Tissue samples
A total of 312 HGSOC and 108 normal fallopian tube tissues were obtained in the Department of Obstetrics and Gynecology of Qilu Hospital, Shandong University, between 2003 and 2015. All the pathological results were confirmed blindly by two professional pathologists. Tumour stage was identified according to the International Federation of Gynecology and Obstetrics 2013 staging system [18]. A total of 108 normal fallopian tube (FT) tissues were collected from patients who underwent surgery with benign neoplasms at Qilu Hospital. The last date of follow-up was June 29, 2018. All patients within the study were informed and provided written consent. Platinum resistance was defined as tumour relapse or progression within 6 months. The study was approved by the Ethics Committee of Shandong University Qilu Hospital.
Cell culture and reagents
OVCAR3 cells were purchased from American Type Culture Collection (ATCC). A2780 and A2780/DDP cells were gifts from Jianjun Wei’s laboratory. UWB1.289 and HEK293T cells were obtained from China Type Culture Collection. A2780, A2780/DDP and UWB1.289 cells were cultured in RPMI 1640 medium (Gibco, USA) with 10% foetal bovine serum (FBS) (Gibco, USA). OVCAR3 cells were cultured in RPMI 1640 medium with 20% FBS. HEK293T cells were cultured in DMEM (Gibco, USA) with 10% FBS. All cells were cultured at 37 °C under 5% CO2 in an incubator. Cisplatin was obtained from Sigma-Aldrich.
Tissue microarray (TMA) construction and immunohistochemistry (IHC)
Sections of 4 μm were cut from each TMA receiver block, made by our laboratory. After deparaffinization in xylene and rehydration in a decreasing series of ethanol, slides were immersed in boiled 10 mmol/L EDTA buffer for antigen retrieval. Endogenous peroxidase was inactivated by 3% hydrogen peroxide for 15 min and nonspecific binding was blocked by goat serum for 30 min. Then the slides were covered with a kallistatin antibody (dilution 1:300, Abcam, USA, ab1544597) at 4 °C overnight, followed by incubation with an anti-rabbit antibody for 20 min. Finally, staining in the cytoplasm was evaluated by two pathologists who were blinded to the research. The four different scores used were defined as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong), and the staining proportion ranged from 0 to 100 based on the percentage of stained cells. Kallistatin expression was graded by calculating the product-sum of the staining intensity and the proportion. The samples were divided into the low expression group if the product-sum was less than or equal to 110 and the high expression group if it was more than 110.
Plasmid, lentivirus production, siRNA and transfection
The CDS sequence of kallistatin was purchased from Genechem (Shanghai, China) and inserted into the EcoRI/Nhel sites of the Plenti-C-Myc-DDK-IRES-Puro (PCMV) vector (Origene, USA). Lentivirus was produced by HEK293T cells with the psPAX2 (Addgene, USA) and pMD2.G (Addgene, USA) plasmids and Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s protocol. After transfection with lentivirus for 24 h, the ovarian cancer cells were selected for a week in medium containing 4 μg/ml puromycin (Merck Millipore, USA) to obtain stable expressing cells. Small interfering RNA (siRNA) for silencing kallistatin was designed by Biosune (Shanghai, China) (sequence: 5′-CCAGCUUCGCGAUCAAAUUTT-3′). Ovarian cancer cells were transfected transiently with Lipofectamine 2000 (Invitrogen, USA).
Protein extraction and western blot
The tissue samples and cells were placed on ice and treated with RIPA lysis buffer (Beyotime, China) containing NaF and PMSF. The concentration of proteins was quantified with a BCA Protein Assay kit (Merck Millipore, USA). A total of 60 μg of protein per well was separated with SDS-PAGE (5% stacking gel and 10–12% separation gel) and transferred to 0.22-μm PVDF membranes (Merck Millipore, USA) with the Bio-Rad Trans-blot system (16 V, 90 min). The membranes were blocked with 5% skim milk for 1 h and incubated with primary antibodies overnight at 4 °C. On the following day, the membranes were incubated with secondary antibodies for 1–2 h. The bands were detected with Western Lightening Plus-ECL reagent (GE, USA). GAPDH and ACTB were used as internal controls. ImageJ was used to analyze the bands.
Cell proliferation assay
Cell proliferation was monitored by 4-[4,5-dimethythiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assays. A total of 1000 ovarian cancer cells per well were seeded in 96-well plates in quintuplicate. Cell proliferation was measured at different times (1–6 days). For the assay, 20 μl of MTT (Sigma-Aldrich, USA) was added to each well at a designated time every day and incubated for 4 h at 37 °C. After careful removal of the supernatant, 100 μl of DMSO (Sigma-Aldrich, USA) was added per well. Then the absorbance values at 490 nm were measured by a microplate reader (ThermoScientific, USA). The experiment was performed in triplicate.
Cell migration and invasion assay
For the assays, 1 × 105–2 × 105 cells were suspended in FBS-free medium and seeded into the upper chambers (8-μm pores, BD Biosciences, USA) of 24-well plates, and 700 μl of medium containing 20% FBS was added into the lower compartment. After an appropriate incubation time, we wiped away the cells adhered to the upper surface of the chambers. The cells adhered to the lower surface were fixed in methanol for 15 min and stained with 0.5% crystal violet for 15 min. The invasion assay was conducted in the same way except the filter membrane was covered with Matrigel (BD Biosciences, USA).
Cell viability detection
A total of 3000 cells were seeded in 96-well plates in quintuplicate, and exposed to cisplatin at a series of concentrations (0, 2, 4, 8, 16, and 32 μg/ml) for 24 h after adhesion to plates. Then, 20 μl of MTT was added to each well and incubated for 4 h. The supernatant was exchanged with 100 μl of DMSO. The absorbance values at 490 nm were measured by a microplate reader. The experiment was performed in triplicate.
Apoptosis
Ovarian cells were cultured in medium with cisplatin at a concentration of 2 μg/ml for 24 h. Then, the cells were trypsinized without EDTA, washed with 1 × phosphate buffer saline (PBS), centrifuged and resuspended in 1 × Annexin buffer and then stained with Annexin V-FITC and propidium iodide (PI) (BD Biosciences, USA). After 15 min of incubation, the cells were analysed with flow cytometry (BD Biosciences, USA). The experiment was performed in triplicate.
In vivo nude mouse tumorigenesis
Four-week-old female BALB/c nude mice were purchased from NBRI of Nanjing University (Nanjing, China). UWB1.289 cells were transfected with PCMV-NC or PCMV-Kallistatin vector. To induce tumorigenesis, 5 × 106 cells in 200 μl of 1 × PBS were injected subcutaneously into either side of the mouse axilla. After 3 weeks, the mice were sacrificed under anaesthesia and tumour weights were measured. All procedures performed in studies involving animals were in accordance with the National Institutes of Health guidelines for the care and use of Laboratory animals (NIH publication no. 8023, revised 1978). All animal experiments were approved by Shandong University Clinical Medical College Animal Experiment Ethics Committee.
Statistical analysis
SPSS version 18.0 (Chicago, IL, USA) was used for the statistical analysis. Student’s t test was applied to assess the significance between two groups. The correlation between kallistatin expression and clinicopathologic parameters was analysed by the chi-squared test. Survival rates were calculated using the Kaplan-Meier method and the difference was calculated using log-rank test. Multivariate analysis of OS and PFS was performed by the Cox proportional hazard regression model. Additionally, p < 0.05, p < 0.01, and p < 0.001 were considered significant *, very significant ** and extremely significant ***, respectively.
