Ethical statement
All patients provided signed informed consent and the experimental study protocol was approved by the Ethics Committee of Suzhou Science & Technology Town Hospital. The experimental protocol involving animals was approved by the Animal Ethics Committee of Suzhou Science & Technology Town Hospital.
Bioinformatics analysis
A microarray dataset GSE102686 was downloaded from the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), which used the GPL19978 platform, and included 5 cervical cancer tissue and paired adjacent tissue samples. Differential analysis was performed to screen differentially expressed circRNA in cervical cancer using the “limma” package in R (http://www.bioconductor.org/packages/release/bioc/html/limma.html) with |logFoldChange|> 1 and p value < 0.05 as the threshold.
Clinical samples and cell culture
Fifty-two pairs of primary cervical cancer tissues and adjacent normal tissues were collected from patients (aged from 34 to 70 years with a mean age of 49.21 ± 10.16 years old) from June 2015 to November 2018 at Suzhou Science & Technology Town Hospital and Huzhou Central Hospital. All patients were diagnosed using pathological examination, and the included cases were not complicated by pelvic inflammatory diseases or immune-related diseases. There were 46 cases of stage I–II and 6 cases of stage IIIa based on the International Federation of Gynecology and Obstetrics criteria.
Cervical cancer cell lines (SiHa, HT-3, Hela, SW756 and ME-180) obtained from the American Type Culture Collection (Rockville, MD, USA) were cultured in Dulbecco's modified Eagle medium (DMEM, HyClone, Thermo Scientific, USA) containing 10% fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 IU/mL penicillin at 37 °C with 5% CO2. Cells at 90% confluence were subcultured and the culture medium was removed. The expression of hsa_circ_0000520 in 5 cervical cancer cell lines was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR), and the two cell lines with the highest expression of hsa_circ_0000520 were screened for subsequent experiments.
RT-qPCR
Trizol (15596026, Invitrogen, Carlsbad, CA, USA) was applied to extract total RNA, and RNA was reverse transcribed into complementary DNA (cDNA) according to the instructions of PrimeScript RT reagent Kit (RR047A, Takara, Japan). PCR was performed using SYBR Premix EX Taq kit (RR420A, Takara) in a real-time fluorescent qPCR instrument (ABI 7500, ABI, Foster City, CA, USA). Primers (Table S1) for hsa_circ_0000520, ribonuclease P RNA component H1 (RPPH1), miR-1296, CDK2, U6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized by GeneChem (Shanghai, China). U6 was used as the internal reference gene of miR-1296 whereas other genes were normalized to GAPDH. The relative expression of the products was calculated by the 2−△△Ct method.
Western blot analysis
Total protein was extracted by radio immunoprecipitation assay (RIPA) lysis buffer (R0010, Solarbio, Beijing, China) containing phenylmethylsulfonyl fluoride, incubated on ice for 30 min and centrifuged at 12,000 r/min at 4 °C for 10 min followed by collection of supernatant. The protein concentration of each sample was determined by bicinchoninic acid kit (23225, Pierce, Boston, MA, USA). A total of 50 μg protein sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis for 2 h and transferred to polyvinylidene fluoride membranes (ISEQ00010, Millipore, Billerica, MA, USA). The membrane was blocked with 5% skimmed milk powder for 2 h and incubated with primary antibodies (Abcam, Cambridge, UK) of rabbit anti-human CDK2 (1: 500, ab194868), CyclinD1 (1: 500, ab61758), P21 (1: 1000, ab227443), P27 (1: 1000, ab75908), B-cell lymphoma-2 (Bcl-2) (1: 500, 59348), Bcl-2 associated protein X (Bax) (1: 500, ab53154) and GAPDH (1: 2500, ab9485) overnight at 4 °C. Horseradish peroxidase labeled goat anti-rabbit immunoglobulin G (IgG) (1: 2000, ab6721, Abcam) secondary antibody was added to the membrane and incubated at room temperature for 1 h. Color was developed by enhanced chemiluminescence reaction solution (WBKLS0100, Millipore). With GAPDH as the internal reference, the relative expression of proteins was presented as the value of the target band to the internal reference band.
Dual-luciferase reporter gene assay
Circ_0000520 transcripts containing the putative miR-1296 binding sites as hsa_circ_0000520-wild type (Wt) and hsa_circ_0000520-Wt mutated at the putative miR-1296 binding sites as hsa_circ_0000520-mutant type (Mut) were inserted into the luciferase reporter vector, respectively. CDK2-Wt and CDK2-Mut were developed similarly. Well-designed reporter vectors were delivered into 293T cells with miR-1296 mimic, respectively. The luminescence of firefly luciferase was determined using dual-luciferase reporter assay system kit (K801-200, BioVision, USA) and normalized to that of renilla luciferase detected following the instructions of Glomax20/20 luminometer (Promega, USA).
Immunohistochemistry (IHC)
Primary cervical cancer tissues and the matched adjacent normal tissues were fixed with 4% neutral formaldehyde buffer (DF0113, Solarbio), paraffin-embedded, and sectioned at a thickness of 4 μm. The sections were dewaxed with xylene (YB-5485, YBi, Shanghai, China), hydrated with gradient alcohol, and soaked in 3% hydrogen peroxide for 20 min at room temperature to remove endogenous peroxidase activity. Meanwhile, the antigen was thermally retrieved twice and sections were blocked with 10% goat serum for 15 min, which were incubated with rabbit anti-CDK2 primary antibody (1: 500, ab194868, Abcam) overnight at 4 °C. Biotin-labeled goat anti-rabbit IgG (1: 1000, ab6721, Abcam) secondary antibody working solution was added into the sections for 40-min incubation at 37 °C. Color was developed with diaminobenzidine (DA1010, Solarbio) for 10 min, followed by the counterstaining of sections with hematoxylin (H8070, Solarbio) for 1 min. Phosphate buffer saline (PBS) was used instead of primary antibody as negative control (NC). The final results were scored by two pathologists in a blinded-manner. Sections were observed under a light microscope (CX41-12C02, Olympus, Japan) with 5 visual fields randomly selected. The positive cells were seen as brown-yellow. The percentage of positive cells among the total cells indicated CDK2 protein staining results with > 10% considered as positive expression (+) and < 10% as negative expression (−).
RNase R linear RNA digestion experiment
Cells were seeded into a 6-well plate (2 × 105 cells/well), trypsinized and centrifuged at 2000×g for 2 min. Cytoplasm and nuclear RNA were each recovered using a nuclear-cytoplasm separation kit according to standard instructions. RNA was divided into RNase digestion group and non-digestion group. Linear RNA was digested by 10 × reaction buffer with 1 μg RNA digested by 1 unit (U) RNase at 37 °C for 10 min. Subsequently, the digested products were extracted by phenol/chloroform and ethanol precipitation, and reverse transcribed into cDNA.
RNA fluorescence in situ hybridization (FISH)
Cell slides were cultured at the bottom of the 24-well plate (6 × 104/well). FISH assay was performed when cell confluence reached 60–70%. Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, added with 1 mL of precooled permeate each well and stood at 4 °C for 5 min. Furthermore, each well was blocked with 20 μL pre-hybridization solution for 30 min at 37 °C with hybridization solution preheated at 37 °C simultaneously. After removal of pre-hybridization solution, each well was hybridized with hybridization solution containing probes overnight at 37 °C in the dark. Slides were then stained with 4',6-diamidino-2-phenylindole staining solution for 10 min and mounted for fluorescence detection.
Cell transfection
Human cervical cancer cells were transfected with short hairpin RNA targeting hsa_circ_0000520 (sh-hsa_circ_0000520), sh-CDK2, CDK2 overexpression vector (oe-CDK2), miR-1296 mimic, miR-1296 inhibitor or relevant NC alone or in combination. All plasmids were purchased from Dharmacon (Lafayette, CO, USA). Cervical cancer cells were seeded in 6-well plates at a density of 3 × 105 cells/well and transfected with the aforesaid plasmids following the instructions of Lipofectamin 2000 kit (Invitrogen). The culture medium was renewed with complete culture medium 6 h post transfection. Cells were further cultured at 37 °C in 5% CO2 and then collected after 48 h.
Colony formation assay
Cells were detached with 0.25% trypsin, triturated into single cells, and suspended in DMEM containing 10% FBS. The cell suspension was diluted and cultured in a dish with 37 °C preheated culture solution at the gradient density of 50, 100, or 200 cells/dish, respectively. After 2–3 weeks of culture, the supernatant was discarded when clones were visible. A total of 5 mL cells were fixed with 4% paraformaldehyde for 15 min and stained with GIMSA’s staining solution for 10–30 min. Clones were counted directly.
Cell counting kit-8 (CCK-8) assay
After a single cell suspension was prepared, cells were seeded into 96-well plates with a volume of 200 μL cell suspension per well and incubated in an incubator with 6 repeated holes. The plates were removed at 24 h, 48 h and 72 h during incubation, followed by the addition of 10 μL CCK-8 solution (Sigma) and another 2 h-incubation. The absorbance values of each well were measured at 570 nm by enzyme-linked immunoassay (NYW-96 M, NYAW Instrument, Beijing, China). A cell viability curve was plotted with time points as the X-axis and optical density (OD) value as the Y-axis.
5-ethynyl-2′-deoxyuridine (EdU) assay
Cells in the logarithmic growth phase were seeded into 96-well plates (5 × 104 cells/well). Each well was incubated with 50 μmol/L EdU medium (a total of 500 μL) for 2 h. Plates were then fixed with 40 g/L paraformaldehyde for 20 min, incubated with 2 mg/mL glycine for 10 min, and permeabilized with 500 μL 0.5% TritonX each well. Cells were further incubated with Apollo staining reaction solution for 30 min in the dark and then Hoechst 33,342 reaction solution for 30 min in conditions void of light. After washing with 0.5% Triton twice, the cells were observed under an inverted fluorescence microscope with the number of cells counted using Image-Pro Plus 6.0 Professional Image Analysis Software (IPP, Texas, USA).
RNA binding protein immunoprecipitation (RIP) assay
Cells were lysed in an ice bath with RIPA lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) for 5 min, and centrifuged at 14,000 rpm for 10 min at 4 °C to remove the supernatant. One part of the cell extract was taken as input whereas the other part was incubated with antibody for co-precipitation. The antibodies used for RIP involved rabbit anti-Argonaute 2 (Ago2) (1: 50, ab32381, Abcam) with IgG antibody as NC.
Flow cytometry
The density of trypsinized cells was adjusted to 3 × 105 cells/mL, which were seeded in 6-well plates and cultured for 48 h. The single cell suspension was centrifuged at 1000 r.min−1 × 5 min followed by removal of the supernatant. Cells were fixed with 70% ice ethanol solution at − 20 °C or 1 h and then added with 20 μL RNA enzyme and reacted at 37 °C for 30 min. Subsequently, cells were incubated with 400 μL propidium iodide (PI) (C0080, Solarbio) for 15 min in the dark, on ice. The cell cycle was detected by a flow cytometer (BDLSR II, BD, FL, NJ, USA) with the excitation light wavelength at 488 nm. For detection of cell apoptosis, the cells were resuspended with 400 μL of Annexin V binding solution (CA1020, Solarbio) to 3 × 105 cells/mL and then incubated with 50 μL Annexin V-Fluorescein Isothiocyanate staining solution on ice, in the dark, for 15 min. After the addition of 10 μL PI staining solution, cell apoptosis was detected by flow cytometer also with the excitation light wavelength as 488 nm.
Xenograft tumor formation in nude mice
Thirty-two BALB/C nude mice (aged 4 weeks and weighed 18–22 g; Animal Experimental Center of Southern Medical University) were treated with SiHa cells overexpressing CDK2 (by transfection of oe-CDK2) and/or silencing miR-1296 (by transfection of miR-1296 inhibitor) or hsa_circ_0000520 (by transfection of sh-circ_000052) with eight mice in each group. Specifically, SiHa cells were prepared into a single cell suspension by suspension in the mixture (PBS: Matrigel = 1: 1) with the final cell concentration as 1 × 106 cells/200 μL. Additionally, nude mice were anesthetized with sodium pentobarbital and injected with SiHa cells subcutaneously at the back of the right hind leg, and were raised in the same environment as the untreated mice. Tumor volume was monitored once a week. At the end of the fourth week, the nude mice were euthanized and tumors were removed.
Statistical analysis
SPSS 21.0 software (IBM Inc., Armonk, NY, USA) was used for statistical processing of the measurement data derived from experiments. All experiments were repeated at least three times independently. Measurement data were presented as mean ± standard deviation. Cervical cancer tissues were compared with adjacent normal tissues using paired t test. Independent sample t-test was used for comparison between two groups, whereas one-way analysis of variance (ANOVA) was applied to data comparison between multiple groups followed by Tukey’s post hoc test. Data at different time points were analyzed using repeated measures ANOVA followed by Bonferroni’s post hoc test. The statistical significance was set at p < 0.05.