Cell culture and cell knockdown
The cells involved in the experiment were immortalized human trophoblast HTR8/SVneo cell line obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and incubated in the RPMI 1640 medium containing 10% fetal bovine serum（FBS, PAN, Germany）and 1% penicillin and streptomycin. The cells were cultured in a standard cultivation environment (in a humidified environment with 37℃ and 5% CO2). Lentiviral vectors (GenePharma, China) were used to transfect HTR8/SVneo cells for 48 h to knock down the expression of FoxO3a, and there were two cell groups: control group (Normal) and scrambled shRNA group (sh-FoxO3a). Sodium nitroprusside (SNP) was selectively added into the complete medium to construct oxidative stress in the cells, and the optimal concentration has been explored in previous studies.
Cellular protein from the transfected HTR8/SVneo cells was extracted using RIPA lysis buffer (Beyotime Biotechnology, China) containing PMSF (1:100, Beyotime Biotechnology, China). The concentration of the extracted protein was determined by using a BCA assay kit (Beyotime Biotechnology, China). Each protein sample was loaded onto SDS-PAGE for electrophoresis and then transferred to a piece of PVDF membrane. TBST containing 5% skimmed milk was used to block the PVDF membrane for 1 h. Next, the membrane was incubated with various primary rabbit antibodies, including anti-FoxO3a (1:800, Catalog#:12829, Cell Signaling Technology) and β-actin (1:5000, Catalog#: GB11001, Servicebio) at 4 °C overnight. After incubated with goat anti-rabbit IgG for 1 h, bands density was detected using the Quantity One System image analyzer (Bio-Rad, USA).
Cell migration assay
Wound Healing Assay was performed to assess the ability of migration between the normal group and the sh-FoxO3a group. 5´105 cells were plated into a 6-well plate. A 200 µl sterile pipette tip was applied to scratch the cell monolayers when cells reached 90% confluence. Floating cells were removed with PBS, then cells remained were incubated in fresh complete mediums for 24 h. Images were taken by microscopy (EVOS FL Auto Imaging System, Life Technologies, USA) at the time point of 0h and 24 h after the scratch, and ImageJ software was used to measure the wound healing rate.
Cell invasion assay
The invasiveness of the two group cells was detected using Matrigel invasion assay. After the diluted matrigel (BD BioScience) was added to the invasion chamber for 4 h, about 5´104 cells were seeded into the upper compartment. After incubation for 24 h, a cotton swab was used to wipe the residual cells in the upper chamber. The lower chamber cells were fixed using 4% paraformaldehyde, washed with PBS, and stained using crystal violet (Beyotime Biotechnology, China). We used microscopy (EVOS FL Auto Imaging System, Life Technologies, USA) to determine the amount of the cells on the lower chamber. ImageJ software was used to evaluate the invasion rate.
Metabolic flux analysis of the mitochondrial
Seahorse XFp Analyzer (Agilent, Santa Clara, CA) was used to evaluate the oxygen consumption rate (OCR), which reflected the mitochondrial function. HTR8/SVneo cells (Normal group, sh-FoxO3a group, Normal+SNP group, sh-FoxO3a+SNP group) were seeded in Seahorse XFp plates and cultured in a complete medium overnight. The next day, XF assay medium was added to replace the complete medium, and then the cells were incubated at 37℃ without CO2 input atmosphere. FCCP (working concentration:10 mM), Oligomycin (working concentration: 2.5 mM), and Rotenone (working concentration: 0.5 mM) were added into the probe separately. At the time point of 26 min (basal respiration detection lasted for 26 min), 50 min and 70 min, oligomycin (2.5 mM), FCCP (10 mM), antimycin A (2.5 mM) /rotenone (2.5 mM) were injected into the chamber respectively. Mitochondrial parameters (basal respiration, proton spill, maximal respiration, and ATP turnover rate) were evaluated by various OCR indexes. Seahorse XFp software was used to analyze the OCR index.
Intracellular, extracellular, and biomass metabolite extraction from cell culture
2 ml of each culture medium of normal and knockdown group HTR8/SVneo cells was used for extracellular chemical derivatization. For the intracellular metabolite extraction, 10 ml of liquid nitrogen was added to each plate of HTR8/SVneo cells. Then cold methanol/chloroform (9:1), containing the standard internal 2,3,3,3-d4-alanine (0.3 µmol), was used to extract metabolite from HTR8/SVneo cells. The collected samples were centrifuged at 15000 g for 15 min at 4℃, and the supernatant and the biomass were obtained. The supernatant attained was dried in the SpeedVac (Labconco Corp., Missouri, USA) for 5 h at room temperature and stored at -80℃ for intracellular chemical derivatization. For the biomass metabolite extraction, the fraction of biomass was dissolved in 100 ml sodium hydroxide, and then samples were kept at 98℃ for 10 min. 100 ml ddH2O and 200 ml methanol were added to each heated sample. The collected specimens were centrifuged at 15000 g for 15 min at 4℃, and the supernatant was obtained for chemical derivatization.
Chemical derivatization of metabolites and GC-MS assay
The samples from Intracellular, extracellular, and biomass were derivatized using the methyl chloroformate (MCF) method as previously described (Smart et al. 2010). The chemical derivatives were analyzed by a system of Agilent GC7890B coupled to an MSD5977A mass selective detector (EI) set at 70 eV. The ZB-1701 GC capillary column (30 m × 250 µm id ×0.15 µm with 5 m guard column, Phenomenex) was used for metabolite analysis. The parameter analysis was previously described (Smart et al. 2010).
GC-MS data analysis
The software, which is based on MassOmics XCMS R, was applied to extrapolate the relative abundance of the metabolites through the peak height of the most enriched ion mass (https://zenodo.org/record/4961895). Aiming to achieve stable repeatability and instrumental deviations and minimize sample preparation, the corresponding concentration of the identified metabolites normalized by an internal standard (D4-alanine), total ion concentration of the cellular metabolome, and revised by quality control of pooled samples. Before the HTR8/SVneo metabolome was analyzed, each metabolite concentration was transformed by log10 scale and Pareto scaling set up Gaussian distribution for this data. Model validation and partial least squares discriminant analysis (PLS-DA) were operated through MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/). The Student's t-test and false discovery rate were implemented to calculate the significance of HTR8/SVneo metabolites between two groups by using R software. Only two-tailed P-values less than 0.05 were regarded as statistically significant. Receiver operating characteristic (ROC) curves were conducted using the pROC R package (Robin et al. 2011). Pathway enrichment analysis was performed by blasting our identified metabolites to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. The chord plot illustrated how the GOplot R package rendered metabolites participating in KEGG metabolic pathways.
Isotope Tracer Experiment
The effect of 13C-labeled tracer (U-13C6 glucose) on flux estimation precision was measured in previous research (reference). There were two types of culture mediums used in 13C-glucose isotope labeling and metabolomics experiments: 1. RPMI 1640 medium containing 30% 13C6-labelled glucose (U-13C6 glucose); 2. RPMI 1640 medium containing 30% 12C6-labelled glucose. Subsequently, as described above, metabolite extraction, chemical derivatization, and GC-MS analysis were performed.
Cellular oxidative stress detection
ROS in the HTR8/SVneo cells was measured using a ROS Detection Assay Kit (Beyotime, China). After knockdown of FoxO3a by lentiviral vectors (GenePharma, China) for 2 days, the HTR8/SVneo cells seeded in six-well plates were incubated with 1.5 mL of 0.1% DCFH-DA (diluted with a-MEM) at 37℃ for 25 min, and then complete medium-BSA free was used to wash the cells for three times. Subsequently, green fluorescence was evaluated by using a fluorescence microscope.
According to the manufacturer's protocol, total RNA was extracted from HTR8/SVneo cells when it reached 90% fusion with TRIzol (Invitrogen, Carlsbad, CA, USA). Then, Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) was used to assess the quantity of RNA, and then qualified RNA was detected by performing agarose gel electrophoresis (RNase free). Next, beads of Oligo(dT) were applied to enrich mRNA. The enriched RNA fragments were broken by ultrasound and then as templates to synthesize cDNA with random primers. Before the purified cDNA came to Illumina, sequencing adapters, end-repaired, and poly(A) added were performed using a PCR extraction kit (Qiagen, Venlo, The Netherlands). RNase-free agarose gel electrophoresis was operated for size selection of the ligation products. Next, fragments amplified by PCR were sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology company (Guangzhou, China). Differential expression of the obtained RNA (two groups) was conculcated by DESeq2 and edgeR software. Only the false discovery rate (FDR) below 0.05 and fold change ≥ 2 of these transcripts were considered differentially expressed genes.
According to the manufacturer's instructions, RNA of the two groups of cultured cell lines was extracted using TRIzol reagent (Invitrogen, USA). The obtained RNA concentration was assessed using ultraviolet spectroscopy (Nano Drop 2000, Thermo, USA). Subsequently, Roche Reverse Transcription Kit (#07912455001, Roche, Germany) transcribes 1 mg RNA of each sample to cDNA reversely. GAPDH (housekeeping gene) was used to control relative gene expression analysis. The primer pairs of GAPDH were: forward: 5' GGAAGCTTGTCATCAATGGAAATC 3', reverse: 5' TGATGACCCTTTTGGCTCCC 3'. Primers for the target gene were as follows: COX-2: forward: 5' AAGACAGATCATAAGCGAGGGC 3', reverse: 5' AAACCGTAGATGCTCAGGGACT 3'; MMP9: forward: 5' TCGACGTGAAGGCGCAGAT 3', reverse: 5' AGAAGCGGTCCTGGCAGAAATA 3'.