Patient tissue samples and cell lines
This study included 44 patients with cervical cancer who were treated at the Shanghai First Maternity and Infant Hospital from January to December 2017. None of the patients received chemotherapy or radiotherapy before biopsy or surgery. After surgical resection, samples of cervical cancer tissues and matched adjacent tissues were immediately frozen in liquid nitrogen and then transferred to a -80 °C refrigerator for stable storage until use. This study was approved by the Ethics Committee of the Shanghai First Maternity and Infant Hospital. All the included patients signed informed consent forms. The histopathological and clinical data came from pathology reports and medical records.
A human cervical immortalized squamous cell line (Ect1/E6E7) and human cervical cancer cell lines (HeLa, SiHa and CaSki) were provided by the American Type Culture Collection (ATCC, USA). DMEM (Gibco, USA) was used to cultivate HeLa and SiHa cells, and RPMI-1640 medium (Gibco, USA) was used to cultivate CaSki cells. Ten percent foetal bovine serum (Gibco, USA) was added to the above two types of media. Ect1/E6E7 cells were cultured in keratinocyte serum-free medium with 0.1 ng/ml human recombinant EGF, 0.05 mg/ml bovine pituitary extract and 44.1 mg/L calcium chloride. All cell lines were cultured at 37 °C in a humidified incubator containing 5% CO2.
Quantitative real-time polymerase reaction (qRT-PCR)
TRIzol reagent was used to extract total RNA from the tissue or cell line according to the manufacturer’s instructions. For circRNA and mRNA, the PrimeScript RT reagent Kit (Takara, Japan) was used to synthesize total RNA into cDNA, and TB Green Premix Ex Taq II (Takara, Japan) was used for cDNA amplification. For miRNA, the microRNA Reverse Transcription Kit (EZBioscience, USA) was used for reverse transcription, and Probe qPCR Master Mix for microRNA (EZBioscience, USA) was used for cDNA expansion. β-Actin, GAPDH and U6 were used as internal controls. The expression of each gene was normalized to that of the internal control and quantified using the 2−ΔΔCT method. Related primer sequences are shown in Additional file 1: Table S1.
Cell transfection and stable transfection strain construction
Circ_0087429 was overexpressed by pcDNA5.1-ciR integrated with the full-length sequence of circ_0087429 (Geneseed, China). OGN and EIF4A3 was overexpressed by PGMLV-6395 integrated with the full-length sequence of OGN or EIF4A3 (Genomeditech, China). According to the manufacturer’s protocol, Lipofectamine 3000 Reagent (Thermo Fisher Scientific, USA) was used for transfection. After successful transfection, cell lines were screened with 2 μg/ml puromycin for 30 days. In addition, the silencing of circ_0087429, OGN and EIF4A3 were achieved by si-circ_0087429, si-OGN and si-EIF4A3 respectively, and si-NC was a negative control. The overexpression and inhibition of miR-5003-3p were achieved by miR-5003-3p mimics and miR-5003-3p inhibitors, and NC mimics and NC inhibitors were used as controls (provided by Genomeditech, China). The above oligonucleotide sequences are shown in Additional file 1: Table S2.
RNase R treatment
Two micrograms of total RNA with or without 5 U/μg RNase R (Epicentre Technologies, USA) was incubated at 37 °C for 30 min. Then, qRT-PCR was used to detect the expression of circ_0087429 and the parental gene SPIN1.
Actinomycin D assays
Cells were seeded in six-well plates (4 × 105 cells per well). Twenty-four hours later, 2 μg/ml actinomycin D (Sigma, USA) was added to the cells. Then, total RNA was collected at the specified time, and the expression of circ_0087429 and SPIN1 was detected by qRT-PCR. The half-life was the time required for the RNA level to reach 50% of that at 0 h.
Separation of cytoplasmic and nuclear RNA in SiHa and HeLa cells was carried out using the Cytoplasmic and Nuclear RNA Purification Kit (Norgenbiotek Corporation, Canada). qRT-PCR was used to detect the relative expression levels of circ_0087429 and SPIN1. U6 was used as the internal reference for the nuclear fraction, and GAPDH was used as that for the cytoplasmic fraction.
Fluorescence in situ hybridization (FISH)
FAM-labelled circ_0087429 and Cy3-labelled miR-5003-3p were used to observe the cellular localization of the two. According to the manufacturer’s instructions, circ_0087429 and miR-5003-3p probes were used for hybridization overnight, and then the nuclei were counterstained with DAPI. The pictures were acquired by a Zeiss LSM710 laser scanning confocal microscope (Zeiss Instrument Inc., Germany).
Cell Counting Kit-8 (CCK-8) assay
CCK-8 reagent (Dojindo, Japan) was used to evaluate the proliferation ability of cervical cancer cells. Cells were seeded in 96-well plates at 1 × 103 cells per well. After culturing for 0, 24, 48, 72, and 96 h, CCK-8 solution (10 µL) was added to each well, incubated for 2 h in an incubator, and then a spectrophotometer was used to evaluate the absorbance at 450 nm.
5-Ethynyl-20-deoxyuridine (EdU) Assay
The Cell-Light EdU DNA cell proliferation kit (RiboBio, China) was used for EdU detection. Cells were incubated with EdU for 2 h and then fixed with 4% paraformaldehyde, followed by dyeing and sealing with Apollo dye solution and Hoechst 33,342. An inverted fluorescence microscope (Carl Zeiss, Germany) was used to take pictures to evaluate the proportion of EdU-positive cells.
Colony formation assay
The cells to be tested were seeded in a 6-well plate at 800 cells per well. After 2 weeks of culture, the cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.5% crystal violet solution for 15 min. Finally, the colonies were counted.
Wound healing assay
The cells were cultured in a 6-well plate to 90% confluence. A 200-µl pipette tip was used to create a scratch of the same width. A microscope was used to acquire images immediately. The cells were then incubated in serum-free medium for 24 h and 48 h and photographed again. The wound width was recorded at each time point.
Transwell invasion assay
The transwell chamber (Corning, USA) was paved with matrigel mix (BD Biosciences, USA) for invasion assays. According to the manufacturer’s protocol, 200 µl of the cell suspension was placed in serum-free medium in the upper chamber, and 20% FBS medium was added to the lower chamber. After 24 h of incubation, cotton swabs were used to remove the cells inside the upper chamber. The cells that invaded the bottom of the membrane were fixed and stained. A microscope was used to take pictures and then evaluate the number of cells with ImageJ software.
Tube formation assay
HUVECs were plated in 96-well plates coated with Matrigel (Corning, USA) at a density of 4 × 104 cells per well. After incubating for 3–4 h in the incubator, calcein AM (Sigma, USA) was added to the plate. A fluorescence microscope (Carl Zeiss, Germany) was used to take pictures and then analyse the tube-forming ability of each group of cells.
One percent PMSF added to RIPA lysis buffer (Beyotime, China) was used to extract total proteins. The same amounts of protein were separated by 10% SDS-PAGE, transferred to PVDF membranes (Millipore, USA), and then sealed with skim milk. The blots were incubated with the primary antibody at 4 °C for 12 h and then with the secondary antibody at room temperature for 1 h. An electrochemiluminescence kit (Millipore, USA) was used for the visualization of protein signals. β-Actin was the internal control. Primary antibodies were anti-E-cadherin, anti-N-cadherin, anti-Claudin-1, anti-Vimentin, anti-Snail, anti-OGN, anti-MMP2 and anti-EIF4A3 from Abcam (Cambridge, UK).
Luciferase reporter assay
The sequences that bind to miR-5003-3p in the circ_0087429 region and the OGN-3’UTR and their corresponding mutant sequences were synthesized and inserted into the luciferase reporter vector GM-1013FL02 and named circ_0087429-WT, circ_0087429-MUT, OGN-WT and OGN-MUT, respectively (Genomeditech, China). These plasmids were cotransfected with miR-5003-3p mimics or mimics NC. Then, according to the manufacturer’s protocol, the relative luciferase activity was checked with the Pierce Renilla-Firefly Luciferase Dual Assay Kit (Thermo Fisher Scientific, USA).
Cell immunofluorescence (IF) staining
A 4% formaldehyde solution was used to fix the cells for 15 min. Triton X-100 (0.2%) was used to permeabilize the cell membrane for 30 min. Then, the cells were blocked in blocking solution at room temperature for 1 h, and the primary antibodies were added and incubated overnight at 4 °C. Then, the fluorophore-tagged secondary antibodies were incubated for 1 h at room temperature. After that, the nuclei were counterstained with DAPI for 15 min and photographed with a confocal microscope.
RNA immunoprecipitation (RIP) assay
RIP assay was performed using an EZMagna RIP kit (Millipore, USA) according to the manufacturer’s protocol. The magnetic beads were incubated with anti-EIF4A3 antibody or IgG negative control antibody. Cell lysates were then incubated with the corresponding antibody-coated beads. Finally, the immunoprecipitated RNA was extracted and detected by qRT-PCR.
Immunohistochemistry (IHC) examination
Tissue sections were deparaffinized with xylene and dehydrated with a graded alcohol series. Afterwards, the slices were treated with 3% hydrogen peroxide and pressure-cooked for antigen retrieval. Then, the slices were incubated with anti-OGN, anti-E-cadherin, anti-N-cadherin, anti-CD31 and anti-Ki67 primary antibodies from Abcam (Cambridge, UK) at 4 °C overnight and then incubated with the appropriate secondary antibodies for 1 h at room temperature. Finally, the sections were treated with 3′-diaminobenzidine tetrahydrochloride and counterstained with haematoxylin.
All animal care and experiments were conducted in accordance with the guidelines of the National Institutes of Health and approved by the Animal Care Committee of Tongji University. We chose 4-week-old female BALB/c nude mice for tumour xenografts experiments. In tumour growth assay in vivo, HeLa cells stably transfected with pcDNA5.1-NC or pcDNA5.1-circ_0087429 were injected subcutaneously into the upper back of nude mice (1 × 107, 100 μl). The size of the tumours was measured with a calliper every week. The tumour volume was calculated as length x width2 × 0.5. Twenty-eight days after the injection, the mice were euthanized, and the tumours were dissected for further analysis. In tumour metastasis assay in vivo, HeLa cells stably transfected with pcDNA5.1-NC or pcDNA5.1-circ_0087429 were injected into nude mice through the tail vein (5 × 106, 150 μl). After five weeks, the mice were euthanized, and the livers and lungs were dissected, embedded in paraffin, and finally validated by haematoxylin and eosin (H&E) staining.
Statistical analyses were performed using SPSS 22.0 (IBM, SPSS, Chicago, USA) and GraphPad Prism 9.1.0 (GraphPad, La Jolla, CA, USA). Student’s t test (two-tailed) was used to compare the differences between the two groups, and one-way ANOVA was used to compare the differences among multiple groups. Pearson correlation analysis was used to analyse the correlations. The results of quantitative data are expressed as the mean ± SD. All experiments were repeated at least 3 times. For all the above analysis results, p < 0.05 was considered to indicate a significant difference.
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