Tissue samples and patient data
The ethics approval for present study was obtained from the Ethics Committee of the Fujian Medical University Union Hospital. We collected 109 formalin-fixed paraffin-embedded ovarian tissue, including 13 normal ovarian tissue chips, 26 ovarian cystadenoma chips, 8 border ovarian tumour chips, and 62 ovarian malignant tumour chips from patients treated initially at the Fujian Medical University Union Hospital between 2016 and 2019. Relevant clinical parameter data were collected from the hospital medical record system and the definite histological diagnosis and grading came from the pathological reports. The clinical-stage were determined based on the International Federation of Gynecology and Obstetrics, 2009 (FIGO, 2009).
IHC and quantitative analysis
Serial 3-μm sections from all samples were deparaffinized and rehydrated through xylenes and serial graded ethanol to water followed by antigen retrieval. These samples then were incubated overnight at 4 °C with TCP1 alpha primary antibody (Abcam Corporation; 1:200). The washed tissue samples were incubated with secondary antibody IgG (Merck Millipore; 1:300) for 30 min at room temperature (RT). Tissue slices were stained with 3,3’-diaminobenzidine and hematoxylin, and observed under an optical microscope. Finally, all images were analyzed integrated optical density (IOD) to calculate the average IOD /TCP1 positive staining area (µm 2) using Image-pro plus software.
Survival analysis using KM Plotter
The correlation between TCP1 expression and prognosis of OC was analyzed using the KM Plotter database (http://kmplot.com). In the database, 1657 OC cases with clinical data and mRNA expression were downloaded from Gene Expression Omnibus. Patients were divided into two subgroups according to TCP1 low or high expression using the optimal cut-off value. The overall survival (OS), progression-free survival (PFS), harzad ratio (HR), and log-rank P of TCP1 in OC patients were analyzed online using the Affymetrixmetrix ID (222010_at) approach.
Cell culture
The human EOC cell line A2780 and normal ovarian cell IOSE-80 were purchased from the Bena Culture Collection (Kunshan, Jiangsu Province, China) and cultured in 5% CO2 at 37℃ in dulbecco's modified eagle medium (DMEM; Gibco) added with 10% fetal bovine serum (FBS; Gibco). Another EOC cell SKOV3 was obtained from Guangzhou Cellcook Biotech Company and cultured in 5% CO2 at 37℃ in McCoy’s 5a supplemented with 10% FBS. Cell line authentication by short tandem repeat (STR) profiling
Western blot assay
Total protein (20 μg/lane) was separated by polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. Then, the membrane was blocked with 5% non-fat dry milk solution and incubated with the various primary antibodies at 4 ℃ overnight. Next day, the washed membrane using tris buffered saline tween (TBST) was incubated with horse radish peroxidase (HRP) conjugated secondary antibodies at RT for 2 h followed by visualized using chemiluminescent HRP substrate (Merck Millipore) on a Western blot imaging system. The band intensity was detected using Image Lab software. The protein expression was normalized to glyceraldehydes 3-phosphate dehydrogenase (GAPDH) expression. The primary antibodies used were anti-TCP1 alpha Rabbit Monoclonal (Abcam Corporation; 1:1,000), anti-mTOR Rabbit antibody (Abcam Corporation; 1:1,000), Akt antibody (CST Corporation, 1:1,000), phospho-Akt(Ser473) Rabbit mAb (CST Corporation, 1:1,000), and anti-GAPDH Mouse (TransGen Biotech, 1:2,000). Anti-mouse (Merck Millipore, 1:20,000) and anti-rabbit (Merck Millipore, 1:20,000) secondary antibodies were used.
Construction of stable TCP1-knockdown cell line
The pLKO.1 Puro vector was used to construct lentiviruses for TCP1 RNA interference (shTCP1) and negative control (shCtr) experiments. The sequences targeting TCP1 were designed based on the human TCP1 gene (Table 3) and synthesized according to the pLKO.1 Puro vector specification. To prepare lentiviral particles, 8 μg of the shTCP1 vector (pLKO.1 Puro) and the packaging plasmids (5 μg pMDL, 3 μg pVSVG, and 2 μg pREV) were cotransfected into 293T cells. The TCP1-NC group was transfected with negative lentivirus. Lentivirus-containing medium was collected after 48 h of transfection and used to culture A2780 cells. After 48 h of transfection, the medium was replaced with complete medium. Then, puromycin with a final concentration of 2.0 μg/mL was added for stable cell line screening for 72 h. Then, the survived cells were collected for TCP1 expression analysis. The stable cell lines were constructed in A2780/TCP1- negative control group (NC) and A2780/TCP1- knockdown group (KD), which were used for subsequent experimentation.
MTT assay
The proliferation of A2780/TCP1-NC and A2780/TCP1-KD cells was detected by MTT assay. The cells were firstly inoculated to 96-well plates (1,000 cells/well) and cultured in a humidified 5% CO2 incubator at 37°C. Then, the plates were added with methyl thiazolyl tetrazolium (MTT, 0.5 mg/mL, 10 µL/well) at 24, 48, 72, and 96 h. After 4 h of normal culture, the supernatant was removed and purple formazan crystals were dissolved using a 150 µL dimethyl sulfoxide (DMSO) solution. The plate oscillated for 10 min at RT. The optical density at 490 nm (OD490) of each well was measured by microplate reader using wells without cells as blanks. The cell viability curve was drawn by the abscissa of the time point and the ordinate of OD value. Each experiment was performed in triplicate.
Colony formation assay
Infected cells were routinely harvested, resuspended, and then placed in 6-well plates (1000 cells/well) to analyze cell colony formation. After 10 days of incubation with each 3-day medium changes, the surviving cells were washed using cold phosphate buffered solution (PBS),fixed by 4% polyformaldehyde,and dyed with 1% crystal violet. The colonies with more than 50 cells were counted. We divided colony number by plated cell number to calculate the colony forming efficiency (CFE, %). The experiments were repeated three times.
Wound-healing assay
Approximately 2×106 cells were seeded in 6-well plates. The cell monolayers were scratched using sterile 200-µl pipette tips after reaching 80% confluence. Serum-free medium was added into the plates after washing the floating cells. Cells were cultured at 37°C for 48 h. The wound width was photographed and recorded every 24 h. The results were observed using the Image J software. Wound closure was computed according to the ratio of districts uncovered by cells before and after wound scratching.
Cell invasion and migration assay
Transwell (8-µm pore size) chambers (Falcon) were coated with matrigel, placed in 24-well cell culture plates, and then air-dried in the incubator for 4h. The 50 μL complete medium was added into each pore at 37°C for 30 min. A2780/TCP1-NC and A2780/TCP1-KD cells were cultured with 5% FBS medium. Suspended cells (15×104 cells/200 µL) were added into the upper chamber and 600 µl 15% FBS medium as a chemoattractant was put into the lower chamber. The invasive cells on the outside of the chamber were stained with 0.5% crystal violet after 48h incubation. The cell slides were photographed under an inverted microscope in 5 randomly-selected fields at ×200 magnification.
For migration assays, the Transwell chambers were not coated with matrigel and the follow-up procedure was consistent with the invasion assays. Each experiment was conducted three times.
Statistical analysis
All the data were represented as the mean ± standard deviation (SD). Statistical analyses were carried out by the SPSS software (version 20.0; SPSS) and Graph Pad Software (Graph Pad Prism 8.0.1). The independent t-test was used to the comparison of two groups. The one-way analysis of variance (ANOVA) followed by post-hoc test was applied for comparing multiple groups. P<0.05 was considered as statistically significant.