Cell lines and cell culture
OVCAR3 and SKOV3 cells were obtained from Cell Bank of Chinese Academy of Sciences (Shanghai, China) and maintained in RPMI-1640 medium (HyClone, USA) supplemented with 10% FBS. Cells were transfected with pCMV-MIR-miR-454 (Ribobio, Guangzhou, China) using Lipofectamine 2000 (Invitrogen, CA, USA), pCMV-MIR vector was used as negative control (NC). The E2F6 cDNA sequences were cloned into pcDNA3.1 vector and the pcDNA3.1-E2F6 was transfected into cells using Lipofectamine 2000.
Clinical samples
Seventy-five cases of ovarian cancer tissues and fifteen cases of tumor-adjacent tissues were obtained from Beijing Anzhen Hospital, Capital Medical University. All patients gave informed consent in written, and this study was approved by the ethics committee of Beijing Anzhen Hospital, Capital Medical University. Venous blood was collected from 13 patients with ovarian cancer and 6 normal subjects from Beijing Anzhen Hospital, Capital Medical University. All blood samples were taken before patient treatment.
Quantitative RT-PCR analysis
Total RNA extracted from OVCAR3 and SKOV3 cells or serum samples using TRIzol Reagent. The SYBR PrimeScript miRNA RT PCR kit (Takara, Shiga, Japan) was performed for RT-PCR reaction to examine the expression of miR-454. For detection of E2F6 mRNA expression, a HiFiScript cDNA Synthesis Kit (CWBIO, Beijing, China) was used to reverse transcribe RNA to cDNA, and a SYBR Premix Ex Taq II kit (Takara) was performed for RT-PCR reaction. Primers were obtained from Ribobio. The relative expression of miR-454 or E2F6 mRNA was calculated using the 2-ΔΔCT method and normalized to NC group.
CCK8 assay
Cells were seeded in a 96-well plate at a density of 1000 cells/well and cultured for 0, 24, 48, and 72 h, respectively. Then, 10μl of CCK8 reagent (Beijing Solarbio Science & Technology, Beijing, China) was added into each well. Following incubation for 1.5 h, the absorbance was detected at 450 nm.
Colony formation assay
Cells were cultured in 60-mm dishes (500 cells/dish) and incubated for 1-2 weeks. After that, cell colonies were fixed with 4% paraformaldehyde for 30 min and dyed with 0.1% crystal violet for another 30 min. Finally, the number of cell colonies was counted.
Transwell migration and invasion assay
Transwell chambers (Millipore, MA, USA) were used to measure cell migration and invasion. About 1×105 cells were seeded in the upper chamber and the lower chamber was filled with RPMI-1640 medium containing 20% FBS. Following incubation of 12 h, the migrated or invaded cells were fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet for 20 min. In invasion experiment, the upper chamber was coated with Matrigel (BD Bioscience, CA, USA) before seeding cells.
Flow cytometry assay
Cells transfected with plasmids of 24 h were cultured in serum-free medium for 24 h, and re-suspended in loading buffer after PBS washing. An Annexin V/FITC-propidium iodide kit (BioVision, USA) was used to stain cells. The apoptotic rate was examined by a flow cytometer (BD FACSC anto II, BD Biosciences, USA).
Western blot analysis
Total proteins were extracted from transfected cells by RIPA lysis buffer, and quantitated by a BCA kit. Proteins were subjected to 10% SDS-PAGE and transferred to the membrane of PVDF. After blocked with 5% dried skimmed milk for 1 h, the member was probed with primary antibodies (1:1000; Proteintech Group, USA). Then, the blots were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000), and developed using the Enhanced Chemiluminescence kit (CWBIO, Beijing, China).
Dual-luciferase reporter assay
The wild-type of E2F6 3′-UTR (E2F6-wt) or mutant E2F6 3′-UTR (E2F6-mut) was cloned in pmir-GLO fluorescein enzyme vector. 293T cells were co-transfected with luciferase reporter plasmids (wt or mut) and pCMV-MIR-miR-454 or pCMV-MIR. Luciferase activity of each group was measured by the dual-luciferase assay system kit (Promega, WI, USA).
Immunohistochemical staining analysis
Immunohistochemical staining was performed to evaluate E2F6 expression as described previously [21]. E2F6 expression levels were scored by staining intensity and percentage of positive stained cells [22]. The staining intensity was graded as follows: 0, no staining; 1, pale yellow staining; 2, buffy staining; 3, intense brown staining. The percentage of positive stained cells was scored as follows: 0=0–10%, 1=11–25%, 2=26–50%, 3=51–75%, 4=> 75%. Samples were scored by multiplying synchronically the two sections, and score > 6 as E2F6 high expression level.
Statistical analysis
Data was presented as Mean± SD and statistical analyses were conducted using GraphPad Prism 7.0 software (GraphPad, CA, USA). The chi-square test was performed for continuous or discrete data analysis, and the Student’s t test or one-way ANOVA analysis was used for comparisons between groups. Differences were considered significant when P value was less than 0.05.