Patients and tissue preparation.
A total of 45 epithelial malignant EOC specimens, 15 benign epithelial ovarian tumour specimens, and 15 normal ovarian tissues were acquired between 2012 and 2017 in the First and Fourth Affiliated Hospitals of Chongqing Medical University. All specimens were authorised with pathological diagnoses. No patients had been subjected to chemotherapy or radiotherapy. The clinical characteristics of patients were gathered, for instance, age and grade. The consent and approval of the patients were acquired for research purposes from the Institutional Research Ethics Committee of Chongqing Medical University.
Cell culture and reagents.
HO8910 and HO8910PM cell lines and the packaged retrovirus containing the KIAA1456 expression plasmid used in this study were produced in our previous work. The EOC cell lines were placed in RPMI 1640 complemented with 100 U/ml penicillin, 100 µg/ml streptomycin (Hyclone) and 10% FBS and incubated in 37°C with 5% CO2 atmosphere. Antibodies against KIAA1456 and SSX1 were purchased from Abcam, and phospho-AKT, AKT, PCNA, Bcl-2, MMP9 and CyclinD1 were procured from Cell Signaling Technology (Danvers, MA, USA). β-actin and GAPDH were obtained from Beyotime (China).
Paraffin-embedded tissue sections were deparaffinised with xylene and rehydrated with graded ethanol. Antigen retrieval was accomplished by incubating the specimens with boiled citrate buffer for 5 min at 100°C. Hydrogen peroxide (3%) and serum were used to block endogenous peroxidase activity and non-specific antigens. The specimens were then incubated with KIAA1456(1:100) and SSX1(1:100) antibodies overnight at 4°C. Afterwards, the specimens were incubated with goat anti-rabbit secondary antibody (1:500). After each treatment, the specimens were developed using 3,3-diaminobenzidine solution and counterstained with hematoxylin. After staining, the slides were critiqued by two independent observers using a microscope (DM6000B). The immunohistochemical staining of KIAA1456 and SSX1 was computed based on the population of positive cells and staining intensity. Positive expression (The staining scores were calculated by both percentage of positive cells and color intensity) was graded as follows: <10% (negative staining), 10–20% (weak staining), 20–30% (moderate staining), > 30% (strong staining).
KIAA1456 lentivirus (LV- KIAA1456) used for KIAA1456 overexpression, SSX1 lentivirus (LV-SSX1) used for SSX1 overexpression, both lentiviral constructs expressing KIAA1456 shRNA (RNAi- KIAA1456), matched control lentivirus (shcon) and negative control lentivirus (con) were obtained from Genechem Co. Ltd (Shanghai, China). For transfections, HO8910PM and LV-KIAA1456 cells were cultured in media to probably 50% clustering in six-well plates. These lentiviruses were introduced into HO8910PM or LV-KIAA1456 cells with 8 µg/ml polybrene (Genechem) and complete medium. A fluorescence microscope was applied to detect the transfection effects after 48 h. The transfected cells were purified with puromycin. The effective forced expression of KIAA1456 and SSX1 was tested by real-time PCR and Western blot analysis after 72 h.
Quantitative real-time PCR.
RNAiso Plus (TaKaRa, Beijing, China) was used to extract total RNA in cells and tissues. The Reverse Transcription Kit (Takara, Dalian, China) was used to reverse-transcribe RNA into cDNA. Real-time PCR analyses were performed using Power SYBR Green (Takara, Dalian, China). The results were standardised to the expression level of GAPDH. The relative fold-changes of mRNA levels were counted according to the 2 - ΔΔCT method. All qRT-PCR experiments were done in triplicate. Student’s t-test was used to compare differences in gene expression levels between groups. A p value < 0.05 was considered to indicate a significant difference. Primers were synthesised by Shanghai Sangon Biological Engineering Technology.
Western blot analysis.
The tissues and cells were lysed by RIPA Lysis Buffer (Beyotime, China) containing PMSF and a phosphatase inhibitor. The BCA protein assay kit (Beyotime, China) was used to measure protein concentration. Cellular protein lysates were separated via SDS-PAGE and transferred to PVDF membranes using a standard protocol. The membranes were incubated with primary antibodies (including KIAA1456(1:500), SSX1(1:500), AKT (1:1000), p-AKT (1:2000), PCNA (1:1000), Bcl2(1:500), CyclinD1(1:1000) and MMP9(1:500)) overnight at 4°C after blocking with 5% non-fat milk. Subsequently, the membranes were incubated with the the appropriate secondary antibodies. HRP-conjugated anti-mouse/rabbit IgG (1:5000) was applied to the secondary antibody. Protein bands were detected using an enhanced chemiluminescence system. GAPDH (1:2000) and β-actin (1:1000) were used as loading controls. Digital images were quantified using the Quantity-One software (Bio-Rad, USA).
Cell viability assay.
To detect cell growth ability, we cultivated cells with a density of 2000 cells/well into 96-well plates and incubated them at 37°C. For 24, 48, 72 and 96 h; cells in each well were added with 10 µl of Cell Counting Kit-8 (Beyotime) and then incubated for 1 h at 37°C. Absorbance values were read at 450 nm employing an enzyme-labelled instrument. Data were analyzed using GraphPad Prism 5.0. The assay was
performed three times in triplicates, and the data presented as mean ± SD.
Plate colony formation assay
For a plate colony formation assay, the same amounts of different cells were cultivated in a six-well plate (500 cells/well) for 10–14 days. The plate was replenished with fresh medium every 3 days. Finally, the cells were fixed with 4% paraformaldehyde and stained with freshly prepared crystal violet for 20 min. Colony formation was assessed under a microscope (Nikon, Japan).
Cell cycle and apoptosis analysis with flow cytometric assay
HO8910PM, LV-KIAA1456, LV-SSX1, RNAi-KIAA1456 and their control cells were seeded in a six-well plate (5×105 cells/dish). Cells were harvested after 24 h of incubation, washed with PBS and then fixed in 70% ice-cold ethanol overnight at -20°C. Cells were cultivated at 37°C for 30 min with 10 mg/ml RNase (Sigma, St. Louis, MO, USA) and 50 mg/ml propidium iodide (Sigma). Flow cytometry (BD Bioscience, San Jose, CA, USA) was used to analyse the cell cycle. Conforming to the manufacturer’s protocol, reactants from the Annexin V-FITC apoptosis kit (BioVision, Wuhan, China) were used to cultivate and incubate the same treated cells to test for cell apoptosis. We conducted three independent experiments to determine the Cell cycle and apoptosis, and the data indicated as mean ± SD.
Cells were attached to slides, fixed with 4% paraformaldehyde, incubated in 0.3% Triton X-100 for 15 min and then blocked with 5% goat serum. The cells were then incubated with primary antibodies against KIAA1456 and SSX1 at 4°C overnight and then with suitable secondary antibodies. DAPI (Beyotime) was used to counterstain the nuclei. Immunofluorescent signals were measured using a microscope (DM6000B).
Wound-healing cell migration analysis
Different cells with a concentration of 6×105 cells/well were seeded into six-well plates filled with complete RPMI 1640 medium, which then formed a confluent monolayer. Thereafter, a 200 µl pipette tip was used to scratch the plate to form perpendicular wounds in the monolayers. Floating cells were washed with PBS and continuously incubated in complete medium. At 0, 24, and 48 h after wounding, scratched areas were photographed with a microscope, and the width of the wound was measured using the Image J software. All assays were performed in triplicate.
Transwell invasion assays
The invasion ability of LV-KIAA1456, RNAi-KIAA1456 and LV-SSX1 cells was investigated with 6.5 mm chambers with 8 µm pores (Corning, USA). The same amounts of cells were plated on the top side of polycarbonate Transwell filters lidded with 1 µg of Matrigel in the upper chambers with serum-free RPMI 1640 medium (5×104 cells). The bottom chambers were appended with 600 µl of RPMI 1640 medium complemented with 10% FBS. Invaded cells were fixed with methanol and dyed with hematoxylin and eosin after 12 h of incubation. Images of each membrane were captured in five random fields (×100 magnification) in a microscope. Statistics were done using GraphPad Software.
Xenograft tumor model.
The female nude mice (4 weeks old) used in this research were supplied by the experimental animal centre of Chongqing Medical University. All animal experiments were authorised by the Ethics Committee of Chongqing Medical University. The LV-KIAA1456 and control cells at a density of 2×106 cells per 50 µl were re-suspended in RPMI 1640 and injected subcutaneously into the nude mice. The tumours were measured when they were grown out every 7 days, and the volume was measured by the formula length × width2/2. The nude mice were sacrificed after 35 days of inoculation. Tumour tissues were resected, weighed and then used for IHC.
Statistical analyses were implemented using SPSS 22 and GraphPad Prism 5.0 software. Differences between the two groups were evaluated by Student t-test. One-way ANOVA was used when comparing multiple groups. Clinical data were analyzed using the chi-square test. Association between KIAA1456 and SSX1 expression in epithelial ovarian cancer tissue was assessed using Spearman’s rank correlation test. The prognostic significance analysis was executed using Kaplan-Meier analysis and log-rank tests. Data were represented as mean ± S.D. of at least three independent experiments. The criteria of statistical significance was P < 0.05.