Patient specimens
In the total of 62 paired samples of CC and neighboring non-tumorous tissues were harvested from CC patients with prior written informed consent from the patients at The Affiliated Hospital of Qingdao University. All tissues were confirmed as CC samples by two pathologists following surgery and then frozen in liquid nitrogen. Sample tissues were transferred to a -80℃ refrigerator until total RNA or protein extraction. Patients with CC were assigned to two groups (I+II group and III+ IV groups) according to the patient’s clinical stage. In addition, CC patients were divided into high (n=31) and low (n=31) circ_0000069 expression groups with median circ_0000069 expression value as the cutoff. The correlations between hsa_circ_0000069 expression and clinical characteristics of cervical cancer patients were listed in Table 1. All the procedures were permitted by the Ethics Committee of The Affiliated Hospital of Qingdao University.
Cell culture
The normal cervical epithelial cells (HcerEpic) were bought from the Chinese Academy of Sciences Cell Bank (Shanghai, China). In addition, CC cell lines (HeLa and SW756) were acquired from the American Type Culture Collection (Rockville, MD, USA). The above cells were maintained in RPMI1640 media (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and antibodies (penicillin/streptomycin; Invitrogen) in a humidified atmosphere containing 5% CO2 at 37℃.
RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR)
RT-qPCR assay was conducted to determine RNA level in cells and tumor tissues. In brief, total RNA was isolated by TRIzol Kit (Vazyme Biotech, Nanjing, China) as instructed by the manufacturer. For quantitation with circRNA and mRNA, complementary DNA (cDNA) was synthesized by High-capacity cDNA Reverse Transcription kit (Bio-Rad, Hercules, CA, USA). The transcript levels of target genes were measured by SYBR Fast qPCR Mix (Thermo Fisher Scientific, Waltham, MA, USA) under AB7300 thermo-recycler (Applied Biosystems, Carlsbad, CA, USA) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by using 2−ΔΔCt method. As for miR-4429, total RNA was reversedly transcribed to cDNA using TaqMan Reverse Transcription Kit (Applied Biosystems) according to the manufacturers’ instructions. TaqMan MicroRNA Assays Kit (Applied Biosystems) was used to quantify the expression level of miR-4429 in cells or tissues, with small nuclear RNA U6 as an internal control. Moreover, partial total RNA was treated with RNase R (3U/mg: Epicentre Technologies, Madison, WI, USA) at 37℃ for 15 min for RNase R treatment assay.
The sequences of partial primers were listed:
circ_0000069 (Forward (F)-5’-CTACTTCAGGCACAGGTCTTC-3’; Reverse (R)-5’-CTGACTCACTGGATGAGGACT-3’);
STIL (F-5’-CCCAACGCCAACTGGAGATTT-3’; R-5’-AGTCGGATGGTCTTCTCAGTC -3’);
miR-4429 (F-5’-GCCGAGAAAAGCTGGGCTGAG-3’; R-5’- CTCAACTGGTGTCGTGGA-3’);
ZIC2 (F-5’-GCGCAACTCCACAACCAGTA-3’; R-5’-TGCCGCATATAGCGGAAAAAG-3’);
GAPDH (F-5’-TCCCATCACCATCTTCCAGG-3’; R-5’-GATGACCCTTTTGGCTCCC-3’);
U6 (F-5’-AACGCTTCACGAATTTGCGT-3’; R-5’- CTCGCTTCGGCAGCACA-3’).
Transfection assay
Small interfering RNA (siRNA) against circ_0000069 (si-circ_0000069) and siRNA control (si-NC), miR-4429 mimic (miR-4429) and its control (miR-NC), and miR-4429 inhibitor (anti-miR-4429) and its control (anti-miR-NC) were generated by Sangon (Shanghai, China). Furthermore, circ_0000069-overexpression vector (circ_0000069), ZIC2-overexpression vector (ZIC2) and their control (vector) were obtained from Genomeditech (Shanghai, China). For transfection assay, 1 × 105 cells were seeded into 12-well plates and allowed to attach. After culture overnight, Lipofectamine 2000 (Invitrogen) reagent and 150 ng of siRNA, 1 μg of plasmids, or 80 nM of miRNA mimic/inhibitor were mixed and then added to the cells.
Cell proliferation assay
For 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) assay, HeLa and SW756 cells were seeded in 96 wells (3000 cells per well). After incubation for the indicated time, 20 μL of MTT (Promega, Madison, WI, USA) was added to cells and then incubated for another 4 h. After discarding the medium, 150 μL of dimethyl sulfoxide (DMSO) was administrated to cells. We reported the cell viability every 12 h by detecting optical density at wavelength of 490 nm each well on microplate reader (Bio-Rad). For colony formation assay, briefly, HeLa and SW756 cells were seeded in 96 wells (500 cells per well) and routinely cultured for two weeks. Then the colonies were fastened with 4% formaldehyde for 10 min and dyed with 0.1% crystal violet. Cell colonies were counted and photographed.
Apoptosis and cell cycle
For cell apoptosis analysis was conducted by using an Annexin V labeled with fluorescein isothiocyanate (FITC) Apoptosis Detection Kit I (Thermo Fisher Scientific) following the manufacturer’s recommended instructions. HeLa and SW756 cells were collected by trypsin and then re-suspended in binding buffer supplemented with 5 μL of Annexin V labeled with FITC and 5 μL of propidium iodide (PI). After incubation for 20 min, apoptotic cells were monitored by flow cytometry (Applied Biosystems). Apoptosis rate = (Annexin V+PI+ cell numbers + Annexin V+PI- cell numbers)/total cells×100%). For cell cycle detection, 100 uL of cell suspension (1×106/mL) was treated with PI staining solution contained RNase R and TritonX-100 for 30 min at 4℃, then flow cytometry was conducted to detect cell cycle distribution.
Western blot assay
Briefly, proteins were segregated on 10% sodium dodecyl sulfate polyacrylamide gels. The isolated proteins were subjected to wet electrophoretic transfer method and then transferred onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were incubated with 5% non-fat milk followed by incubation with the primary antibodies at 4℃ overnight. After being washed, membranes interacted with horseradish peroxidase-conjugated Goat polyclonal Antibody to Rabbit (ab150077; 1:3000 dilution; Abcam, Cambridge, MA, USA) for 1 h. Antibody binding was visualized with Western Blotting Detection Kit (Solarbio, Beijing, China) under Alpha Innotech Imaging System (Protein Simple, Santa Clara, CA, USA). The primary antibodies were listed as followed: anti-B-cell lymphoma-2 (Bcl-2; ab32124; 1:1000 dilution; Abcam), anti-Bcl-2-associated x (Bax; ab32503; 1:1000 dilution; Abcam), anti-cyclin-dependent kinases2 (CDK2; ab32147; 1:1000 dilution; Abcam), anti-cyclin-dependent kinases4 (CDK4; ab108357; 1:1000 dilution; Abcam), anti-ZIC2 (ab150404; 1:1000 dilution; Abcam), and anti-GAPDH (ab181602; 1:3000 dilution; Abcam).
Transwell assay
For the migration assay, 5×104 CC cells were plated in the top chamber of 24-well transwell chamber (6 well insert, 8 μm pore size). Notably, the top chamber of transwell chamber was pro-adhered with Matrigel (Becton Dickinson, San Jose, CA, USA) for transwell invasion assay. The complete medium contained with 10% fetal bovine serum was added into the lower chamber of transwell chamber as chemoattractant. After 24 h of incubation, cells that migrated or invaded were fastened with 4% formaldehyde and dyed with 0.1% crystal violet. Eventually, a microscope (Olympus Corp, Tokyo, Japan) was used to count cell numbers of migrated or invaded cells in five random visual fields.
Biotinylated RNA pull-down assay
The magnetic beads (Life Technologies, Carlsbad, CA, USA) pro-covered with circ_0000069 probe were incubated with cell lysates from HeLa and SW756 cells at 4℃overnight. After pull-down assay, immunoprecipitated RNA was extracted by TRIzol Kit and then subjected to RT-qPCR assay. In particular, for analysis of the relationship between miR-4429 and circ_0000069, HeLa and SW756 cells were infected with biotinylated miR-4429 mimics or mutant (Biotin-miR-4429-WT and Biotin-miR-4429-MUT; designed by Sangon) using Lipofectamine 2000. After 48 h, HeLa and SW756 cells were collected, lysed, and then incubated with streptavidin-coupled magnetic beads to generate biotin-coupled RNA complex. The expression level of circ_0000069 was analyzed by RT-qPCR assay.
Dual-luciferase reporter assay
The bioinformatics databases starbase (http://starbase.sysu.edu.cn/) and circBank (http://www.circbank.cn/) bioinformatics databases were utilized to forecast target miRNA of circ_0000069. Furthermore, the complementary sites between miR-4429 and 3’UTR of ZIC2 were predicted by bioinformatics databases starbase. The fragments of circ_0000069 or 3’ UTR of ZIC2 were amplified by RCR and then cloned into pGL3 vectors (Promega), named as circ_0000069 WT and ZIC2 3’-UTR-WT, respectively. In addition, mutant of circ_0000069 or 3’ UTR of ZIC2 was constructed with KOD-plus-mutagenesis kit (Toyobo, Osaka, Japan). After that, 80 nM of miR-4429 mimic or miR-NC were co-transfected with 0.5 μg of reporter vector using Lipofectamine 2000. The relative firefly luciferase activity was checked by Dual-Luciferase Assay Kit (GeneCopoeia, Rockville, MD, USA) and standardized to renilla luciferase signal.
In vivo experiment
For the animal experiments, HeLa cells were stably transfected with lentiviral vectors with short hairpin RNAs (shRNAs) targeting circ_0000069 (sh-circ_0000069) or control (sh-NC) constructed by GeneCopoeia. The 12 BALB/c nude mice (Shanghai Experimental Animal Center, Shanghai, China) were randomly divided into 2 groups. 2×106 HeLa cells in 200 μL of FBS-free culture medium were hypodermically vaccinated into left flank near the forelimb of BALB/c nude mice, while sh-NC was served as control group. Tumor volume was measured every 3 d based on V = 1/2× ab2 method (length (a) and width (b) length of the tumor). All mice were sacrificed by cervical dislocation on 21 d, and the tumors tissues were collected for subsequent experiments. The animal experiments were approved by the Institutional Animal Care and Use Committee of The Affiliated Hospital of Qingdao University.
Statistical analysis
All data were exhibited as mean ± standard deviation from at least 3 times independent experiments, and P <0.05 was considered statistically significant. All analyses were carried out using the SPSS 21.0 software (IBM, Somers, NY, USA) based on Student’s t-test and one-way analysis of variance. In addition, the survival curves of CC patients were plotted using the Kaplan-Meier method and the log-rank test. The correlation between clinicopathological characteristics of CC patients and circ_0000069 expression was assessed by Chi-square test. Pearson’s correlation analysis was used to analyze the relationship between miR-4429 and circ_0000069, or ZIC2 mRNA in CC tissues.