2.1 Bioinformatic analysis of TCGA data and GEO data
The TCGA visual online database cBioPortal (https://www.cbioportal.org/) was used to analyse the survival of patients with OvCa and 19q13.2 AMP, the genes contained within 19q13.2, and the change in MED29 expression after gene CNAs. The Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) is a national public database, including high-throughput gene expression and array- and sequence-based data . In this study, qualified data were obtained from the GEO dataset GSE29450 (Affymetrix Human Genome U133 Plus 2.0 Array; 10 samples of OvCa and 10 samples of normal tissue), and then the data were pre-processed and analysed with R software. A boxplot was built to show the differential analysis of specific gene expression between OvCa tissues and non-tumour tissues. Kaplan-Meier Plotter (http://kmplot.com/analysis/) was used to certificate the relationship between the dysregulated genes and patient survival. The pathway mapping of candidate genes by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (http://www.webgestalt.org/website.pathway) was also executed.
2.2 TMA and IHC
A tissue microarray (TMA) comprising 80 formalin-fixed paraffin-embedded primary OvCa tissue dots with corresponding clinical annotations was purchased from Taibsbio.com (Xi’an, China).
Immunohistochemistry (IHC) was performed to detect MED29 protein expression in tumour and normal tissues in the microarray. The TMA was sent to Servicebio Company (Hunan, China) for immunohistochemical analysis. Quantitative evaluation and automatic scoring of immunohistochemical images were performed with ImageJ software. This method is based on the research of Varghese F et al. . The score was evaluated into 4 grades: 1 (almost negative), 2 (weakly positive), 3 (positive), and 4 (strongly positive).
2.3 Cell culture
OVCAR3 cells (Cell Resource Center, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 medium (Solarbio, Beijing, China) complemented with 20% FBS (Gibco), penicillin (100 U/ml) and streptomycin (100 ng/ml). A2780 cells (Cell Resource Center, Shanghai Academy of Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 medium (Solarbio) complemented with 10% FBS (Gibco), penicillin (100 U/ml) and streptomycin (100 ng/ml). ES-2 cells were cultured in DMEM (Gibco) complemented with 10% FBS (Gibco), levofloxacin penicillin (100 U/ml) and streptomycin (100 ng/ml). All cells were incubated at 37°C in 5% CO2.
2.4 siRNA-mediated MED29 gene silencing
Two MED29 gene-specific siRNAs (295 and 426) and negative control siRNA (NC) were obtained from GenePharma, Shanghai, China. The siRNA sequences targeting MED29 were as follows: siRNA-295-MED29, 5′-CAGAACACUAACAUCGACATT-3′; siRNA-426-MED29, 5′-GUCACAGAGUUGUGACAGUTT-3′; and siRNA-NC-MED29, 5′-CAGAACACUAACAUCGACATT-3′. In all, 5 nM siRNAs were transfected into 60% confluent cells using Thermo Turbofect transfection reagents (Thermo Fisher Scientific, UAB) accordance with the manufacturer’s protocol. Then the cells were incubated in 5% CO2 at 37°C for 48 h until subsequent analysis.
2.5 RT-PCR analysis
Total RNA from OvCa cells was extracted with an AxyPrep Multisource Total RNA Miniprep Kit (Axygen Scientific, Union City, CA, USA) accordance with the standard protocol. cDNA (20 µl) was synthesized from 1 µg of total RNA by a Takara PrimeScript™ RT reagent kit and gDNA Eraser (Cat# RR047A, Lot# AK2802). Quantitative real-time PCR (RT-qPCR) by TB GreenTM Premix Ex TaqTM II (TakaRa Code: DRR820A) was fulfilled following the standard protocol on a 7900HT Fast Real-time System (Applied Biosystems, Foster City, CA, USA). GAPDH was used as the reference gene. The relative gene expression levels were determined using the critical threshold (Ct) number and calculated through the 2−ΔΔCt method.
2.6 Western blot
At 48 h following siRNA transfection, cells were lysed with RIPA buffer and centrifuged at 12000× g for 10 min in order to collect total protein. A PierceTM BCA protein assay kit (Thermo Fisher Scientific, UAB) was used to determine the protein concentrations in each sample. In all, 25 µg of total protein was separated via SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After they were enclosed in Tris-buffered saline (TBS) with 5% non-fat milk for 1 h to saturate additional protein binding sites, the blots were incubated with the following primary antibodies: anti-Med29 (clone sc-502, 1:500 dilution, Santa Cruz Biotechnology, Santa Cruz, CA), anti-Fibronectin (1:1000, Proteintech, cat15613-1-AP), anti-E-cadherin (1:5000, Proteintech, cat20874-1-AP), anti-N-cadherin (1:2000, Proteintech, cat22018-1-AP), anti-Vimentin (1:2000, Proteintech, cat10366-AP), anti-cyclinD1 (1:5000, Proteintech, cat60186-1-Ig), anti-cyclinE (1:1000, Proteintech, cat11935-1-AP), and anti-cyclinB1 (1:1000, Proteintech, cat55004-1-AP) antibodies at 4°C for 12 h, then incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary IgGs at room temperature for 2 h or at 4°C for 4 h. The proteins were visualized through using a BM chemiluminescence Western blotting kit (Roche Diagnostics GmbH). To guarantee equal loading and accuracy for assessment of the changes in protein abundance, the protein levels were standardized to those of GAPDH as a housekeeping control.
2.7 Colony formation assay
At 48 h after siRNA transfection, cells were inoculated in 6-well tissue culture plates at a density of 50 cells/cm² and cultured for 14 d, after which colonies were fixed with ethanol, stained with 2% crystal violet, washed with PBS to remove excess dye, and imaged on a scanner. To determine the quantitative changes in clonogenicity by counting the number of colonies with ImageJ software.
2.8 EdU cell proliferation assay
Using incorporation of 5-ethynyl-2′-deoxyuridine (EdU) with an EdU Cell Proliferation Assay Kit (RIBOBIO, Guangzhou, China, Cat# C10310-3) to detect cell proliferation. At 48 h following siRNA transfection, 5×103 OVCAR3 and A2780 cells were plated in 24-well plates, and the cells were incubated in complete medium under standard conditions. Images viewed under a fluorescence microscope were obtained.
2.9 CCK8 assay
Using the CCK-8 kit (US EVERBRIGHT INC, China, Cat: C6005-500T) to detected cell proliferation. Cells were inoculated in 96-well plates in 100 µl of medium at approximately 3000 cells per well, and three independent parallel experiments were established. After cells were incubated at 37°C in 5% CO2 for 24 h, 10 µl of CCK8 was added into the wells after an additional 1, 2, 3, 4, 5 and 6 days and cultured for 2 h. The absorbance in each well at a wavelength of 450 nm was measured.
2.10 Cell migration assay
Serum-starved OVCAR3 cells (2×104 cells) in 200 µl of 1640 medium complemented with 5% FBS were plated into the upper chamber of prepared transwells; the lower chamber contained medium supplemented with 20% FBS. A2780 cells (1×105 cells) in 200 µl of serum-free 1640 medium were plated into the upper chamber of prepared transwells; the lower chamber contained medium supplemented with 10% FBS. After plates were incubated in 5% CO2 at 37°C for 24 h, the migratory or invading cells in the lower chamber were fixed and stained with 2% crystal violet. Five representative fields of view of each membrane were selected, microscopy images by were captured, and the number of migrating cells or invasive cells was counted with ImageJ.
2.11 Cell cycle analysis by flow cytometry
Forty-eight hours after transfection with siRNA targeting MED29, cells were fixed and stained with propidium iodide (PI) (50 µg/ml, Sigma). The tests were executed in triplicate. To perform accurate cell cycle analysis, the cells subjected to siRNA transfection were centrifuged at 1500 rpm for 5 min and afterwards resuspended in 500 µl of PBS, and 1.5 ml of 95% ethanol (-20°C precooled) was added to fix the cells at -20°C for 10 minutes. The cells were vortexed before they were centrifuged at 1500 rpm for 10 min, after which the supernatant was discarded and the cells were resuspended in 500 µl of PBS for 10 min before subsequent treatment with RNase A at 37°C for 10 minutes. Ultimately, PI (2 µg/ml) was added to the cells for 15 min to stain the DNA, and then the BrdU test was performed. BrdU analysis and cell cycle distribution were executed with BD CellQuest Pro™ on a BD FACSCalibur flow cytometer (BD Biosciences, New Jersey, USA) through obtaining a minimum of 2×104 mononuclear cells.
2.12 Statistical analyses
Statistical analyses were performed using SPSS 22.0 and GraphPad Prism 5 software. Each experiment was independently repeated at least triplicate. All data are expressed as the means ± standard deviation (means ± SD). One-way analysis of variance (ANOVA) was conducted to compare multiple groups, whereas Student’s t-test was used for comparisons between two groups. For all analyses, two-tailed p values below 0.05 were considered significant and are indicated as follows: *p < 0.05, **p < 0.01.