Bioinformatical analyses
Data for bioinformatics analysis were obtained from Chinese Glioma Genome Atlas (CGGA; http://www.cgga.org.cn/) and The Cancer Genome Atlas (TCGA; https://cancergenome.nih.gov/). Using GEPIA (http://gepia.cancer-pku.cn), we examined NRBP1 mRNA expression levels in gliomas with CNS WHO grade, 1p/19q co-deletion status, IDH1 mutation status, CDKN2A/2B deletion, overall survival (OS), and disease-free survival (DFS). The R package "cluster Profiler" was used to conduct GO and KEGG enrichment analysis.
Clinical tissue sample collection
From January 2021 to June 2022, 10 normal tissue samples and 20 low- and 20 high-grade glioma samples were harvested from the First Affiliated Hospital of USTC. The freshly collected samples were immediately frozen at − 80°C. In accordance with the WHO Classification of Central Nervous System Tumours (5th Edition), two pathologists classified 305 surgically-removed paraffin-embedded intracranial glioma tissues collected between October 2019 and July 2022. These samples included 30 pilocytic astrocytomas (CNS WHO grade 1), 34 IDH-mutant astrocytomas (CNS WHO grade 2), 30 IDH-mutant astrocytomas (CNS WHO grade 3), 47 oligodendrogliomas (CNS WHO grade 2), 32 oligodendrogliomas (CNS WHO grade 3), and 132 IDH wild-type glioblastomas (CNS WHO grade 4). Patients’clinical data were collected and summarized. The hospital’s ethics committee granted approval for the experimental procedures of this research (approval number: 2022-RE-298).
Quantitative real-time PCR (qRT-PCR)
Total RNA extraction from human glioma tissue samples was carried out with the TRIzol reagent (Thermo Fisher Scientific, USA). A reverse transcription kit from TaKaRa, Japan was employed to generate cDNA, which was subsequently subjected to qRT-PCR assays for quantification of the relative mRNA levels of NRBP1 based on the 2−△△Ct algorithm. The reaction systems for these qRT-PCR assays were established with the SYBR Green Real-Time PCR Master Mix (Roche, Switzerland), and all the reactions were performed with the 7500 Fast Real-Time PCR System. The following oligonucleotide primers were used in these assays: β-actin, sense primer: 5'-CATGTACGTTGCTATCCAGGC-3' and antisense primer: 5'-CTCCTTAATGTCACGCACGAT-3' and NRBP1, sense primer: 5'-GGACTCATCAAGATTGGCTCTG-3' and antisense primer: 5'-TCTTCTGCTCTTCTCGACAAGT-3'. β-actin served as the internal reference gene.
Immunohistochemistry and staining
Paraffin-embedded intracranial glioma specimens were sectioned at 4-µm thickness. The sections were removed from xylene and immersed in graded alcohol for rehydration. Citric acid buffer (pH 6.0) was used for antigen fixing. The activity of endogenous peroxidase was stopped by washing the slides with phosphate-buffered saline (PBS). Then, the sections were subjected to overnight incubation in an NRBP1 primary antibody (GeneTex, Beijing, China) solution at 1:250 dilution at 4°C, then in a horseradish peroxidase-labeled goat anti-mouse IgG antibody (Beyotime, Beijing, China) for 30 min at ambient temperature. As per the proportion of stain-positive tumor cells, staining extent was calculated as 0% (0), 1–5% (1), 6–25% (2), 26–75% (3), and 76–100% (4). For staining intensity, scores of 0–3 were respectively assigned to negatively, weakly, mediumly, and strongly stained samples. The scores from 0 to 12 were determined with staining extent score×staining intensity score. For the statistical analysis of cytoplasmic expression, low expression was indicated by a score of 0–4 indicated, and high expression by a score of 5–12.
Cell line culturing
We purchased human GBM cell lines A172, SHG44, U87, and U251, and the normal astrocyte cell line HA1800 from the Institute of Biophysics, Chinese Academy of Sciences (Beijing, China). All cells were maintained at 37°C in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, China) added with fetal calf serum (10%) and antibiotics (streptomycin and penicillin at 1%, Amimed, Switzerland) in a moisturized incubator filled with 5% CO2.
Western blotting
Cell lysis was induced by incubating the samples for 30 min in ice-cold radioimmunoprecipitation assay lysis buffer. The bicinchoninic acid assay was conducted for measuring total protein concentrations (Beyotime, Beijing, China). All protein samples underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis before their electrotransfer to a polyvinylidene difluoride (PVDF) membrane. The blots were immersed for 60 min in 5% BSA in Tris-buffered saline with Tween-20 (TBST, pH 7.0) and then subjected to overnight incubation (at 4°C) with primary antibodies. The primary antibodies included those raised agaist NRBP1 (1:1000) from GeneTex, China. Bcl-2, Bax, PI3K, p-PI3K, Akt, and p-Akt (1:1000) from Thermo Fisher Scientific, USA, and N-cadherin, E-cadherin, MMP-7(1:1000) and β-actin (1:5000) from Cell Signaling Technology, USA. The blots were visualized by HRP-labeled goat anti-mouse or goat anti-rabbit IgG (1:5000) from CoWin Biosciences, China, with enhanced chemiluminescence kits from Millipore, USA. β-actin bands served as the loading control.
Lentiviral infection
We purchased the lentivirus plasmid from Beyotime Biotechnology (Shanghai, China). For lentiviral infection, 1×106 U87 cells were grown in the presence of 4 µL lentivirus with NRBP1 for 12 h, and lentivirus without NRBP1 was the negative control. After culturing for 48 h at 37°C, puromycin (4 µg/mL) was utilized to sift the cells that were successfully transferred into the plasmid. We constructed three short hairpin RNAs (shRNAs) targeting NRBP1 mRNA, with the following sequences: shNRBP1-1, GACCTTGAACAAGTTCAATTT; shNRBP1-2, GCAATGGAGAGTCCTCATATG; and shNRBP1-3, CCAACACATGATCCCAGAGAA. Western blotting was carried out to determine the transfection rate. For subsequent experiments, the two groups of cells with superior transfection efficiency were chosen. NRBP1-overexpressing (pcDNA-NRBP1) and empty plasmids were bought from GenePharma (Shanghai, China) and transfected into cells that maintained in Opti-MEM by utilizing Lipofectamine 2000. 48-h after transfection, the cells were harvested for transfection efficiency determination.
Cell proliferation assay and colony formation
Cell counting kit-8 (CCK-8) assays were implemented to appraise cell proliferation ability. Briefly, logarithmically growing cells were inoculated in 96-well plates at a density of 3000 cells per well and grown for 0, 24, 48, 72 or 96 h. In each well, the CCK-8 reagent (Dojindo Laboratories, Kyushu Island, Japan) was supplemented. After incubation for 4 h. Optical density at 450 nm was recorded with a Synergy H1 microplate reader (BioTek, USA).
Five hundred cells were maintained in one well of a 6-well plate, with the medium refreshed once on the fourth day of culture for two weeks. Cloned cells were fixed in 4% paraformaldehyde for 15 min, dried, and subjected to crystal violet staining. Six randomly picked fields were observed under an Olympus optical microscope for colony number counting (colonies with ≥ 50 cells were considered a single clone).
Wound healing assay
2×106 cells were allowed to proliferate for 12 h in each well of a 6well cell culture plate to achieve 90–100% confluence. Afterwards, scratch wounds were generated with a sterilized pipette tip. After two PBS washes, the cells were maintained in a culture medium without serum for evaluating wound closure. A phasecontrast microscope (×100) was utilized to visualize the cultured cells immediately and 24 after the wounds were created to assess the percentage of would healing that was determined as [(Ai − At)/Ai] × 100, in which Ai is the wound area determined immediately while At denotes the wound area after cell culture.
Analysis of migratory and invasive abilities of glioma cells
The migratory and invasive abilities of glioma cells were appraised through Transwell assays, for which the Matrigel was applied in the upper chamber only in the cell invasion analysis. Cells that had undergone starvation treatment were inoculated into an even distribution in the upper chamber containing culture medium without serum. Then the lower chamber was added with cell culture medium plus 10% FBS, which served as a chemoattractant. The Transwell chambers were then incubated for 24 h at 37°C, after which cells in the lower chamber were subjected to fixtion and crystal violet (1%) staining. Five fields of view were used for cell counting.
Cell apoptosis assay
A TUNEL kit from Beyotime, China was used to assess DNA fragmentation, which is a hallmark of apoptosis, in accordance with the provider’s protocol. 2×106 cells were placed onto a glass slide in each well of a 6-well plate and grown for 4 days. The cells were then mounted in a solution that prevents quenching of fluorescence and observed with an Olympus fluorescence microscope. We randomly picked five high-power fields (×200 magnification) to determine the numbers of TUNEL- and Hoechst-positive cells, the proportion of which reflects the amount of apoptotic cells.
AKT phosphorylation experiment
NRBP1-overexpressing U251 cells (pcDNA-NRBP1) were incubated with 10 µM MK-2206 (Selleck, Houston, USA), and U87 cells with NRBP1 knockdown (sh-NRBP1) were incubated with 10 µg/mL SC79 (Calbio, La Jolla, CA). U251 and U251 (pcDNA-NRBP1) cells without MK-2206 incubation served as controls, whereas U87 and U87 (sh-NRBP1) cells that were not incubated with SC79 were used as controls. Next, CCK-8, Transwell assay, and western blotting were conducted to determine cell proliferation, invasion, and EMT in GBM.
Statistical analysis
We employed GraphPad Prism 8.0 (GraphPad Software, USA) for analysis of statistical data. The data from three assays performed in triplicate are displayed as mean ± SEM. The Kaplan–Meier method was employed for plotting DFS and OS curves, and intergroup differences were analyzed by the log-rank test. One-way ANOVA or two-tailed Student’s t-test was carried out to decide whether differences were statistically significant. Values with ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 indicate statistical significance.