Human tissue samples
Totally 25 normal cervical epithelium, 34 HSIL, and 36 cervical cancer tissues were obtained for qRT-PCR assay. Cervical cancer samples were obtained from patients who underwent radical hysterectomy, HSIL samples were obtained from patients who underwent Colposcopy biopsy and normal cervical tissues were collected from patients who underwent hysterectomy because of benign gynecological diseases. All the samples were collected from September 2015 to December 2018 at Women’s hospital, Zhejiang University School of Medicine, China. Patients were informed consent for obtaining samples and the study was subject to approval by the Hospital Ethical Committee. Patient information for all specimens was shown in Additional file1: Table S1. Tissue samples were stored in RNA store solution at 4 degrees overnight and stored at -80 degrees centigrade until use.
Identification of HPV type
HPV type of tissue samples was identified using 21 HPV GenoArray Diagnostic innovation technologies of Hybribio Company. With the use of PCR principle to amplify extracted HPV DNA from cervical samples, amplified DNA amplicons are then hybridized with immobilized specific HPV probes on the HybriMem under the patented flow-through hybridization technique. Enzyme immunoassay method is applied for color development in order to obtain test results. HPV negative specimens used for RNA sequencing were amplified by the common primer my09/11 PCR of HPV L1 to further determine the HPV negative status.
RNA extraction and qRT-PCR
RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. For those tissue samples used for circRNA expression validation, RIN was assessed to confirm RNA integrity. qRT–PCR analyses were performed using PrimeScript RT reagent kit and SYBR Premix Ex Taq (TaKaRa, Japan). All the primers were presented in Additional file2: Table S2.
Cell line culture
Human cervical cancer cell SiHa cell line and human embryonic renal cell HEK293 cell line was obtained from the American Type Culture Collection (ATCC, USA). Human cervical cancer CaSki cell line was obtained from Cell resource center, Shanghai institute of life sciences, Chinese academy of sciences (China), where it was tested and authenticated. It was not cultured continuously for more than 3 months. SiHa cell line was cultured in DMEM (BI, Israel), 10% FBS and maintained at 37 °C in 5% CO2, whereas cervical cancer cell line CaSki and Human embryonic renal cell HEK293 cell line were cultured in RPMI-1640 (BI, Israel) containing 10% FBS.
RNA-FISH assay
CY3 labeled probes targeting the junction point sequence were used to visualize circCDKN2B-AS1 in situ (Details of Probes were shown in Additional file2: table S2). SiHa and CaSki cells were planted on the slides and incubated overnight. 0.5% tritonx-100 was added after washing slides with PBS for 3 times, then incubated at room temperature for 15 min.Then the cells were fixed with 4% paraformaldehyde for 5 min, then incubated with100% ethanol. After discarding ethanol, the denatured probe was added to immerse the slides. Then the slides were incubated at 37℃ overnight. Nuclei were counterstained with DAPI-Antifade and images were taken by Laser confocal microscope (TCS SP2 AOBS).
RNaseR resistance assay
RNA samples were incubated with 2U/ug RNaseR or without at 37℃for 15 min.
Northern Blot
The probe targeting the junction point of circCDKN2B-AS1 was synthesized and labeled with digoxigenin (DIG, details of Probes were shown in Additional file2: Table S2). 15 µg of total RNA was separated on 1% formaldehyde denatured gel electrophoresis at 25V overnight and transferred to Hybond-N + membrane (Amersham, UK, RPN303B). Prehybridization was carried out at 50 °C for 2 h in DIG Easy Hyb solution (Roche). Hybridizations were performed at 50 °C overnight. The membrane was washed stringently, blocked in blocking solution for 1 h. Blots were detected by anti-DIG antibody staining, and recorded using X-ray films with chemiluminescence substrate CSPD (Roche).
Western Blot
Cellular proteins were extracted using lysis buffer. Totally 20 µg of total protein was separated using 10% SurePAGE (GenScript, USA, M00665) and transferred to PVDF membrane (Bio-Rad, USA,1620177). The antibodies used are as following: IMP3(EMD Millipore), HK2(Abcam), and GAPDH (Diagbio).
Gene knockdown and overexpression
Human circCDKN2B-AS1 linear sequence (380 bp) was inserted into plasmid vector pLent-EF1a-circRNA-CMV-RFP-P2A-Puro (Weizhen, Shandong, China). A non-fluorescent circCDKN2B-AS1 overexpression plasmid was constructed for apoptosis detection using the plasmid vector (Weizhen, Shandong, China). The full length and junction point of the circCDKN2B-AS1 overexpressed products were determined by sanger sequencing after RT-PCR. ShRNAs specific against circCDKN2B-AS1 were inserted into lentiviral vector GV334(Genechem, Shanghai, China). Stable cell lines were obtained by puromycin resistance. Human HK2 overexpression plasmid was purchased from Genechem Company. All of the siRNA and primer sequences used in this article were presented in Additional file2: table S2.
Cell viability, migration, invasion and apoptosis assays
In vitro cell viability was detected using Cell Counting Kit-8 (Dojindo, Japan). Transwell assay was performed at 48 h after transfection, 1 × 104 cells were resuspended with serum-free opti-mem medium and cell invasion/ migration was examined in Transwell cell culture chamber filters coated on the upper side with/without Matrigel (Corning Biocoat) as described [33]. Apoptotic rates of SiHa and CaSki cells were detected at 72 h after transfection using Annexin V-FITC/PI apoptosis kit (Mutisiences, China, AP101-100-kit). For the cells transfected with circCDKN2B-AS1 overexpressed or empty plasmids, apoptosis was induced by incubation with Serum-free medium for 6 h.
Extracellular Acidification Rate(ECAR)
Assays were performed using the Seahorse XFe96 analyzer (Seahorse Bioscience, Agilent) according to the manufacturer’s instructions. ECAR was measured using Seahorse XF Glycolytic Rate Assay Kit(Seahorse Bioscience, Agilent,103044-100) and Seahorse XF Glycolysis Stress Test Kit(Seahorse Bioscience, Agilent༌103020-100). Glycolytic capacities cells were analyzed by the Seahorse XF Glycolysis Rate/ Stress Test Report Generator package. %PER from glycolysis was calculated by subtracting the acidification from CO2 produced by the mitochondria.
Biotin-labeled RNA pull-down and mass spectrometry analysis
Biotin-labeled RNA pull-down assay was performed using the Pierce™ Magnetic RNA-Protein Pull-down Kit (Thermo Scientific, USA,20164). Briefly, Cell lysates were prepared using standard lysis buffers (Thermo Scientific, USA). Biotin-labelled DNA probes (Details of Probes were shown in Additional file2: table S2) were incubated with streptavidin magnetic beads for 30 minutes at room temperature with agitation. Cell lysates were incubated with the streptavidin magnetic beads at 4 °C overnight.
The magnetic bead was thoroughly washed. The proteins bound to the magnetic bead were separated with SurePAGE (GenScript, USA, M00665), and the specific strip is cut and recycled for mass spectrometry analysis (Lumingbio, Shanghai, China).
RIP
RIP assay was performed with Magna RIP™ Quad RNA-Binding Protein Immunoprecipitation Kit (Sigma-Aldrich, USA, 17–704). The experiment was conducted according to the instructions of this kit.
Determination of mRNA half-life
To assess the half-life of HK2 mRNA, actinomycin-D (5 µg/ml, Sigma A4262) was added to block mRNA synthesis. Total RNA was collected at different time points and subjected to RT-qPCR analysis. The expression level of HK2 mRNA was normalized with 18S rRNA and plotted as a percentage of the value at time 0 (set at 100%).
Design and synthesis of inhibitory peptides
The inhibitory peptides for blocking the interaction between circCDKN2B-AS1 and IMP3 were designed and synthesized (ChinaPeptides Co. Ltd, Shanghai, China).The inhibitory peptides were synthesized by linking the biotin-labeled 11 amino acid cell-penetrating peptide (YGRKKRRQRRR) of Tat protein transduction domain with the core amino acids of IMP3 at N-terminus, with purity larger than 95%.
Biotin peptide pulldown assay
Biotin-labeled peptide were incubated with streptavidin magnetic beads (Thermo Scientific, USA,88817) for 4 h at 4 °C. Then, incubation of beads-peptide complex with total RNA was undertaken at 4 °C overnight. Beads were extensively washed, and RNAs pulled down were measured by qRT-PCR.
Nude mice xenograft experiments
This study was conducted in full accordance with the ARRIVE Guidelines for reporting animal research [34].All animal experiments were approved by the Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University under an Affidavit of Approval of Animal Ethical and Welfare license (No. IACUC-20190128-01) and in accordance with the Animals (Scientific Procedures) Act, 1986 (UK) (amended 2013).The BALB/c nude mice(4 ± 1 week old, 18 ± 5gram weighted,female) were ordered from Shanghai SLAC Laboratory Animal Company, Ltd(China) .Nude mice were randomly divided into two groups and anesthetized by inhalation of isoflurane gas (2–3%, also had the effect of muscle relaxation). Totally 107 stable SiHa cells were resuspended in 100µL PBS, injected subcutaneously under the left armpit of 4-week old nude mice(n = 10 per group).The length and width of the subcutaneous tumor were measured once a week for 5 weeks, and the volume of the subcutaneous tumor was calculated according to the formula: volume (cm3) = (length × width2)/2. Five weeks after the injection, 6 mice in each group were randomly selected and sacrificed by intravenous injection of pentobarbital (100 mg/kg) and subcutaneous tumors were harvested. A portion of the tumor was stored in RNA store Reagent solution to extract RNA, and the rest was fixed with paraformaldehyde for Immunohistochemistry and HE staining. The other 4 mice in each group were scanned by microPET/CT.
MicroPET/CT scanning of nude mice
MicroPET/CT imaging of nude mice was performed using a SuperArgus PET/CT 4R (Sedecal, Spain) after five weeks after the cell injection. Briefly, 4 tumor-bearing nude mice in each group were anesthetized by inhalation of isoflurane gas (2–3%). Then the nude mice were injected with 7.4 MBq (200 µCi) of18F-FDG via the abdominal cavity and Scanned at 120 min after injection. Nude mice were subjected to a 10-min microCT scan and then to a 15-min microPET scan. The scan was performed under isoflurane (1-1.5%) inhalation to maintain anesthesia. Images of nude mice were reconstructed Manually. The PET/CT camera Vista explore was used to calculate average SUVs (SUVAVG). Then the mice were sacrificed by intravenous injection of pentobarbital (100 mg/kg).
Zebrafish embryo model
Zebrafish(AB, wild type, 48-hour embryo) embryos were ordered in Core facilities,Zhejiang University, School of medicine and cultured in petri dishes with appropriate amount of water and placed in 28 ℃ incubator under constant temperature and light. 0.003%1-phyenyl-2-thiourea (PTU) (Sigma) were added Within 24 hours after the embryo being produced. Totally 200 SiHa cells after transfection were harvested and resuspended with PBS. Plasma membranes of the cells were stained with red-fluorescent Alexa Fluor® 594 wheat germ agglutinin of Image-iT™ LIVE Plasma Membrane and Nuclear Labeling Kit (Molecular Probes, USA, I34406). Zebrafish embryos were anesthetized with 0.016% Tricaine (Sigma). Then the cells were injected to the yolk sac of zebrafish embryo. Then we took photos with a fluorescence microscope at 48 h after injection. The area of red fluorescence in zebrafish yolk sac were calculated using image J software [32]. Then the zebrafish embryos were anesthetized with overdose Tricaine.
Immunohistochemical staining
Immunohistochemical staining and quantitative evaluation were performed as previously described [35], with antibodies specific for HK2 (Proteintech, USA).
Statistical Analysis
Statistical significance was calculated by using GraphPad Prism 5 software in this study. All experiments were repeated at least three times.