Ethics statement
This study was approved by the Ethical Committee of The Second Hospital of Shanxi Medical University and in accordance with Helsinki Declaration. The animal experiments were approved by the Experimental Animal Welfare and Ethics Committee of The Second Hospital of Shanxi Medical University. All patients had signed the written informed consent. All the animal experiments were implemented based on the Guide for the Care and Use of Laboratory Animals [30].
Clinical sample collection
We collected the tumor tissues of 40 patients (at an average age of 54.35 ± 9.52; 21 males and 19 females) with primary glioma and corresponding adjacent tissues in The Second Hospital of Shanxi Medical University from June 2016 to June 2018 and stored in liquid nitrogen at –80 °C. None patients received chemotherapy or radiotherapy before. The histological features of collected tissues were independently diagnosed by two pathologists.
Cell culture
Human glioma cell lines (T98G, LN229, A172, and LN18) and healthy glioma cell line (HEB) were obtained from American Type Culture Collection (Manassas, Virginia, USA). All cells were subjected to short tandem repeat (STR) identification and cultured in RPMI-1640 medium (HyClone, South Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C with 5% CO2.
Cell treatment
Three siRNAs specifically targeting circDLC1, circDLC1 pcDNA 3.1, three siRNAs specifically targeting METTL3, METTL3 pcDNA 3.1, three siRNAs specifically targeting catenin beta interacting protein 1 (CTNNBIP1), CTNNBIP1 pcDNA 3.1, miR-671-5p mimic, miR-671-5p inhibitor, and their negative controls (NCs) were designed and synthesized by GenePharma (Shanghai, China). Finally, these vectors were transfected into A172 or LN229 cells using Lipofectamine 3000 (Thermo Fisher Scientific).
Cell-counting kit-8 (CCK-8) assay
A172 and LN229 cells were seeded into 96-well plates (2000 cells/well) and cultured for 0, 24, 48, and 72 h. Each well was added with 10 μL CCK-8 reagent (Beyotime, Beijing, China) for 2-h incubation at 37 °C. The absorbance at the wavelength of 450 nm was measured using a microplate reader (Bio-Rad, Philadelphia, PA, USA).
Colony formation assay
A172 or LN229 cells (200–300 cells) were seeded into the petri dish in humidified air at 37 °C with 5% CO2 for 10 days. After the removal of the complete medium, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 20 min. Colony cells were counted under an optical microscope (Nikon, Tokyo, Japan).
5-ethynyl-2ʹ-deoxyuridine (EdU) staining
A172 and LN229 cells were seeded into 96-well plates (20,000 cells) and cultured in the medium (100 μL/well, ThermoFisher) containing 50 μM EdU for 2 h (5% CO2, 37 °C). Briefly, the cells were treated with 4% paraformaldehyde for 30 min and stained with 100 μL Apollo dye solution (Ribobio) for 30 min. The nuclei were stained with DAPI (ThermoFisher) for 30 min, followed by observation under the fluorescence microscope (Olympus, Tokyo, Japan). The final results are presented as a percentage.
Actinomycin D treatment
A172 or LN229 cells were seeded into 24-well plates (5 × 105 cells/well). After 24 h, the cells were treated with 1 μg/mL actinomycin D. The relative RNA expressions of circDLC1 and DLC1 were analyzed using reverse transcription quantitative polymerase chain reaction (RT-qPCR).
RNase R treatment assay
The total RNA of A172 and LN229 cells (3 μg) was incubated with 10 U RNase R (20 U/μL, Epicentre, Madison, WI, USA) at 37 °C for 30 min and at 75 °C for 10 min to deactivate the RNase R, followed by RT-qPCR.
m6A level detection
The m6A level in total RNA was measured using m6A RNA methylation quantitative kit (ab185912, Abcam Inc., Cambridge, MA, USA). Firstly, RNA was coated in the detection well at 37 °C for 90 min, then added with capture antibody (50 μL), detection antibody (50 μL), and enhancer solution (50 μL) respectively, and incubated at room temperature for 60 min. Finally, the chromogenic solution was added for signal detection and absorbance measurement. The m6A level was measured using the colorimetry method (450 nm).
m6A RNA immunoprecipitation (MeRIP) assay
MeRIP was performed using Magna MeRIP m6A kit (Millipore, Billerica, MA, USA) to detect the m6A level of circDLC1. Briefly, 3 μg anti-m6A antibody (ab208577, Abcam) was incubated with protein A/G magnetic beads at 4 °C overnight. Then, the conjugate was incubated with RNA in the IP buffer containing RNase inhibitor and protease inhibitor. RNA was isolated and detected using RT-qPCR.
Nuclear/cytosol fractionation assay
After rinsing with pre-cooled phosphate-buffered saline (PBS), 1 × 106 A172 or LN229 cells were resuspended in 1 mL RLN buffer (50 mM Tris-HCl pH 7.4, 0.14 M NaCl, 1.5 mM MgCl2, 0.5% IGEPAL CA-630, and 1 mM DTT), incubated on ice for 5 min, and then homogenized. The cytoplasm was the supernatant after centrifugation. The precipitate was resuspended in 1 mL RSB buffer, homogenized, and centrifuged to remove the residual cytoplasm. The preliminary nucleus was resuspended in 3 mL 2 M RSB (2 M sucrose, 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1 mM DTT, and 0.5 mM PMSF) and centrifuged at 3000 × g for 45 min. The isolated circDLC1 was subjected to RT-qPCR analysis, with U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as nuclear and cytoplasmic controls, respectively [31].
Dual-luciferase assay
Wild-type (WT) circDLC1 or CTNNBIP1 containing miR-671-5p binding sequence was synthesized and inserted into the pmirGLO vector (Promega, Madison, WI, USA) and named circDLC1 WT or CTNNBIP1 WT. Mutant-type (MUT) circDLC1 or CTNNBIP1 containing miR-671-5p mutant binding sequence was synthesized and inserted into the pmirGLO vector, named circDLC1 MUT or CTNNBIP1 MUT. The constructed plasmids were co-transfected with miR-671-5p mimic or mimic NC into A172 and LN229 cells using Lipofectamine 3000, respectively. The luciferase activity was measured after 48 h using a luciferase detection kit (Promega).
RNA pull-down assay
miR-671-5p and miR-NC were biotinylated using the biotin RNA Labeling Mix (Roche, Indianapolis, IN, USA) and transcribed using T7/SP6 RN polymerase (Roche). Then, biotin-labeled miR-671-5p (bio-miR-671-5p) and bio-miR-NC were treated with RNase free DNase I (Promega) and RNeasy Mini kit (Qiagen, Redwood City, CA, USA). Next, M-280 streptavidin magnetic beads were incubated with bio-miR-671-5p or bio-miR-NC at room temperature for 4 h, and then with A172 and LN229 cell lysates. The RNA binding to bio-miR-671-5p or bio-miR-NC was extracted with TRIzol reagent for RT-qPCR.
Xenograft tumor experiments
Male BALB/c nude mice (aged 6 weeks old; weighing 20 g) purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing, China) [SYXK (Beijing) 2017-0033] were raised in a standard animal room and maintained under a 12-h light/dark cycle, with food and water provided ad libitum. LN229 cells were infected with the lentiviral overexpression vector of METTL3 (LV-oe-METTL3) or NC (LV-oe-NC) obtained from Genechem (Shanghai, China). Afterward, puromycin was used to screen the stably expressed cells. Before the experiment, the nude mice were weighed and numbered. The researchers divided them into two groups using the random number method. LN229 cells (5 × 105) were injected into the posterior flank of mice subcutaneously and tumor growth was observed. The health status of mice was detected every 3 days and the the tumor size was measured. The tumor volume was calculated: volume (mm3) = (length × width2)/2. The mice were sacrificed by intraperitoneal injection of 1% pentobarbital sodium (150 mg/kg) 21 days after injection. The tumors were removed and weighed. During the experiment, the researchers tried their best to reduce the number of nude mice and the pain of nude mice, and there was no animal death in the whole process. To ensure the reliability of experimental data, the tumor tissues of 6 mice in each group were fixed with 4% paraformaldehyde and embedded in paraffin for immunohistochemistry. The tumor tissues of the remaining six mice were used to detect the expression of cytokines in the tumor homogenate using RT-qPCR.
Immunohistochemistry
The tumor tissue of mice was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. After dewaxing and dehydration, the sections were incubated with METTL3 antibody (ab195352, Abcam) and Ki67 antibody (ab15580, Abcam) at 4 °C overnight. After PBS washing, the sections were incubated with the secondary antibody (ab6721, Abcam) at 37 °C for 20 min. After 2,4-diaminobutyric acid development (10 min) and hematoxylin counterstaining (3 min), the sections were dehydrated with gradient alcohol, cleared with xylene, and sealed with neutral resin. The sections were observed by professionals under a microscope using a double-blind method. Five visual fields were randomly selected from the section, and the positive rate of each visual field was calculated [32].
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using the TRIzol reagent (Invitrogen) and 2 μg RNA was reverse transcribed into cDNA using SuperScript RT kit (Invitrogen). RT-qPCR was performed using SYBR Premix ExTaqTM (Takara, Kyoto, Japan) and Applied Biosystems 7300 system (Applied Biosystems, Inc., Carlsbad, CA, USA). PCR primers are shown in Supplementary Table 1. The relative expression was calculated using the 2–ΔΔCt method [33], with GAPDH and U6 as internal references [34].
Western blot
Total protein was extracted using radio-immunoprecipitation assay buffer. The extract was centrifuged at 4 °C and 12,000 × g for 15 min and the protein concentration was determined using bicinchoninic acid kits (Beyotime). The protein was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk for 1 h and incubated with the primary antibodies METTL3 (ab195352, 1:1000, Abcam) and GAPDH (ab9485, 1:2000, Abcam) at 4 °C overnight. Following tris-buffered saline-tween buffer washing, the membranes were incubated with the secondary antibody (ab6721, 1:2000, Abcam) at 37 °C for 1 h. NIH Image J (National Institutes of Health, Bethesda, Maryland, USA) was used to analyze the gray value, with GAPDH as internal reference.
Bioinformatics analysis
The downstream miRNAs of circDLC1 were predicted through the Starbase database (http://starbase.sysu.edu.cn/index.php) [35]. The downstream genes of miR-671-5p were predicted through the Starbase database, Targetscan database (http://www.targetscan.org/vert_71/) [36], RNA22 v2 database (https://cm.jefferson.edu/rna22/Precomputed/) [37], and miRDB database (http://mirdb.org/index.html) [38].
Statistical analysis
Data analysis and map plotting were performed using the SPSS 21.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). The data complied with the assumption of normality and homogeneity of variance. Data are expressed as mean ± standard deviation. The t-test was adopted for comparisons between two groups. One-way or two-way analysis of variance (ANOVA) was employed for the comparisons among multiple groups, following Tukey’s multiple comparison test. A value of p < 0.01 indicated a significant difference.
