Clinical human samples
Thirty-six pairs human samples were collected from cervical cancer patients who underwent surgery in the department of obstetrics and gynecology, Renmin Hospital of Wuhan University from 2018 to 2019. The cervical cancer patients who received chemotherapy, immunotherapy, or radiotherapy were not enrolled. All specimens (2 cm away from the tumor tissues) were exanimated and approved by two pathologists, respectively. All samples were immediately froze and stored at -80°C after operation. All patients or their families were informed the approach of sample collection, and informed consent were obtained. All experiments in this study were approved by the Ethics Committee of the Renmin Hospital of Wuhan University.
Cell culture and transfection
All cervical cancer cell lines C33A, HT-3, HeLa, SiHa, normal cervical cell H8, and HEK-293T cells were commercially obtained from American Type Culture Collection (ATCC, USA). Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Gibco, USA) and 1% streptomycin double-antibody at 37°C with 5% CO2. Sh-circCCDC66 and its normal control shRNA were constructed by GeneChem (Shanghai, China). Circ-CCDC66 or REOX1 sequences were subjected to pcDNA3.1 vector to generate circCCDC66 or REXO1 overexpression vectors by Genepharma (Suzhou, China). MiR-452-5p mimic and its normal control mimic were constructed and commercially procured from Sangon Biotech (Shanghai, China). Lipofectamine 3000 (Invitrogen, USA) were applied to carry out all transfections.
The 8-week old nude mice (28 for study) were purchased from Vital River Inc. (Beijing, China). The ethics committee of the Renmin Hospital of Wuhan University approved animal experiments in this study. HeLa cells were stably transfected with Sh-circCCDC66 and Sh-NC. Then, about 1 × 107 treated cells (per tumor) were subcutaneously injected into nude mice. After 28 days, mice were sacrificed, and tumors were harvested. The animal experiments were followed with the ethical standard of Helsinki Declaration of 1975 (1983 reversion).
Total RNAs from cervical cancer human samples and cells were collected by Trizol kit (Invitrogen, USA). A PrimerScript RT Reagent kit (TaKaRa, Japan) was used to reversely transcribed RNA into cDNA. The amplification was conducted using SYBR Green dye. GPADH was used as an internal reference. The reliability of PCR results was confirmed by the comparative CT(2−ΔΔCt) method.
The information about primers used in study is showed in the supplementary Table 1.
Total proteins from cervical cancer tissues and cells were collected using a RIPA lysis buffer (Beyotime, Shanghai, China). Proteins were separated by 10% SDS-PAGE, and then transferred to PVDF membranes (Millipore, USA). Membranes were incubated with primary antibodies over night at 4°C, and then with secondary antibodies at room temperature for 2 hours. Protein bands were visualized by the ECL Western Blotting Detection Kit (PA, USA). Primary antibodies as following: AGO2 (1:1000, CST, 2897S), IgG (0.5 µg/ml, Abcam, ab190475), REXO1 (0.2 µg/ml, Abcam, #ab243536), GAPDH (1:2000, Abcam, #ab8245).
RNA fluorescence in situ hybridization (FISH)
Fluorescent In Situ Hybridization Kit (RiboBio, China) was used to carry out RNA FISH assay according to manufacturer’s instruction. Cy3-labeled circ-CCDC66 probes were procured from GeneChem (Shanghai, China). 4% paraformaldehyde was adopted to fix cells. Subsequently, cells were treated by permeabilization with 0.5% Triton X-100, then subjected to hybridization solution. After that, treated cells were incubated with Cy3-labeled circ-CCDC66 probes overnight. The results were detected by Fluorescent In Situ Hybridization Kit and visualized with a confocal microscopy.
Cell proliferation experiment
Cell proliferation ability was detected by cell account kit 8 (CCK-8, Dojindo, Japan) according to a previous study . Collectively, HeLa and SiHa cells (1× 104 per well) were seeded into a 96-well plate and housed for 3 days. CCK‐8 solution (10 μl) was added into well, two hours before detection. The absorbance at a wavelength of 450 nm was recorded, and repeated three times.
Cell migration and invasion experiment
Transwell chamber with 8.0 μm pores (Corning, USA) was used to detect cell migration and invasion ability. For migration, the upper chambers were seeded with cells (1× 104 per well) with DMED, and the lower chambers were added with DMED with 10% FBS. after two days, the cells on the lower surface were fixed with 4% paraformaldehyde, and visualized under a microscope. For invasion, Matrigel (BD, USA) was used to cover upper chambers, cells were incubated on the upper chambers with Matrigel and DMEM, lower chambers were filled with DMEM with 10% FBS. After 2 days, cells attached on lower surface were fixed with 4% paraformaldehyde, and visualized under a microscope. Experiments were carried out three times.
The biotinylated RNA pull-down assay was carried out followed by previous studies[22,23]. Biotinylated CCDC66 and miR-452-5p probes were synthesized and commercially obtained from Sangon Biotech (Shanghai, China). Briefly probe-coated beads were generated using C-1 magnetic beads (Life Technologies, Carlsbad, CA, USA). Cell lysates were collected using lysis buffer (Sigma, Germany), subsequently, incubated with C-1 magnetic beads at 4°C for 120 min. Then, elution buffer (Sigma-Aldrich) was used to elute precipitated RNAs from beads. RNA bands were analyzed by qRT-PCR.
RNA immunoprecipitation assays using anti-AGO2 and anti-IgG antibodies were performed using a Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA, USA) according to manufacturer’s protocol. Then, RNA complexes were subjected to qRT-PCR assay. Experiments were repeated three times.
Dual luciferase assay
The sequences of CCDC66 and REXO1 3′UTR containing wide-type or mutant-type miR-452-5p binding sites were subjected to pGL3-Basic luciferase vector (Promega, USA). Then, vectors were transfected into 293T and HeLa cells with pRL-TK vector (Promega), miR-452-5p mimic and its normal control mimic. After two days, firefly activity was detected in comparison with the Renilla control by a Dual-Luciferase Reporter assay system (Promega).
All data from experiment were presented as mean± Standard Deviation (SD). SPSS 17.0 software (SPSS, USA) was used to carry out results data. All experiments were repeated at least three times. The differences between two groups were calculated by student t-test. The differences between multiple groups were analyzed by Analysis of Variance (ANOVA). The statistical correlation between miR-452-5p and circ-CCDC66 or REXO1 were calculated by spearman analysis. p < 0.05 was considered statistically significant.