At the First Affiliated Hospital of Nanjing Medical University, glioma patient specimens (n = 45) were collected by surgical resection. Normal brain tissue samples (NBT) from severe traumatic brain injury patients (n = 8) undergoing craniotomy decompression and cerebrovascular malformations were also collected as negative controls. After surgical excision, samples were promptly frozen in liquid nitrogen and preserved there. Experienced pathologists identified glioma samples using World Health Organization (WHO) diagnostic standards. Patients and their families granted informed consent for all tissue collection, and the ethics committee of Nanjing Medical University authorized our study technique. Specific patient information is provided in Additional file 5: Table S2.
Cell lines and cell culture
Glioma cell lines U251, LN229, U118, U87, and T98G were bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). In a DMEM media with 10% FBS, the primary GBM cell line (pGBM-1) was maintained after being generated from surgical specimens of GBM. The American Type Culture Collection (ATCC) provided normal human astrocytes (NHAs). In the laboratory of the common platform of the Wutai Campus of Nanjing Medical University, all cell lines were kept in liquid nitrogen containers.
GenePharma (Shanghai, China) created mimics of siRNAs and miRNAs. Following is a list of the oligonucleotide sequences.:
#sh-RNA-2: 5′-AGTAAAGTGTATTGAATGCAA -3′;
The aforementioned sequence was cloned into the RioBio pcDNA3.1 vector (Shanghai, China) to make pcDNA-cNDC80. This allowed for the regulation of the expression level of cNDC80. GenePharma provided the miR-139-5p inhibitor (anti-miR-139-5p), miR-139-5p mimic (miR-139-5p), and matching controls (anti-NC and miR-139-5p-NC) (Shanghai, China). GenePharma supplied lentiviral vectors with anti-miR-139-5p or anti-NC and shRNA or shCONT (Shanghai, China).
Quantitative RT–PCR and RNase R treatment
TRIzol (Invitrogen) reagent was utilized to harvest total RNA from tissues and cells. Following the manufacturer's instructions, PrimeScript RT Master Mix (TaKaPa) was applied to reverse-transcribe circRNA, miRNA, and mRNA. Using the 2–ΔΔCT technique and 18S rRNA as the internal reference, the relative quantification of circNDC80, mNDC80, miR-139-5p, and ECE1 were calculated. CircNDC80, mNDC80, ECE1, and miR-139-5p were standardized to GAPDH and U6, respectively. Three separate runs of these responses were made. On a StepOnePlus Real-Time PCR System (Thermo Fisher, USA), quantitative PCR tests were run, and the results were assessed on AGE (agarose gel electrophoresis). The Additional file contains a list of all primer sequences (Additional file 4: Table S1).
The RNeasy MinElute Cleaning Kit (Qiagen, Dusseldorf, Germany) was used to purify RNA after it had been subjected to 3 U/mg of RNase R. After that, qRT–PCR was employed to ascertain the level of mNDC80 and cNDC80 expression.
RNA fluorescence in situ hybridization (FISH)
RiboBio (Guangzhou, China) produced the miR-139-5p FAM-labeled and cy3-labeled probes for cNDC80. Following 4% paraformaldehyde fixation for twenty minutes at room temperature and PBS washing, cell lines were grown in 10% fetal bovine serum. After that, the membranes were dissolved in 0.5% triton-X-100 PBS for 5 min at room temperature. Prehybridization buffer was immediately applied to the cells, and then the probe was added to the hybridization buffer overnight. Following that, cells were washed three times with saline-sodium citrate (SSC) at 42 °C for 5 min. After staining the nuclei with DAPI for ten minutes, the signal from the probes was examined using a fluorescence in situ hybridization kit (RiboBio, Guangzhou, China).
The proliferation of U87 and pGBM-1 was discovered using the CCK-8 kit (RiboBio, China). A 96-well plate with 100 μl of media each well and six duplicate wells included was filled with 1000 cells. A culture system was created by mixing 10 μl of CCK-8 reagent with 90 μl of DMEM media. Then, each well received 100 μl of this system, and it was cultured for 1.5 h. This experiment was carried out over 1, 2, 3, and 4 days.
Colony formation assay
Before being frozen in 4% paraformaldehyde for a further 10 min, cells (3 × 103) were cultured for 14 days at 37° C in an incubator with 5% CO2 before being seeded on cell culture plates (35 mm, Corning, USA). The number of clones was recorded on the clear film on the grid after staining with crystal violet dye (Beyotime, China) for 20 min.
5-Ethynyl-20-deoxyuridine (EdU) assay
Cells (2 × 104) were seeded onto 96-well plates as previously reported and cultured overnight at 37° C in an incubator with 5% carbon dioxide for the EdU experiments . Cells were fixed using 4% paraformaldehyde fixator, the Apollo dye solution (RiboBio) and the nuclei were stained with DAPI (Invitrogen). Images were then captured using a fluorescence microscope (Olympus) after culturing for 2 h in EdU (RiboBio, Guangzhou, China).
As reported earlier, glioma cell invasion and migration were measured using Transwell [31, 32]. The main change was the adoption of a modified 8-m pore size culture method and matrigel coating for the top chamber. Serum-free media and 10%FBS medium were layered at the bottom of Transwell Inserts before transfected cells were planted inside. Cells were stained with crystal violet after being in development for 24 h before being fixed with 4% paraformaldehyde solution. The number of cells infiltrating the holes was determined using ImageJ software after pictures taken under an Olympus microscope were obtained. Three times each were done for every experiment.
In order to compare the effects of transfection on GBM cell lines, several were seeded in 6-well plates and grown to confluence. Utilizing a 200 µL pipette tip, cell monolayers were scraped. At 0 h, 24 h, and 48 h, respectively, cell movement was recorded using an Olympus microscope (Olympus, Tokyo, Japan).
Western blot assay
Different sets of GBM cells were lysed with RIPA buffer (KeyGEN, Jiangsu, China) below the ice. The protein lysates were then centrifuged at 12,000 RPM, and the supernatant was collected and electrophoresed on a 10%SDS polyacrylamide gel, which was subsequently transferred to a PVDF membrane (Millipore, USA) and blocked with 5% skim milk. Subsequently, primary antibodies were added at 4℃: anti-ECE1 (abcam, #ab71829,1:1000), anti-Sox2 (#ab97959); Cell Signaling Technology: anti-N-cadherin (# 13116,1: 1000), anti-Vimentin (# 5741, 1:1000), anti-CyclinD1 (# 55506, 1:1000), anti-Oct4 (# 2750, 1:1000), anti-Nanog (#4903, 1:1000), anti-GAPDH (#5174,1:1000) was used overnight. Subsequently, anti-rabbit IgG secondary antibody (Cell Signaling Technology, #7074, 1:1000) was applied to the membranes for 2 h at room temperature. Finally, the gel imaging system (Bio-Rad, USA) was used for exposure.
Glioma stem cell viability assay
The pGBM-1-GSCs and U87-GSCs that were in excellent condition were gathered, digested, resuspended, spread into large dishes, and incubated for 48 h at 37 ℃ in a 5% CO2 incubator. GSCs cells were harvested and organized into a centrifuge tube at 600 rpm for 5 min to remove dead cells. After discarding the supernatant, 2 ml of trypsin was added and resuspended for 5 min. Subsequently, the supernatant was removed by centrifugation again, and 2–3 ml of the medium was added and resuspended. Cells were counted (1 × 103 cells per well), and 1000 cell volumes were calculated. Then 200 ul of medium and the corresponding 1000 cell volumes were added to each well, and the control and experimental groups were repeated for 6 wells each. Finally, 30 ul of the reagent (CellTiter-Glo, Promega, USA) was added, shaken for 5 min with a shaker, and placed in a microplate reader (Thermo Scientific, USA) for activity detection.
GSCs sphere formation assay and extreme limiting dilution assay
The cells in good condition (U87-GSCs, pGBM-1-GSCs) were digested and collected by centrifugation to remove the serum-containing medium. They were then washed twice with PBS. Using an Olympus microscope (Olympus, USA), sphere formation was examined for 10 days while cells were cultivated in ultra-low adhesion cell culture plates (about 1 × 103 cells per well in 6-well plates). The sphere formation rate was then estimated. For extreme limiting dilution assay, GSCs with the specified alterations or treatments were divided into individual cells and plated in 96-well plates at densities of 5, 10, 20, 40, or 100 cells per well for limit dilution tests. Each well's tumor sphere development was evaluated after seven days of incubation. Determining the frequency of stem cells by using the extreme limit dilution analysis (ELDA) (http://bioinf.wehi.edu.au/software/elda/).
All the animal experiments used in this research by the Chinese Ministry of Health were conducted in accordance with the Animal Control Regulations and according to the standards and experimental designs that Nanjing Medical University has authorized. Six-week-old nude mice (Charles River, Beijing, China) and pGBM-1 cell line were used to construct an orthotopic tumorigenesis model in nude mice. Two sets of nude mice were randomly assigned, and lentiviruses carrying the shcNDC80 and sh-CONT sequences were used to transplant pGBM-1 cells. To gauge the size of intracranial tumors, a total of 5 × 105 pGBM-1 cells were stereotactically injected intracranially and regularly put in the imaging system. Nude mice were given an intraperitoneal injection of fluorescein potassium salt at a concentration of 50 mg/ml, given general anesthesia, and then put into an IVIS imaging system (Caliper Life Sciences, USA) for 10–120 s before to each experiment. The fluorescence intensity of the region where the cerebral tumor formed was measured and recorded using Living Images software (Caliper Life Sciences, USA).
RNA immunoprecipitation (RIP) assay
For RNA-bound protein immunoprecipitation analysis, the Magna RIP kit (Millipore, USA) was obtained and used following the manufacturer’s guidelines. After lysing the cells in RIP lysis buffer, the lysates were treated with AGO2 (abcam ab32381) or IgG antibody magnetic beads and spun for an extended period at 4° C. The immunoprecipitated RNA was purified and normalized to the input control before being used to examine the cDNA using qRT–PCR.
CircRNA in vivo precipitation (circRIP) assay
Using GenePharma (Shanghai, China), biotin-labeled circNDC80 probes were created and produced. In a 10-cm petri dish, cells were sown, and they were cultivated for 48 h at 37° C and 5% CO2. After that, cells were transfected for 24 h with either a particular biotin-labeled probe or a control probe (200 nM). The formaldehyde fixation process was then completed by equilibrating the cells with glycine for 5 min after they had been fixed for 10 min with 1% paraformaldehyde fixative. The cells were cleaned three times with precooled PBS, scraped clean with 1 ml of lysate. The samples underwent sonication (50% amplitude, continuous pulse for 30 s, halt for 30 s, and cycle for 10 min), 10000 g centrifugation, and the supernatant was transferred to a 2 mL centrifuge tube while 50uL was preserved as the Input control. The leftover supernatant was combined with magnetic beads that had been streptavidin-labeled by Invitrogen (USA) and let to sit at room temperature for the night. The mixture was then cleaned using lysis buffer containing proteinase K, and the crosslinking reaction was reversed. Enrichment levels were determined by qRT–PCR after total RNA was obtained with the miRNeasy mini kit (Qiagen, Germany).
Dual-luciferase reporter assay
In order to create the luciferase reporter gene, the 3′untranslated region (3′UTR) sequence of circNDC80 or ECE1 containing the predicted and mutated binding sites was inserted into the vector pGL3. These constructs included circNDC80-WT, circNDC80-MUT, ECE1 3′UTR-WT, circNDC80-WT, circNDC80-MUT, and ECE1 3′UTR-WT and ECE1 3′UTR-MUT. After that, Lipofectamine 3000(Invitrogen, USA) was used to co-transfect the luciferase reporter gene with either miR-139-5p or miR-NC into cells. Finally, the dual-luciferase reporter gene assay kit (Vazyme, China) was employed in line with the instructions to measure the luciferase activity.
Immunohistochemistry and Hematoxylin–eosin staining
The mouse brain was fixed with 4% paraformaldehyde (BOSTER, Wuhan, China), embedded in paraffin, and cut into 3.5–4 µm thick sections. Section stained with antibody (N-cadherin, Vimentin and SOX2). Brain tissue slices in paraffin blocks were extracted in xylene and hydrated in alcohol and distilled water before being stained with HE. The samples were then stained with hematoxylin (Sigma, USA) for 5 min after being rinsed three times with PBS for 5 min each. Eosin (USA, Sigma) was used to stain sections for 2 min so that researchers could see how clear the cytoplasm and nuclei were under a microscope. Images were examined and gathered under a microscope after regular dehydration and sealing.
Three times each were done for every experiment. The program GraphPad Prism 9(La Jolla, USA) was used to plot all data analysis, which were all carried out using SPSS 17.0 (SPSS, USA) tool. A two-tailed Student’s t-test was utilized to determine if the distinctions between the two groups were statistically significant. The connection between circNDC80 and miR-139-5p was examined using the Spearman’s test. The Kaplan–Meier method was used to estimate overall survival.