Pathological Samples
Ovarian cancer specimens embedded in paraffin were obtained from 60 patients who underwent surgery in the Department of Obstetrics and Gynecology at Affiliated Hospital of Nantong University from June 2009 to April 2013. All tumors were from patients newly diagnosed with ovarian cancer and patients did not receive any treatment before surgery. Among them, four patients’ survival information was missing. All investigations described in this study were performed after informed consent was obtained. Approval for the study was obtained from the Research Ethics Committee of Affiliated Hospital of Nantong University.
Immunohistochemistry (IHC) and quantification
Formalin-fixed, paraffin-embedded sections were prepared for all tissues and reviewed by 3 pathologists. The protein expression of cyclin H, Ki67 and p-CDK2 in ovarian cancer was determined by IHC. In short, ovarian cancer sections were dewaxed in xylene and rehydrated through gradient ethanol, and then heated in Tris-EDTA buffer (pH 8.0) for 15 min in a microwave oven to retrieve the antigen. After cooling naturally and rinsing with phosphate-buffered saline, slides were then incubated with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity and 2% bovine serum albumin (Beyotime Biotechnology, Shanghai, China) was applied for 2 h at room temperature to block nonspecific reactions. Tissue sections were then incubated with cyclin H monoclonal antibody (1:50, MA5-32331, Thermo Fisher Scientific, Waltham, MA, USA), Ki67 monoclonal antibody (1:300, ab92742, Abcam, Cambridge, MA, USA) or p-CDK2 polyclonal antibody (1:100, PA5-38128, Thermo Fisher Scientific) at 4℃ overnight. All slides were finally visualized using the Envision kit (Dako, Glostrup, Denmark) according to the instructions and counterstained with hematoxylin, dehydrated, and cover slipped. Sections were imaged under an optical microscope and evaluated in a blinded manner. More than 500 cells were counted for each slide to determine the mean positive percent and the expression range was scored as follows: 0 (<20%), 1 (20% to 50%), 2 (51% to 75%) and 3 (>75% points).
Cell culture and transfection
Human ovarian carcinoma HO8910 cell line used in this study was obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). HO8910 cell line was cultured in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 0.11g/L Sodium Pyruvate, 100 U/mL penicillin-streptomycin mixture (Gibco) at 37℃ and 5% CO2 incubator. shRNA of cyclin H (sequence: 5’-CCG GCG ACC TGG TAG AAT CTC TCT ACT CGA GTA GAG AGA TTC TAC CAG GTC GTT TTT G-3’) was synthesized by Sangon Biotech (Shanghai, China) and cloned to pLenti-CMV-puro vector and transfected to HO8910 cells using FuGENE transfection reagent (Promega, Madison, WI,USA ). Stable transfected cell lines were obtained after screened with 1 µg/mL puromycin. Empty plasmid was transfected into HO8910 cells as control group.
Cell viability assay
Cells were seeded in 96 well plated (3000 cells per well) and culture in complete RPMI 1640 medium containing 10% fetal bovine serum. Cell viability was measured by cell counting kit-8 (C0038, Beyotime, Shanghai, China) according to the manufacture’s instruction at 0, 12, 24, 48 and 72 h after seeding. Five wells were performed for each time point. Cell viability at each time point was compared to the initial cell viability.
Western blot
HO8910 cells digested with trypsin, washed with PBS and collected by centrifugation. Cell pellets were resuspended with cell lysate and incubate on ice for 20 min. Samples were blotted using SDS-PAGE gel and transferred to polyvinylidene fluoride membrane (0.2 μm, Millipore, USA). After blocking with 5% non-fat milk, membrane was incubated with cyclin H antibody (1:1500, MA5-32331, Thermo Fisher Scientific), MAT1 antibody (1:1000, ab129176, Abcam), CDK7 antibody (1:1000, 2916, Cell Signaling Technology, Danvers, MA, USA), p-CDK2 antibody (1:100, PA5-38128, Thermo Fisher Scientific) or CDK2 antibody (1:1000, 2546, Cell Signaling Technology) and then HRP goat anti-rabbit or mouse IgG (ABclonal, Wuhan, China). Membranes were finally visualized using an enhanced chemiluminescence reagent (34095, Thermo Pierce). GAPDH (1:2000, Abcam) was used as control.
mRNA level of cyclin H detection
The total RNA of HO8910 cells were obtained using Trizol (Invitrogen) and reverse transcription was performed using a commercial kit (RR047B, TaKaRa, Tokyo, Japan) containing gDNA Eraser to eliminate genomic DNA contamination. Relative mRNA level of cyclin H was determined by real time quantitative PCR (RR420L, TaKaRa). β-actin was used as control. Following primers was used: cyclin H forward, 5’-TGT TCG GTG TTT AAG CCA GCA-3’; cyclin H reverse, 5’-TCC TGG GGT GAT ATT CCA TTA CT-3’; β-actin forward, 5’-TCG AGC ACG GCA TCG TCA CCA-3’ ; β-actin reverse, 5’-ATA GCA ACG TAC ATG GCT-3’.
Cell cycle detection
HO8910 cells were synchronized by deprivation of serum over 72 h. The serum-free medium was replaced by complete medium and cells were cultured for another 48 h before collection. Cells were digested with trypsin and single cell suspension was prepared before fixation and staining. Propidium staining was performed using a detection kit (C1052, Beyotime) and detected using BD FACSCanto II (San Diego, CA, USA). The data was analyzed using Modfit software and the percentage of cells in each phase was counted.
Tumor model
Female BALBC/c nude mice (5–6 weeks old, 14–16 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and maintained in specific pathogen-free conditions. HO8910 cells (5 × 105/mouse) in 100 μL PBS were inoculated subcutaneously of the nude mice in the flank. Tumor diameters are measured with digital calipers every other day, and the tumor volume is calculated by the following formula: Volume = (width)2 × length/2.
Statistical Analysis
IBM SPSS was used for all statistical analyses. Chi-square test was used for clinicopathological categorical variables. Overall survival was analyzed by the Kaplan-Meier method. T test was used to study the differences between two groups. Spearman rank correlation analysis was used to measure the association between the expression of cyclin H, Ki67 and p-CDK2. Differences were considered statistically significant at p<0.05.