Tissue samples
A total of 58 glioma tissues and 58 normal brain tissues (normal controls) were obtained from The 980 Hospital of the joint logistics support force of the Chinese people's Liberation Army. All samples were collected with written informed consent from patients or guardians, and the experiments had been approved by the Ethics Committee of The 980 Hospital of the joint logistics support force of the Chinese people's Liberation Army. These samples were stored at -80℃ after freezing by liquid until use.
Cell lines and cell culture
Glioma cell lines (A172, T98G, N18 and LN229) and normal human astrocytes (NHA; control) were all purchased from Bena Culture Collection (Beijing, China). All of these cell lines were cultured at 90% DMEM (GIBCO, Grand Island, NY, USA) containing 10% fetal bovine saline (FBS; GIBCO) in a constant temperature incubator at 37℃ containing 5% CO2.
Quantitative real-time PCR (qPCR)
A Trizol reagent (Invitrogen, Carlsbad, CA, USA) was used for RNA extraction. Subsequently, the one-Step SYBR PrimeScript RT-PCR Kit was used for cDNA synthesis and quantification assays for circRNAs and mRNAs according to the manufacturer’s protocols. The synthesis of cDNA from miRNAs was conducted by using a TaqMan miRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA), and a TaqMan Universal Master Mix II (Applied Biosystems) was applied for miRNA quantification in line with the instructions. Relative expression was obtained using the 2−ΔΔCT method, using GAPDH or U6 as the internal references. All primers were listed as follows:
CircRELN: F, 5’-GACTCTTGTTATGATCTGTCCGT-3’ and R, 5’-CTGAGTAGCCAGGGTCACAT-3’; RELN: F, 5’-CGTCCTAGTAAGCACTCGCA-3’ and R, 5’-TCGCCTAAGTGACCTTCGTC-3’; miR-1290: F, 5’-GCGCGTGGATTTTTGGAT-3’ and R, 5’-AGTGCAGGGTCCGAGGTATT-3’; RORA: F, 5’-CAAAGCACAGCCCCAGTTTC-3’ and R, 5’-GCCTGTCCAGTTCGAAGACA-3’; GAPDH: F, 5’-GTCTCCTCTGACTTCAACAGCG-3’ and R, 5’-ACCACCCTGTTGCTGTAGCCAA-3’; U6: F, 5’-CTCGCTTCGGCAGCACA-3’ and R, 5’-AACGCTTCACGAATTTGCGT-3’.
Actinomycin D and RNase R treatment
Actinomycin D treatment was carried out using actinomycin D (2 mg/mL; Sigma, St. Louis, MO, USA) to treat the experimental cells for 0 h, 4 h, 8 h, 12 h or 24 h at 37℃.
RNase R treatment was carried out using RNase R (3 U/μg; Epicentre, Madison, WI, USA) to treat the isolated RNA samples for 15 min at 37℃.
Cell treatment
Sev (Maruishi Pharmaceutical, Osaka, Japan) was used to treat T98G and LN18 cells. In brief, cells were seeded into 6-well plates and cultured for 24 h. The plates were then placed into a sterile closed container introduced with 95% air + 5% CO2 containing different concentrations of Sev (1.7, 3.4, and 5.1%) [18]. Sev was delivered into a container using an anesthesia vaporizer. The changes of Sev concentrations were monitored using a gas analyzer (Ohmeda 5250 RGM, Louisville, CO, USA).
Cell transfection
Small interference RNA (siRNA) for circRELN knockdown (si-circRELN) and its negative control (si-NC) were provided by Genomeditech (Shanghai, China). The mimics for miR-1290 upregulation (miR-1290), the inhibitors for miR-1290 inhibition (anti-miR-1290) and their negative controls (miR-NC and anti-miR-NC) were all obtained from Ribobio (Guangzhou, China). PCDNA3.1 for RORA overexpression (pcDNA-RORA) and its control blank vector (pcDNA) were constructed by Genepharma (Shanghai, China). Oligonucleotides or vectors were transfected into cells using Lipofectamine 3000 reagent (Invitrogen).
Cell counting kit-8 (CCK-8) assay
After transfection, cells were plated into 96-well plates (2,000 cells/well) and maintained for 24 h. Then, 10 µL CCK-8 reagent (Dojindo, Kumamoto, Japan) was pipetted into each well for incubation for 2 h. The value of 450 nm optical density (OD) was detected using SpectraMax M5 (Molecular Devices, San Jose, CA, USA).
Colony formation assay
After transfection, cells were plated into 6-well plates (300 cells/well) and cultured for 2 weeks. Colonies (over 200 cells) were washed with phosphate buffered saline (PBS; Sigma), fixed with methanol and stained with crystal violet (Sigma). The number of colonies was counted under a microscope (Nikon, Tokyo, Japan).
Flow cytometry assay
Cells with treatment or transfection were cultured for 48 h and collected by Trypsin digestion. Cells were then washed with PBS and used for apoptosis detection using the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (Sigma). In brief, cells were suspended in binding buffer and then treated with 5 µL Annexin V-FITC and 10 µL PI. Cells were incubated in the dark for 15 min, and cell apoptosis was analyzed using a FACScan flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).
Cells with treatment or transfection were cultured for 24 h and collected by Trypsin digestion. Cells were washed with PBS and fixed in 70% ethanol at 4℃ overnight. Next, cells were washed with PBS and stained with PI/RNase A working buffer (BD Biosciences) in the dark for 15 min. Cell cycle distribution at various phases was determined using a FACScan flow cytometry (BD Biosciences).
Wound healing assay
After transfection, cells were plated in 6-well plates until 90% confluence. An artificial wound was created using a 200-μL pipette tip. The photos of wound closure were recorded at 0 and 24 h. Wound healing rate was measured using Image J software (NIH, Bethesda, MA, USA).
Transwell assay
Matrigel-coated transwell chambers (BD Biosciences) were used for cell invasion analysis, while non-treated chambers were used for cell migration analysis. Cells after transfection were suspended by FBS-free DMEM and transferred in the top of chambers, and DMEM containing 10% FBS was added to the bottom of chambers as a chemoattractant. After 24-h incubation, cells still in the upper chambers were removed by cotton swabs, and cells in the lower surface were fixed with methanol and stained with crystal violet (Sigma). The images of migration or invasion were recorded using a microscope (100 × ; Nikon).
Western blot
Total proteins extracted using RIPA buffer (Roche, Mannheim, Germany) were quantified by BCA kit (Roche). Then, 30 µg proteins were separated and membrane-transferred by standard western blot procedures. The primary antibodies, including matrix metalloproteinase-2 antibody (anti-MMP2; ab86607; Abcam, Cambridge, MA, USA), matrix metalloproteinase-9 antibody (anti-MMP9; ab137867; Abcam), anti-RORA (ab70061; Abcam) and anti-GAPDH (ab181602; Abcam), and the secondary antibodies (ab205718 and ab505719; Abcam) were used to probe proteins. The Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA) was utilized to visualize the protein signals. The expression data were quantified by using Image J software (version 1.46; NIH, Bethesda, MA, USA).
Dual-luciferase reporter assay
The binding relationship between circRELN and miR-1290 was analyzed by circinteractome (https://circinteractome.nia.nih.gov/) and circbank (http://www.circbank.cn/). The binding relationship between miR-1290 and RORA was analyzed by Targetscan (http://www.targetscan.org/vert_72/).
First, the wild-type (WT) and mutant-type (MUT) reporter plasmids of circRELN and RORA 3’UTR were constructed, naming as circRELN WT, circRELN MUT, RORA 3’UTR WT and RORA 3’UTR MUT. T98G and LN18 cells were cotransfected with miR-1290 or miR-NC and one of these plasmids. Cells were then incubated for 48 h, and luciferase activity in cells was detected using the dual-luciferase reporter assay kit (Promega, Madison, WI, USA).
RNA immunoprecipitation (RIP) assay
RIP assay was performed to determine the binding relationship using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions. The magnetic beads were conjugated with antibodies against Ago2 (Millipore) or IgG (control; Millipore). Purified RNAs from beads were extracted and analyzed by qPCR.
Xenograft models
The animal experiment was conducted in accordance with the Animal Care and Use Committee of The 980 Hospital of the joint logistics support force of the Chinese people's Liberation Army. Short hairpin RNA (shRNA) for circRELN knockdown (sh-circRELN) and its negative control (sh-NC) were packaged by lentivirus by Genepharma. Nude mice (BALB/c, 6-week-old, female, n = 20) were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China) and randomly divided into 4 groups (n = 5 per group), including control (T98G cell injection), Sev (T98G cell injection and Sev administration), Sev + sh-NC (T98G cell infected with sh-NC injection and Sev administration) and Sev + sh-circRELN (T98G cell infected with sh-circRELN injection and Sev administration). T98G cells were injected into nude mice by subcutaneous injection. For Sev administration, T98G cells were treated with 5.1% Sev as mentioned above and then injected into mice. Tumor volume (length × width2 × 0.5) was measured once a week. Tumor growth was allowed to develop for 35 days. After that, all mice were administrated with anesthetics and then sacrificed by cervical dislocation. Tumor tissues were excised for other analyses.
Statistical analysis
Data were expressed as mean ± standard deviation (SD). Student’s t-test or analysis of variance (ANOVA) was performed to compare differences between two groups or among multiple groups. GraphPad Prism 7.0 (GraphPad, La Jolla, CA, USA) was used for statistical analysis and graph making. P value < 0.05 was considered statistically significant.