Tissue samples collection
Cancerous and adjacent normal tissues of 50 patients with cervical cancer who had not received prior treatment were obtained from the Second Affiliated Hospital of Soochow University from 2015 to 2018. All patients signed the informed consent. The samples were immediately frozen in liquid nitrogen and stored at -80 °C. The pathological classification and clinical stages were performed to the International Federation of Gynecology and Obstetrics (FIGO) criteria. All patients signed the informed content. All the protocols in this study were approved by the Ethics Committee of the Second Affiliated Hospital of Soochow hospital.
Cell culture and processing
Human foreskin keratinocytes (HEKn, Cat. no. C-001-5C) were obtained from the Cascade BiologicsTm (Portland, OR, USA) and cultured in EpiLife medium (Gibco/LifeTechnologies, Waltham, MA; Cat. No. M-EPI-500-CA). The human cervical cancer cell lines (HeLa, CaSki, SiHa, C-33A and MS751) were purchased from Cell Bank of the Chinese Academy of Science (Shanghai, China) maintaining in RPMI1640 medium. All the mediums were supplemented with 10% fetal bovine serum (FBS; Hyclone; Invitrogen, Camarillo, CA, USA) and 100 U/ml penicillin/100 µg/ml streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C with 5% CO2. For the cell processing, 10 mM LiCl or 100 ng/ml Dkk-1, which are served as the Wnt signaling pathway activator and inhibitor respectively, were added in to the medium 36 h after cell transfection.
Subcutaneous xenografts in mice
As previously described [13], 4 × 106 HeLa cells, which were transfected with si-CASC11 and si-NC for 24 h, were subcutaneously inoculated into male athymic nude mice (n = 12, 6–8 weeks old). The tumors’ size was measured every 5 days with calipers and the volume of the tumors were calculated as length × (width2)/2. 35 days after affections, the tumors were removed surgically and measured the weight.
Quantitative real-time polymerase chain reaction (RT-qPCR)
According to the manufacturer’s instructions, total RNA was extracted from cells with RNAiso Plus (Code No. 9108, TaKaRa, Dalian, China). According to the protocols of manufacturer, RNA quality was assessed using a NanoDrop 2000 Spectrophotometer (Thermo Scientific). Then 1 µg total RNA was converted into the first strand cDNA using PrimeScript RT reagent kit (Takara, Tokyo, Japan). Finally, the PCR amplification was performed using a SYBR® Premix Ex Taq™ II kit (Code No. RR820A, TaKaRa). The reaction system (20 µl) included 10 µl TB Green Premix Ex Taq II (Tli RNaseH Plus, 2×), 0.8 µl PCR Forward Primer (10 µM), 0.8 µl PCR Reverse Primer (10 µM), 0.4 µl ROX Reference Dye (50×), 2 µl DNA template and 6 µl sterile water. The PCR reaction conditions as follows: 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and 60 °C for 30 s. After the cycling protocol, the final step was applied to all reactions by continuously monitoring fluorescence through the dissociation temperature of the PCR product at a temperature transition rate of 5 °C to generate a melting curve, and then Cooling 50 °C for 30 s. Quantification was conducted according to the 2−ΔΔct method. The relative expression of CASC11 was analyzed in an Applied Biosystems 7500 Fast real-time PCR system and was normalized with glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). The primers were purchased from Sangon Biotech (Shanghai, China), and the sequences were shown as follows: CASC11 sense 5′-GCTGCAGA AGGTCCGAAGAA-3′, CASC11 antisense 5′-TTCACCACGTCCAGTTGCTT-3′; GAPDH sense 5′-GGAGCGAGATCCCTCCAAAAT-3, GAPDH antisense: 5′-GGCTGTTGTCATACTTCTCATGG-3′.
Cell transfection
For optimal siRNA transfection efficiency, siRNA sequences were designed to target the human CASC11 gene and the CASC11 siRNAs and control siRNAs were obtained from GenePharma (Shanghai GenePharma Co., Ltd., Shanghai, China). The sequences of siRNAs were 5′-GCCCACATCAAGCCTTCAT-3′ (CASC11 siRNAs) and 5′-UUCUCCGAACGUGUCACGU-3′ (control siRNAs). Cells (2 × 105 cells/well) were added in 6-well plates and transfected with CASC11 siRNAs (100 nM) or control siRNAs (100 nM) for 48 h using Lipofectamine™ 2000 (Invitrogen; Thermo Fisher scientific, Inc.) according to the instructions.
A CASC11 overexpression plasmid, pcDNA3.1-CASC11, was commercially constructed by GenePharma (Shanghai, China), and empty pcDNA 3.1 vector (NC) was used as the control. To establish cell lines with transient overexpression of CASC11, and CaSki cells (5 × 105/ml) were seeded into 6-well plates and transfected with 10 µg pcDNA3.1-CASC11 plasmid or control pcDNA3.1 vector in medium using lipofectamine™ 2000 (Invitrogen). And the effect of CASC11 silencing and over-expression is shown in Additional file 1: Fig. S1.
Cell Counting Kit-8 (CCK-8) assay
Cell Counting Kit-8 (CCK-8, Solarbio, China) assay was used to analyze the cell proliferation. Briefly, 100 µl cell suspension were seeded into 96-well plates. 0 h, 12 h, 24 h, 48 h or 96 h after cell transfection, 10 µl CCK-8 reagent was added into the medium incubating in dark at 37 °C for 2 h. Finally, the absorbance at 450 nm was determined with a full wavelength multifunctional enzyme labeling apparatus (TECAN).
Flow cytometric analysis for apoptosis
48 h after cell transfection, the apoptosis of HeLa cells and CaSki cells was measured with an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences) following the manufacturer’s instructions. The apoptosis rate was analyzed using a FACSCalibur™ flow cytometer (BD Biosciences) as previously mentioned [11].
Transwell assay for cell invasion and migration
Transwell Chambers (Corning) uncoated or coated with Matrigel were used for assessing the invasion and migration of cervical cancer cells. The detailed methods were as mentioned as Lan described [14]. The migratory or invasive cells was imaged and counted utilizing an optical microscope (Carl Zeiss, Jena, Germany).
TOP/FOP-Flash luciferase reporter analysis
TOP/FOP-Flash luciferase reporter assay was used to analyze the activity of Wnt/β-catenin signaling pathway. The vector pRL-SV40 was served as the internal reference. Cells in the control groups were co-transfected with pRL-SV40 and TOP/FOP flash (Promega). Cells in NC groups were co-transfected with pRL-SV40, TOP/FOP flash and si-CASC11 NC/pcDNA 3.1. Cells in si-CASC11 group or pcDNA3.1-CASC11 group were co-transfected with pRL-SV40, TOP/FOP flash and si-CASC11 NC/pcDNA 3.1-CASC11. Dual-Luciferase Reporter Assay System (E1910; Promega) was applied to measure the luciferase activity. The ratio of TOP/FOP indicated the activity of Wnt/β-catenin signaling pathway.
Western blot
Total proteins were extracted from cells with RIPA lysis buffer (P0013B, Beyotime) and nuclear extracts were collected with a Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime). An enhanced BCA protein assay kit (P0010, Beyotime) was used to measure the protein concentration. 50 μg protein per well was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferred onto polyvinylidene difluoride membranes (Millipore). After blocked with 5% non-fat milk, the bands were incubated with primary antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (#7074, 1:2000) from Cell Signaling Technology. The primary antibodies were as follows: β-catenin rabbit monoantibody (#8480, 1:1000), GAPDH rabbit monoantibody (#8884, 1:1000), Lamin B rabbit monoantibody (#12255, 1:1000). The target proteins were visualized with a BeyoECL Plus kit (P0018, Beyotime). Densitometric analysis was carried out with Image J software.
Statistical analysis
All the experiments were replicated independently at least three times in triplicate. The data were represented as means ± standard deviation (SD) and analyzed with one-way ANOVA using SPSS version 22.0 software (IBM, Chicago, IL, USA). The relationship between the overall survival (OS) of patients with cervical cancer and CASC11 was evaluated with the Kaplan–Meier test. A value of p < 0.05 was considered statistically significant.
