Tissue samples and cell lines
Human cervical cancer tissue samples (n=39) and normal cervical tissue samples (n=13) were obtained from patients at the Hebei General Hospital (Shijiazhuang, China). The 39 patients with cervical cancer were aged between 27 and 55 years (average age, 46 years) and had an average weight of 54 kg (weight range, 48 to 63 kg). All patients in this group had been diagnosed with stage IA-IVB cervical cancer, according to the FIGO staging system (55). The 13 normal control patients were aged between 30 and 55 years and weighed between 48 and 61 kg. The control patients were undergoing a simple hysterectomy at the Hebei General Hospital due to uterine leiomyomata. All cancer specimens used in the analyses consisted of >90% tumor cells, as examined by a gynecologic pathologist. Cancer specimens from patients with concomitant gynecological problems were excluded from the study. Informed consent was obtained from all patients prior to the surgical procedure, and approval was obtained from the Medical Ethics Committee of the Hebei General Hospital.
Normal cervical epithelial cells and five cervical cancer cell lines, including SiHa, HeLa, CaSki, c-41 and c-33A, were purchased from the American Type Culture Collection (Manassas, VA, USA). Cell culture was conducted according to methods previously described by Liu et al (56). Briefly, the cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; both purchased from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 2 mM L-glutamine, 100 µg/ml streptomycin and 100 U/ml penicillin (all obtained from Gibco; Thermo Fisher Scientific, Inc.). The cells were incubated at 37°C, in an atmosphere containing 5% CO2.
Cell proliferation, apoptosis and colony formation assays
SiHa cells (1×104 cells/ml) were seeded onto 96-well plates, after which cell proliferation and apoptosis were assessed using the MTT Cell Proliferation/Viability Assay kit (cat. no. 11465007001; Sigma-Aldrich Chemie Gmbh, Munich, Germany) and the Annexin-V-FITC Apoptosis Detection kit (cat. no. APOAF-20TST; Sigma-Aldrich Chemie Gmbh) with a flow cytometer (BD FACSCalibur™; BD Biosciences, Franklin Lakes, NJ, USA), respectively, according to the manufacturer's protocols. Subsequently, SiHa cells (1×104) were suspended in 1.5 ml complete medium [consisting of RPMI 1640 (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Logan, UT, USA)] containing 0.45% low melting point agarose (Invitrogen; Thermo Fisher Scientific, Inc.) and then seeded into 35 mm tissue culture plates containing 1.5 ml complete medium and 0.75% agarose on the bottom layer, which were then incubated for 10 days at 37°C. The plates were then stained with 0.005% crystal violet for 30 sec (Sigma-Aldrich Chemie Gmbh) and the SiHa cell colonies (>0.5 mm in diameter) were counted under a microscope (Leica DMI3000; Leica Microsystems GmbH, Wetzlar, Germany), according to a previous study (57).
Cell transfection
In order to analyze the effects of EDD overexpression or knockdown, SiHa cells (1×104) were transfected with adenovirus-expressing Ad-EDD or retrovirus expressing short hairpin (Sh)-EDD, respectively. Ad-EDD/Ad-negative control (NC) and Sh-EDD/Sh-NC were designed and synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). For miR-143 silencing, SiHa cells (1×104) were transfected with the pLL3.7 lentivirus vector (Addgene, Cambridge, MA, USA) encoding a miR-143 inhibitor sponge. The miR-143 inhibitor sponge was synthesized by GenePharma (Shanghai, China). The cells were cultured in six-well plates to 70–80% confluency and transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were harvested 48 h post-transfection for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in order to determine the transfection efficiency.
Tumor xenograft experiments
Xenograft mice experiments were performed as previously described (56). Briefly, SiHa cells (5×106) transfected with Sh-NC, Sh-EDD, Ad-NC or Ad-EDD were injected subcutaneously into the flanks of female athymic nude mice (age, 3–4 weeks; n=6/group), purchased from Shanghai Laboratory Animal Co., Ltd. (Shanghai, China). The mice were maintained under at 21–22°C under a 12-h light/dark cycle. To obtain the tumors, the mice were sacrificed by an overdose of pentobarbital (50 mg/kg; Sigma-Aldrich Chemie Gmbh) 4 weeks following inoculation.
RT-qPCR analysis
Total RNA (100 ng) was extracted from the harvested tissue samples and cell lines using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and was purified using RNase-Free DNase Set (Qiagen, Inc., Valencia, CA, USA), according to the manufacturer's protocols. Subsequently, 5 ng RNA was reverse transcribed into cDNA using SuperScript III Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). qPCR was performed using TaqMan MicroRNA Assays (Thermo Fisher Scientific, Inc.) on an ABI 7300 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.), according to a previous study (44). The primer sequences were as follows: EDD forward, 5′-TTAGGCTTTTGGTAAATGGCTGCG-3′ and reverse, 5′-TGAGGGCATAGGCTGGAATCCTTC-3′; miR-143 forward, 5′-AGTGCGTGTCGTGGAGTC-3′ and reverse, 5′-GCCTGAGATGAAGCACTGT-3′; and β-actin forward, 5′-CATCCTGCGTCTGGACCT-3′ and reverse, 5′-CAGGAGGAGCAATGATCTTG-3′. β-actin was used as the internal control. EDD, miR-143 and β-actin primers were designed as previously described by Liu et al (54) and Clancy et al (30). The PCR cycling conditions were as follows: Pre-heating at 95°C for 5 min, followed by 35 cycles of denaturation for 30 sec at 95°C, annealing for 1 min at 55°C and extension for 1 min at 72°C, with a final extension for 5 min at 72°C. Relative gene expression levels were calculated using the 2−ΔΔCq method (58).
Western blotting
Western blotting was performed as previously described by Zhang et al (44). Briefly, total protein (50 µg) was extracted from the tissue samples and SiHa cell line using lysis buffer (20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 25 mM 2-mercaptoethanol, 0.5 mM AEBSF, 0.5% Triton X-100, 2 µg/ml leupeptin and 3 µg/ml aprotinin) and the protein concentrations were measured using a Bio-Rad Protein Assay kit (cat. no. 5000002; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Subsequently, the membranes were blocked with 10% skimmed milk solution and then incubated overnight at 4°C with goat anti-EDD (cat. no. sc-9562), rabbit anti-B-cell lymphoma (Bcl)-2 (cat. no. sc-492), rabbit anti-Bcl-2-associated X protein (Bax; cat. no. sc-493), goat anti-caspase 3 p11 (cat. no. sc-1224) and goat anti-GAPDH (cat. no. sc-48166) polyclonal antibodies (1:2,000; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following washing with phosphate-buffered saline, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG, F(ab')2 (1:3,000; cat. no. sc-3836) and chicken anti-goat IgG (1:3,000; cat. no. sc-516086; both Santa Cruz Biotechnology, Inc.) for 45 min at room temperature, prior to incubation with an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Inc.). Subsequently, the membranes were visualized by exposure to ECL film and the band intensities were quantified using UN-SCAN-IT gel analysis software, version 5.1 (Silk Scientific, Inc., Orem, UT, USA). GAPDH was used as a reference gene.
Statistical analysis
All experiments were performed ≥3 times. Data were presented as the mean ± standard deviation, and were analyzed using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Statistical differences between two independent groups were determined using the unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference.