Microarray data collection, comparison and meta-analysis
Because the use of small sample sizes and different microarray analyses may lead to errors during the screening process, the results may not be completely reliable. A meta-analysis is a statistical analysis method that combines multiple studies to analyze a larger population to obtain more accurate and comprehensive results. GEO (https://www.ncbi.nlm.nih.gov/geo/) is a national public database that includes high-throughput gene expression, array-based and sequence-based data. In this study, relevant gene expression data were obtained from the GEO database (GSE6008, GSE18520, GSE26712, GSE27651 and GSE29450). Then, a meta-analysis of these datasets was performed to identify DEGs. In addition, the subtype classification data of GSE9891 were collected and analyzed, and the relationship between subtype classification and HNRNPU expression was studied.
TCGA data extraction and analysis
mRNA expression profiling data from TCGA were extracted from the UCSC database (http://xena.ucsc.edu/). Then, the OC samples downloaded from TCGA were categorized into four subtypes, immunoreactive, proliferative, differentiated and mesenchymal, according to the TCGA research team's criteria for classifying OC subtypes.
Cell lines and cell culture
The human OC OVCAR3 and ES2 cell lines were purchased from the Cell Resource Center of Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were grown in RPMI-1640 medium and Dulbecco's modified Eagle's medium (DMEM) (Invitrogen, Carlsbad, CA, USA), respectively. The cells were cultured in medium containing 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 100 U/mL penicillin and streptomycin (Gibco, Grand Island, NY, USA) at 37°C with 5% CO2. Then, the cells were harvested in the logarithmic growth phase for subsequent experiments.
Transfection and RNA interference
Transfections with small interfering RNAs (siRNAs) were performed using TurboFect Transfection Reagent (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's protocol. siRNAs were synthesized by Shanghai Jima pharmaceutical technology (Shanghai, China). The siRNA sequences for human HNRNPU were as follows: siHNRNPU_#1, CUGUGAGACUGAAGAUUAUTTAUAAUCUUCAGUCUCACAGTT and siHNRNPU_#2, GCUGAGUGCUUUGAUGAAATTUUUCAUCAAAGCACUCAGCTT. The sequence of the control siRNA was UUCUCCGAACGUGUCACGUTTACGUGACACGUUCGGAGAATT.
RNA purification and quantitative RT-PCR
We used 1 μg of total RNA extracted with TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for reverse transcription (RT) using the PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takala Biomedical Technology, Beijing, China) according to the manufacturer’s protocol. Quantitative RT polymerase chain reaction (RT-PCR) was performed with TB Green Premix Ex TaqⅡ(Tli RNaseH Plus) (Takala Biomedical Technology, Beijing, China) using an Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA USA). All reactions were conducted in triplicate. We calculated the relative mRNA expression by the comparative Ct method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. The following primers were used for GAPDH: forward, 5'-TGACTTCAACAGCGACACCCA-3' and reverse, 5'-CACCCTGTTGCTGTAGCCAAA-3'. The following primers were used for HNRNPU: forward, 5'-GAGCATCCTATGGTGTGTCAAA-3' and reverse, 5'-TGACCAGCCAATACGAACTTC-3'.
Western blot analysis
Briefly, cells were lysed with radioimmunoprecipitation assay cell lysis buffer (Solarbio Life Sciences, Beijing, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and quantified using a bicinchoninic acid assay kit (Solarbio Life Sciences, Beijing, China). Equal amounts of protein (20 µg) were separated by SDS-PAGE (8% gel) and then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked for 1 h with 5% skimmed milk at room temperature and then incubated with primary antibodies overnight at 4°C. The primary antibodies against HNRNPU (1:1000) and GAPDH (1:1,000) were purchased from ProteinTech Group (Chicago, IL, USA). After three washes in Tris-buffered saline with Tween-20, the membranes were incubated with secondary goat anti-rabbit or goat anti-mouse antibody (ProteinTech Group, Chicago, IL, USA) at a dilution of 1:1000 for 2 h at 37°C. Finally, proteins were visualized with an enhanced chemiluminescent (ECL) detection solution (Millipore, Billerica, MA, USA).
CCK-8 assays were performed using CCK-8 reagent (Solarbio Life Sciences, Beijing, China). A total of 3000 cells were seeded into a 96-well culture plate and incubated for 1-6 days in an incubator at 37°C in 5% CO2. One hundred microliters of prepared solution (10 µl CCK-8 reagent added to 90 µl medium) was added to each well and incubated for 2 h. The absorbance (OD) of each well at 450 nm was measured using a CMax Plus microplate reader (Molecular Devices, San Francisco, CA, USA). The growth curve was generated according to the OD values.
Colony formation assay
A total of 3000 cells were seeded into a 6-well culture plate and incubated for 2 weeks in an incubator at 37°C in 5% CO2 until visible cell clones were observed. The clones were fixed with 4% formaldehyde (Sigma, St Louis, MO, USA) for 30 min and stained with 1% crystal violet (w/v) for 15 min before counting the number of colonies.
5-Ethynyl-2’-deoxyuridine (EdU) staining assay
Cells were further cultured for 48 h after transfection and labeled by EdU DNA Proliferation in vitro Detection (RiboBio, Guangzhou, China). We used phosphate-buffered saline (PBS) containing 4% polyformaldehyde to fix the cells at room temperature for 30 min. Then, we neutralized the cells with 2 mg/mL deoxythymidylate, which was cleared with 0.1% Triton X-100 applied for 10 min, followed by a PBS wash. Apollo 488 was applied for 30 min, and the nuclei were stained with Hoechst 33342 for 30 min. Subsequently, images were captured using a fluorescence microscope. DAPI-stained cells were regarded as total cells. The signal was activated by laser at a wavelength of 350 nm, while the emitted light was collected in the blue channel (bandpass filter of 420-500 nm). We obtained the number of total cells by collecting and analyzing the target signatures at peak intensity, width and depth. The EdU-Apollo 488-stained cells were considered proliferating cells. The signal was activated by laser at a wavelength of 490 nm, and emitted light was captured by the red channel to determine the number of proliferating cells.
Analysis of cell cycle distribution using flow cytometry
OVCAR3 and ES2 cells (1×106) treated with or without siHNRNPU were collected, fixed with 1500 μL of 95% cold ethanol for 2 h or overnight according to the manual for the cell cycle detection kit (Keygen Biotech, Nanjing, China) and stored at 4°C. After removal of the fixative with PBS, the cells were placed in a water bath, 2 μL RNase A (50 μg/mL final concentration) was added at 37°C, and the cells were incubated for 30 min. Furthermore, we incubated the cells with 25 μL propidium iodide (PI) (50 μg/mL final concentration) for 30 min in the dark. The cell cycle was analyzed with a BD Accuri C6 flow cytometer (Beckman Coulter, Fullerton, CA, USA) by red fluorescence set to a wavelength of 480 nm.
Data were assessed using Student’s t-test or one-way ANOVA followed by Fisher’s multiple range test.