Data Collection and Analysis
The expression and clinical data of The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) were downloaded from the UCSC Xena database (https://xenabrowser.net/datapages/). For FOXA1, the DNA copy number and methylation information were obtained from the cBioPortal database (https://www.cbioportal.org/). The survival analysis of FOXA1 in EOC database from GSE26193, GSE26712, and GSE63885 was performed using PrognoScan database.
Gene Set Enrichment Analysis (GSEA)
Correlation analyses between FOXA1 and other genes were performed using data from TCGA-OV cohort, and Pearson’s correlation coefficient was calculated. GSEA was conducted using the R package “clusterProfiler” based on Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway database.
Correlation analysis of FOXA1 and drug response
We downloaded half-inhibitory concentration (IC50) values of 192 anti-cancer drugs and FOXA1 expression profiles of 809 cell lines from the Genomics of Drug Sensitivity in Cancer database (GDSC: https://www.cancerrxgene.org/) and analyzed the Spearman’s correlation between FOXA1 expression and IC50 values of anti-cancer drugs.
OVCAR3, A2780, 3AO, SKOV-3 (ovarian tumor cell lines) and IOSE80 (normal ovarian cell line) were purchased from ATCC of the United States. OVCAR3, 3AO and A2780 were cultured in RPMI 1640 medium supplemented with penicillin (100 U/mL), streptomycin (100 mg/L), 10% v/v fetal bovine serum (FBS). IOSE80 and SKOV3 were cultured in DMEM and McCoy's 5A medium containing penicillin (100 U/mL), streptomycin (100 mg/L) and 10% FBS, respectively. The cells were cultured at 37 ℃ and 5% CO2. (The culture medium and supplements were purchased from Invitrogen)
Silencing FOXA1 by using small interfering RNA (siRNA)
FOXA1 siRNA and its negative control (NC) siRNA were purchased from Shanghai Sangon Co., Ltd. (Shanghai, China). Three siRNA sequences were as follows: siRNA#1 (5′-GCGACUGGAACAGCUACUATT-3′; 5′-UAGUAGCUGUUCCAGUCGCTT-3′), siRNA#2 (5′-CCACUCGCUGUCCUUCAAUTT-3′; 5′-AUUGAAGGACAGCGAGUGGTT-3′, siRNA#3 (5′-GCACUGCAAUACUCGCCUUTT-3′; 5′-AAGGCGAGUAUUGCAGUGCTT-3′). Transfection of siRNA was performed by using Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher). After transfection for 72h, the cells were collected to evaluate the knockdown efficiency of FOXA1 in OVCAR-3 cells (n=6) by Western Blot and qPCR. The siRNA with the best efficiency was used in subsequent experiments.
CCK-8 viability assay
OVCAR3 cells transfected with siRNA-FoxA1 or siRNA-NC were cultured in a 96-well plate with 1×104 cells/well and 6 parallel wells in each group. The cells were cultured for 24, 48 and 72 h respectively before CCK-8 assay. CCK-8 solution was incubated with cells at 37 ℃ for 1-2 h. The cell survival rate was detected at a wavelength of 450 nm.
BiocoatTM Matrigel® Invasion Chamber was rehydrated for 2 h at 37℃ and 5% CO2 using a filter with 8.0-μm pore size (Corning, USA). After transfection with siRNA, the cells were collected and suspended (1× 106 cells/mL) in serum-free culture medium. 200 μL of the cells in serum-free culture medium were added to the upper compartment, and 600 μL RPMI 1640 containing 10% FBS was added to the lower compartment. After 24 h of incubation at 37 ̊C under 5% CO2, the non-migrating cells were removed and culture medium was discarded. Filters were gently rinsed with PBS, and migrated cells were fixed with 4% w/v formaldehyde for 15 min. 0.1% Crystal violet staining solution was used to stain the cells for 30 min. The upper, middle and lower left and right fields were observed under the optical microscope (magnification ×100) for cell counts for each assay. Cell migration was quantified with Image J software. Each group included three independently performed transwell assays.
The immunofluorescence staining was performed on cells grown on 22 × 22 mm coverslips. OVCAR-3 cells (3×104/well) were grown in a 12-well plate and cultured in the 37℃ incubator overnight. At 72 h after the transfection with 80 nM siRNA-FOXA1, 4% w/v paraformaldehyde was used for fixation for 30 min. 0.5% v/v Triton X-100 was used in permeabilization for 5 min and 10% normal donkey serum for blocking for 1 h at room temperature. Then the cells were incubated at 4℃ overnight with the primary antibodies: mouse anti-E-cadherin (1:500, Abcam, USA) and rabbit anti-Vimentin (1:500, Abcam, USA). After washed by PBS for 3 times, secondary antibodies listed as follows were used for incubation for 1 h: Alexa Fluor® 488 Donkey Anti-mouse IgG or Anti-rabbit (1:200, Jackson ImmunoResearch, USA). Cells were finally counterstained with DAPI (Beyotime, China) for 5 min and the coverslips were mounted by using 10 μL of FluroGuard anti-fade solution (Bio-Rad, USA). Images were taken using a confocal microscope (Lieca, Germany).
Quantitative real-time PCR (qPCR)
Cells or Tissues were collected and the RNAs were obtained by using TRIzol® Plus RNA Purification Kit (Thermo Fisher, Carlsbad, CA, USA) following the protocol. SuperScript™ III First-Strand Synthesis SuperMix for qRT-PCR（Thermo Fisher, Carlsbad, CA, USA）was used to synthesize cDNA. Real-time PCR was carried out by applying PowerUp™ SYBRTM Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). The cycling program was set as: 95 °C, 2 min; 40 cycles of amplification (95°C, 15 s; 60°C, 1 min). The sequences of the primers used in this experiment were as follows: FOXA1, 5’-GCATACGAA CAGGCACTGCAATACT-3＇(forward) and 5’-GTGTTTAGGACGGGTCTGGAATA-3＇(reverse); GAPDH: 5’-CCATGACAACTTTGGTATCGTGGAA -3＇(forward) and 5’-GGCCATCACGCCACAGTTTC-3＇(reverse). GAPDH was used as internal control for normalization. The relative expression of the target genes was evaluated by the 2-∆∆Ct method.
Total cytoplasmic and nuclear proteins from OVCAR-3 cells were extracted with RIPA Buffer (Thermo Fisher, USA). NE-PERTM Nuclear and Cytoplasmic Extraction Kit (Thermo Fisher, USA) added with Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher, USA). BCA protein assay kit (Beyotime Biotechnology, China) was used for quantification. Protein (30 μg) was loaded onto SDS-PAGE gel, and transferred to a Hybond-P PVDF membrane (GE Healthcare, USA). 5% fat-free milk in TBST (Tris-buffered saline with 0.1% Tween-20) was used for blocking. Following antibodies were incubated with the membranes at 4℃ overnight: mouse anti-E-cadherin (1:1000, Abcam, USA), rabbit anti-Vimentin (1:1000, Abcam, USA), rabbit anti-Snail (1:1000, Abcam, USA), and GAPDH (1:5000, Abcam, USA) as an internal control. Protein expression was visualized on X-ray films using the HRP-conjugated goat anti- mouse or rabbit secondary antibodies (1:5000, Thermo Fisher, USA) and SuperSignal West Dura Extended Duration Substrate (Thermo Fisher, USA). Band intensities were quantitated using Image Pro Plus 6.0 software. The results were presented as the density ratio of the target protein band to the internal control.
Construction of the CTGF promoter luciferase reporter plasmids
The binding site of FOXA1 with CTGF was predicted on LASAGNA-search. Sequences of the wild type CTGF promoter (-500 to -1) and mutant CTGF promoter (-399 to -390, CAGGGCAAAC to CACCGCTTAC) with recognition sites specific for the enzymes KpnI/XhoI were manufactured by Sangon Biotech (Shanghai). FOXA1 CDS flanked with BamHI/EcoRI was cloned as well. Luciferase reporter plasmids, pGL3-Basic-CTGF-w (wild type) and pGL3-Basic-CTGF-m (mutant), were constructed by having the primers flanked with KpnI/Xho cloned into the KpnI/Xho sites of pGL3-Basic vector (Promega). The FOXA1 overexpression plasmid was created by cloning the FOXA1 CDS sequence with BamHI/EcoRI into pcDNA3.1 (Invitrogen). pRL-TK vector (Promega)，the renilla luciferase plasmid, was adopted as an internal control reporter vector.
Dual-luciferase reporter assays
OVCAR3 cells were co-transfected with a combination of plasmids comprising of either a mutant or wild-type CTGF reporter gene plasmid, a pRL-TK Renilla luciferase reporter plasmid, and a FOXA1 overexpression plasmid. Thus, the cells were transfected with the combination of plasmids as following respectively: pGL3-Basic/pcDNA3.1-FOXA1/ pRL-TK; or pGL3-Basic-CTGF-w/pcDNA3.1-FOXA1/pRL-TK; or pGL3-Basic-CTGF-m/pcDNA3.1-FOXA1/pRL-TK group. Activities of firefly renilla luciferases were measured 48 h after transfection according to the dual luciferase reporter assay system, with six replicas in each group (Promega).
Effects of FOXA1 silencing on CTGF/TGF-β pathway and EMT-associated markers
OVCAR3 cells were transfected with siRNA-NC or siRNA-FOXA1, which was followed by replacing with the serum-free culture medium after 12 h. The TGF-β1 groups were stimulated with 10 ng/ml TGF-β1 accordingly (R&D systems). Cells were collected after 48 h for Western Blot analysis for FOXA1, CTGF, MMP-2, E-cadherin and snail using rabbit-anti-CTGF antibody (1:1000, Abcam, USA), and rabbit-anti-MMP2 antibody (1: 500, Abcam, USA).
Lithium chloride treatment
The cells were grouped as follows: control; siRNA-NC-transfected; siRNA-NC-transfected and LiCl treatment; siRNA-FOXA1-transfected; siRNA-FOXA1-transfected and LiCl treatment. A final concentration of 10 mM LiCl was added to cells after transfection of siRNA-NC or siRNA-FOXA1 for 8 h.
SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The results were expressed as average value ± standard deviation (SD). The paired, two-tailed Student’s t-test was used to compare the results between two groups. Two-sided p value less than 0.05 was regarded as statistically significant. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.