The KGN granulosa cells were obtained from the Stem Cell Bank, Chinese Academy of Sciences; fetal bovine serum (FBS) and 1640 culture medium were purchased from Gibco; MEM and Trizol were obtained from Shanghai Biotech Co., Ltd.; pancreatin from BBI; rabbit anti-human FOXL2 antibody from Beijing Zhongshan Gold Bridge Biotechnology Co., Ltd.; DAB chromogenic reagent kit from Beijing Zhongshan Gold Bridge Biotechnology Co., Ltd.; RT-PCR kit and related experimental reagents from Takara, Japan; DNA Marker from TIANGEN; RIPA lysis buffer from Pulilai Gene Technology Co., Ltd.
Data Analysis
In this study, we manually downloaded the matrix and platform files of dataset GSE34526. Using the GEOquery package in R version 4.3.2, we retrieved the expression matrix and clinical information. The matrix data was then subjected to standardization, and probe IDs were converted to gene names based on the platform file. For genes with the same name in the expression matrix, the average expression level was calculated. Batch effects were removed using the removeBatch function in the limma package. Differential expression genes between ovarian cancer and normal ovarian tissues in the training set were identified using the limma package. Selection criteria of |logFC|>1 and adj.P.Value < 0.05 were applied. The pheatmap package was used to construct a hierarchical clustering heatmap of the top 100 differentially expressed genes, while the ggplot2 package was utilized to create a volcano plot of differentially expressed genes, with annotations for the top 20 differentially expressed gene names. Enrichment analysis of differentially expressed genes was conducted using the clusterProfiler package, including Gene Ontology analysis of biological processes, cellular components, and molecular functions. Visualization of the enrichment results was performed using the ggplot2 package.
Cells Culture
KGN cells (Innovatbio, Beijing, China) were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin solution, maintained at 37°C in a 5% CO2 humidified cell culture incubator. When the cell confluence reached approximately 80%, cells were passaged using 0.05% Trypsin-EDTA and further utilized for experimental procedures.
The detection of FOXL2 mRNA
The cells were randomly divided into control and experimental groups. KGN cells were seeded in a 6-well cell culture plate and allowed to grow to a density of 70%-80%. Different concentrations of JNK inhibitor (0.1, 1, 5, 10, 50µM) were applied to the KGN cells for 12 hours, with DMSO used as the solvent control. RNA extraction was performed using Trizol (Solarbio, Beijing, China) from both total cells and ovarian tissue. RNA concentrations were measured by absorbance at 260nm using a micro-spectrophotometer (ThermoScientific, USA). RNA quality was assessed by electrophoresis in a 1% agarose gel. The cDNA was synthesized using a Fast Super RT Kit cDNA (with gDNase) (B002004018, Bioteke, Beijing, China). Primer pairs were designed for GAPDH (forward primer: 5′-AGCCAAAAGGGTCATCATCTCT-3′, reverse primer: 5′-AGGGGCCATCCACAGTCTT-3′) and FOXL2 (forward primer: 5′-TCACGCTGTCCGGCATCTACCA-3′, reverse primer: 5′-GCGGCACCTTGATGAAGCACTC-3′) using Primer version 5.0 software. Taq DNA Polymerase (9618080604, ABclonal, Wuhan, China) was used for PCR amplification. qRT-PCR was performed using the SYBR Green Real-time PCR Master Mix in a real-time PCR system (TOYOBO, Japan) following the manufacturer’s protocol. The qRT-PCR parameters were as follows: 94°C for 3 minutes, followed by 40 cycles of 94°C for 30 seconds, 60°C for 30 seconds, and a final extension at 72°C for 5 minutes. Data analysis was conducted using the formula: R = 2^-[ΔCt sample - ΔCt control], where R represents the relative expression level, ΔCt represents the difference between the Ct of the gene and the average GAPDH in the experimental sample, and ΔCt control represents the difference between the Ct of the gene and the average GAPDH in the control sample. Experiments were carried out in triplicate with independent experimental samples.
Protein extraction and Western blotting
The KGN cells and tissue utilized for protein extraction underwent grinding in radio-immunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China) supplemented with 1 mM phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitors, symbolizing a meticulous approach to sample preparation. Following centrifugation at 10,000 g at 4℃ for 10 minutes, the protein-enriched supernatant was meticulously collected. The protein concentrations were determined using the Bradford method (Bradford 1976), showcasing a commitment to precise quantification. Subsequently, protein samples (30 µg) were meticulously subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and precisely electro-transferred onto a 0.22 µM nitrocellulose membrane, illustrating a meticulous technique of protein separation and transfer. The resulting membranes were diligently blocked with a blocking buffer (2% BSA in TBS buffer: 10 mM Tris-HCl, 150 mM NaCl, pH = 7.5) for 1 hour at room temperature, demonstrating a thorough experimental procedure. The membrane was then meticulously incubated with the antiserum anti-FOXL2 against human (Abcam, ab5096, USA) (1:10,000 dilution in blocking buffer) overnight at 4°C, underscoring the meticulous attention to detail in antibody incubation. After being washed twice with TBST buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH = 7.5) for 10 minutes each time, the membrane underwent further incubation with an alkaline phosphatase-conjugated goat anti-rabbit IgG secondary antibody (ABclonal, AS009, Wuhan, China) (1:1000 dilution in blocking buffer) at room temperature for 3 hours, emphasizing a methodical secondary antibody incubation process. Following a thorough washing with TBS buffer three times for 10 minutes, the target protein signal was meticulously visualized using 45 µL of nitroblue tetrazolium (NBT) (Sigma, USA) and 35 µL of 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma, USA) incubated in 10 mL of TBS in the dark for 10 minutes, highlighting a precise approach.
MTT assay for measuring cell proliferation
Exponential growth KGN cells were seeded at 3×103 cells per well in a 96-well plate and incubated in a constant temperature incubator for 24 hours. The OD values of each well were recorded at 490nm on the microplate reader. The proliferation curve of cells was plotted with incubation time as the x-axis and OD values as the y-axis, and the cell proliferation rate was calculated. Cell proliferation rate = OD values of experimental groups A and B / OD values of group C. The experiment was repeated three times.