Dada acquisition and processing
The bioinformatics portal UALCAN (http://ualcan.path.uab.edu) was used to access ERRα protein expression levels in normal ovarian tissues and in in ovarian cancer tissues. This resource for expression analysis uses data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) Confirmatory/Discovery datasets (proteomics by mass-spectrometry) (11). Protein data are expressed as Z-values, and representative of standard deviations from the median across samples for the given cancer type. Log2 spectral count ratio values from CPTAC were first normalized within each sample profile, then normalized across samples.
Cell lines and cell culture
Ovarian cancer cell lines HO8010 and its metastatic equivalent HO8910PM were purchased from the Type Culture Collection Centre of Chinese Academic of Science (Shanghai, China), and were cultured in 90% Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin (100 IU/mL), and 1% streptomycin (100 IU/mL) in a 5%-CO2 incubator at 37°C.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated according to the manufacturer's protocol (Invitrogen, USA, Thermal). The quality and content of mRNA were assessed using a DNA Counter NanoDrop2000 (Thermo Fisher Scientific, Inc.). Those samples with an optical density (OD) 260/280 ratio > 1.8 were used in the experiments. The cDNA synthesis was performed by the Reverse Transcription Reagent Kit (abm, Vancouver, BC, Canada) according to the manufacturer’s instructions. Cobas z480 System (Roche, Basel, Switzerland) and EvaGreen 2X qPCR Master Mix (abm, Vancouver, BC, Canada) were used for quantitative real-time PCR analysis. The specific primers used for qPCR were as follows: ERRα (forward: 5′-ACC GAG AGA TTG TGG TCA CCA-3′, reverse: 5′-CAT CCA CAC GCT CTG CAG TACT-3′ (101 bp); glyceraldehyde 3-phosphate dehydrogenase (GADPH, control): forward 5′-GCA CCG TCA AGG CTG AGA AC-3′, reverse 5′-TGG TGA AGA CGC CAG TGGA-3′ (138 bp). The relative mRNA levels were calculated using the 2(-Delta Delta C(T)) Method (12).
Total protein was extracted from ovarian cancer cells using RIPA lysis buffer (Sig-ma-Aldrich, St. Louis, MO, USA) and low-temperature centrifugation at 10,000 × g for 10 min at 4°C. Total protein was quantified using a bicinchoninic acid (BCA) protein assay reagent kit (Pierce, Rockford, IL, USA). 35 µg of whole cell protein was loaded per lane and separated via SDS-PAGE on a 12% gel at 110 V. Proteins were blotted onto nitrocellulose membranes. Blotted membranes were respectively incubated with the anti-human ERRα rabbit monoclonal antibody (1:500 dilution; Cell Signaling Technology, Beverly, MA, USA) and β-Actin rabbit monoclonal antibody (1:1000 dilution; Affinity Biosciences, no.AF7018) overnight at 4°C. The membranes were washed with Tris-buffered saline containing 0.04% Tween-20 (TBST), followed by incubation with HRP-labeled goat anti-rabbit IgG antibody (1:1000; Affinity Biosciences, no. S0001) for 1 h at room temperature. An enhanced chemiluminescence (ECL) detection system (Thermo Fisher, Waltham, MA, USA) was used to visualize the bands.
Construction of stable ERRα-expressing ovarian cancer cells by lentivirus transfection
A customized ERRα-overexpression lentivirus vector and lentivirus-negative control (plasmid sequence: Ubi-MCS-3FLAG-SV40-EGFP-IRES-puromycin) was obtained from Genechem Co., LTD. (Shanghai, China). Ovarian cancer cells (HO8910) were transfected with the ERRα lentivirus and screened/selected using puromycin (1 µg/ml; Genechem; China). The surviving cells were cultured into multiple monoclonal cell lines and were assessed for the expression of ERRα using PCR analysis. Three groups of cells were set: cells infected with ERRα-overexpression lentivirus vector (group OV-ERRα), lentivirus-negative control (group NC), and cells treated with DMSO as the control (group CON) (Fig. 1).
Treatment with ERRα specific antagonist XCT790
The XCT790 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Prior to treatment, the ovarian cancer cells (HO8910PM) were seeded in 6-well plates at a density of 1×105 cells/well and cultured in 3 ml serum-free DMEM for 12 h to achieve adherence, and then in DMED supplemented with 10% fetal bovine serum, 1% penicillin (100 IU/mL), and 1% streptomycin (100 IU/mL). When the cell population reached about 80% (logarithmic phase), as we previously studied (13), ovarian cancer cells were treated with 10 µM XCT790 for 24 h, and then were collected for RNA and protein extraction. Similarly, prior to the cellular wound healing assay and trans-well chamber assay, cells were incubated with final concentrations of 10 µM XCT790 for 24 h.
In vitro wound healing assay
Ovarian cancer cells were seeded at density 1×105 and cultured in 6-well plates to 80–90% confluence and were serum-starved for 24 h. Two scratches were then introduced to the cell layer in each well using a 100–1,000 µl tip. Following washing twice with PBS, the cells (HO8910, HO8910PM, group OV-ERRα, group NC) were incubated in DMED supplemented with 10% fetal bovine serum, 1% penicillin (100 IU/mL), and 1% streptomycin (100 IU/mL). The cells treated with ERRα specific antagonist XCT790 (group XCT790-treated) were incubated in DMED supplemented with fetal bovine serum, penicillin, streptomycin and 10 µM XCT790. Images of the same regions were captured at 0 and 24 h following stimulation with light microscope (TE2000-U; Nikon, Japan); the paired images were analysed (the ratio of the difference in scratches area between 0 h and 24 h to the 0 h scratches area).
Trans-well chamber migration assay
After thawing overnight at 4°C on ice, Matrigel™ Basement Membrane Matrix (50 µL; BD, USA) was added to a Millicell Hanging Cell Culture Insert (Millipore, USA) to coat the membrane and incubated at 37°C for 30 min. 200-µL cell suspensions containing 0.5% FBS (5.0 × 105 cells/mL) were added to the insert, placed in 24-well plates containing 1,300 µL of DMEM supplemented with 10% FBS. The plates were incubated for 24 h at 37°C. Then, non-invading cells on the top of the filter were removed with a cotton swab, and the filters were fixed with methanol and stained with crystalline violet. The filters were removed from the inserts and mounted onto slides for imaging and quantification as described in a previous study (14).
Statistical analysis was performed using the average results of three experiments under identical conditions. Numerical data are presented as the mean ± standard deviation. Differences between two means were compared by Student's t-test. A one-way analysis of variance was performed for multiple comparisons of groups, which was followed by the Fisher's least significant difference post hoc test, and associated parameters were further analysed using the Spearman's correlation test. Data were analysed using 19.0 for Windows (SPSS Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant difference.