels of the TRIM21 and ANXA2 genes were standardized against the expression of GAPDH. The primer sequences used for qRT-PCR are shown as follows: TRIM21, forward, 5′-CCCTTTGCTGGGTATGT-3′, reverse, 5′-AAACTCTGCGTGAATCCT-3′; ANXA2, forward, 5′-AAGGGTAGAAGAGCAGAG-3′, reverse, 5′-ATCCACTTGGGAACATC-3′.
Co-immunoprecipitation (Co-IP) assay
Cells were lysed in Cell lysis buffer for Western and IP (Beyotime). The lysates were centrifuged at 10,000×g for 5 min at 4 °C. The supernatants were incubated overnight with 1.0 μg of Goat Anti-Mouse IgG, Anti-TRIM21, or Anti-ANXA2 antibodies. The mixture was incubated for 2 h at 4 °C with 60 μL protein A/G agarose (Beyotime). The immunoprecipitated samples were washed 3 times with pre-cooled PBS buffer and then subjected to western blotting with the indicated antibodies including Anti-TRIM21, Anti-ANXA2, or Anti-Ubiquitin antibodies.
Immunofluorescence
The cells tested were fixed with 4% paraformaldehyde, and then followed a permeabilized procedure with 0.1% Triton X-100 solution (Beyotime). Proteins were blocked with 1% BSA (Sangon, Shanghai, China). Subsequently, slides were incubated with anti-TRIM21 and anti-ANXA2 overnight, followed by incubation with CY3-conjugated Goat Anti-Rabbit IgG and FITC-conjugated Goat Anti-Mouse IgG antibodies. Slides were washed with PBS, prior to counterstaining with 4′,6-diamidino-2-phenylindole (DAPI, Aladdin, Shanghai, China). Fluorescence microscopy was performed using a fluorescence microscope (Olympus, Japan).
β-catenin-driven transactivation assessed by dual-luciferase reporter gene assay
JEG-3 and Bewo cells were seeded into 12-well plates and then cultured for 24 h at 37 °C in a CO2 incubator. Afterwards, TOPflash-luciferase plasmids (TOPflash-luc) purchased from Beyotime Biotechnology were co-transfected with Renilla-TK-luciferase reporter vectors (pRL-TK, Beyotime) into cells, using Lipofectamine 3000. Forty-eight hours after transfection, luciferase activities were measured using a Synergy™ H1 hybrid multi-mode microplate reader (BioTek). The activity of renilla luciferase was used as an internal control to normalize the firefly luciferase activity.
Nude mice subcutaneous xenograft model
Six to eight weeks-old balb/c nude mice were used in this study. Cultured logarithmic phase JEG-3 cells were collected and injected (5 × 106 cells per mouse) subcutaneously into the flank regions of mice. Then, mice were randomized into 2 groups and treated with vehicle (saline, i.g., QD) or Equol (20 mg/kg, i.g., QD). The tumor volume was assessed by caliper measurements every three days. At day 24 after the first administration, the tumors were collected, weighed, and then used for pathological analysis. All procedures complied with the Guide for the Care and Use of Laboratory Animals. The animal experiments were conducted with the approval of the Ethics Committee of Shengjing Hospital of China Medical University.
Immunohistochemistry
The 4% paraformaldehyde-fixed and paraffin-embedded tumor tissues were cut into 5 μm-thick sections. The sections were deparaffinized in xylene and rehydrated through ethanol. Antigen retrieval was conducted by microwave heating for 10 min with sodium citrate buffer. Endogenous peroxidase activity was blocked by 3% H2O2 for 15 min at room temperature. Then, the sections were incubated at 4 °C overnight with primary antibodies against TRIM21 and ANXA2, followed by incubation for 1 h with the secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG or Goat Anti-mouse IgG) at 37 °C. The reaction products were visualized using the Diaminobenzidine Kit. Finally, the sections were counterstained with hematoxylin and observed under an Olympus-BX53 microscope (OLYMPUS, Japan).
Statistics
The data represented in the results section are presented as means with error bars representing standard deviation (SD). All statistical analyses were conducted utilizing the GraphPad Prism version 8.0 software. Comparisons between two or more groups were analyzed by using Student’s t-test, One-way ANOVA, or Two-way ANOVA. Levels of statistical significance were set at P value < 0.05.
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