Transcriptome sequencing and data analysis
Transcriptome sequencing was performed by YiKe Population Health Research Institute, Nanjing, China. Briefly, total RNA was extracted from the specified tissues using Trizol reagent (Thermo Fisher, MA, USA), and its quality was assessed using by a NanoPhotometer (IMPLEN, CA) and quantified using a Qubit 3.0 Fluorometer C (Thermo Fisher). Poly(A)-tailed mRNA was captured and transcribed to double-strand cDNA, followed by sequencing on a HiSeq3000 system (Illumina, San Diego, CA) after library construction. Raw reads were acquired by CASAVA v1.8 and stored in FASTQ format. The human reference genome (GRCh37) was used to align the aforementioned reads. Statistical results in terms of “Read counts” were output through Htseq v 0.7.2 and corresponding “FPKM” were calculated. Differentially expressed mRNAs were selected according to the criteria of p < 0.05 and |log fold change (FC)| ≥ 2. Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed based on the sequencing data from the indicated groups. The sequencing and data analysis were performed on six samples, including three duplicates each, for the sensitive (S) and resistant (R) tissues with various constructs.
Immunofluorescence (IF)
For IF staining of tissue arrays, an IHC antibody complex containing E2F7 antibodies and an Opal color fluorescent IHC kit (PerkinElmer, USA) were used, as previously described in other studies [17, 18]. The tissues were incubated with primary antibodies, followed by secondary-HRP (Cell signaling, MA, USA), and Opal working solution (PerkinElmer). The slides were mounted with ProLong Gold Antifade Reagent containing DAPI. The slides were scanned using the Vectra® Polaris™ Imaging System (Akoya Biosciences, Boston, USA), and the acquired images were analyzed using Image J software (National Institutes of Health, NIH, USA). The results were evaluated independently by two blinded pathologists.
Cell culture and reagents
The human GBM cell line U87 was purchased from the research institute (Chinese Academy of Sciences, Beijing, China) and was authenticated using short tandem repeat assays by the company GENEWIZ (Suzhou, China). All cells were cultured in DMEM (Hyclon, Los Angeles, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) and 1% penicillin-streptomycin (P/S; Hyclon) in 5% CO2 at 37°C. The S and R cells refer to primary cells derived from indicated tumor tissues previously established in our laboratory [8]. TMZ and doxorubicin (Dox) were acquired from Selleck Chemicals (Houston, TX).
Vector construction and transduction
The full-length cDNA encoding human E2F7 was amplified by RT-qPCR and verified by DNA sequencing. The E2F7-Flag lentivirus was constructed by inserting the cDNA sequence into the lentivirus vector GV492 (Genechem, Shanghai, China) with a Flag-tag. The p53-HA lentivirus was constructed by inserting the cDNA sequence into the lentivirus vector GV348 (Genechem) with an HA-tag. The small interfering RNAs (siRNA) targeting human E2F7 were purchased from Gene Pharma (Suzhou, China). E2F7 siRNA transfection was performed using Lipofectamine3000 (Thermo Fisher) according to the manufacturer's instructions, and cells were collected for the following assays after 48 hours of transfection. The CRISPR/Cas9 system (Genechem) was used to establish p53 KO (knock out) GBM cell lines. Briefly, cells were infected with Lenti-Cas9 lentivirus and screened by puromycin (Sigma-Aldrich, St Louis, MO). Single guide RNAs (sgRNAs) targeting human p53 gene were designed and cloned into the GV371 plasmid. These sgRNAs were delivered into GBM cell lines stably expressing Cas9. The siRNAs and sgRNA were used as follows: E2F7 siRNA1: CAGTC TCCTGCAGGATTAAA, E2F7 siRNA2: TGCTGCCAGCCCAGATATAA, E2F7 negative control siRNA: UUCUCCGAACGUGUCACGUTT; TP53 sgRNA: CAGATGACATAGAGCAGTGG, TP53 negative control sgRNA: CGGACGATAT TGAACAATCG.
Cell Growth and Colony Formation Assay
For cell growth assays, a total of 1000 cells were seeded into 96-well plates and monitored by Cell Counting Kit-8 (CCK8; Vazyme, Nanjing, China) at the designated time points according to the manufacturer's protocol, with TMZ treatment. For the colony formation assays, 1000 cells were seeded into 6-well plates and maintained in a complete medium for 14 days. The resulting colonies were fixed with 4% paraformaldehyde (PFA) (Sangon, Shanghai, China) and stained with 0.1% crystal violet (Sangon). The number of colonies was counted using an inverted microscope and analyzed by Image J software. The TMZ concentration used for both the cell proliferation and clone formation experiments was 200 µM.
EdU incorporation Assay
Cell proliferation and DNA replication were analyzed using the EdU incorporation method as previously described [19]. Briefly, cells were cultured in a 24-well plate for 24 hours, followed by incubation with 10 µM EdU (KeyGen Biotech, China) for 2 hours. Afterward, the cells were fixed with 4% PFA. The staining procedure was performed following the manufacturer's instructions (Invitrogen). After staining, coverslips were mounted with a gel mount containing Hoechst 33342 (5 µg/mL). The quantitative results were expressed as the percentage of EdU-positive cells relative to the total number of cells/nuclei.
Cell cycle assay
Cells were cultured for 24 hours and then synchronized through serum deprivation for another 24 hours. Following this, the cells were cultured in a complete medium for another 24 hours. They were then harvested and fixed using 70% ethanol at -20°C for 24 hours. The fixed cells were suspended in PBS containing 100 ng/ml of RNaseA (Boehringer Mannheim, Indianapolis, IN) and 50 µg/ml of propidium iodide (PI; Sigma-Aldrich). The cell suspension was incubated for 0.5 hours at room temperature in the dark. The cell cycle fractions were measured by FACS Canto II (BD, USA). The percentages of cells in the G1, S, and G2 phases were calculated using ModFit LT software (VSH, USA).
Cell Apoptosis Analysis
Cell apoptosis was detected by flow cytometry using the Annexin V-FITC Apoptosis Detection Kit (KeyGen Biotech, China), which includes AlexFluor647 labeled Annexin V according to the manufacturer's instructions. Briefly, cells were treated with Dox (2 µM) for 48 hours. Then the cells were harvested and rinsed with cold PBS and resuspended in 200 µl binding buffer. 5 µl AV-AlexFluor647 stock solution and 10 µl PI (Sigma-Aldrich) were added. The mixture was incubated for 15 mins at room temperature in the dark. Cells were further rinsed with 400 µl cold PBS and immediately analyzed by a FACScan flow cytometry (BD, FACSCanto II, CA, USA).
Cell growth inhibition assay
Cytotoxicity studies were performed in 96-well plates, and optimal seeding densities for each cell line were determined to ensure exponential growth during the assay. Cells were cultured with a medium containing a gradient concentration of drugs for 48 hours. Cell viability was determined by CCK8, and the optical density (OD) at 450 nM was measured using a microplate reader (Thermo Fisher). The percentage of cell survival at each concentration was calculated using the formula: (OD treated/OD untreated) ×100. The IC50 value represented the drug concentration that reduced cell growth by 50%.
Cellular uptake assay
Cellular uptake profiles were performed following previously established protocols [8]. Briefly, cells were seeded into 6-well plates and incubated for 24 hours. Subsequently, cells were treated with or without Dox (5 µM) for 3 hours. Afterward, cells were harvested and washed with cold PBS. Cells that were not exposed to Dox were used as background controls. Dox uptake was detected by a flow cytometer and analyzed with FlowJo VX software (RRID:SCR_008520, FlowJo, LCC, OR, USA). The relative mean fluorescence intensity (MFI) was determined by calculating the ratio of the MFI of Dox-positive cells to that of blank control.
RNA extraction and quantitative RT-qPCR analysis
Total RNA was extracted using TRIzol reagent (Thermo Fisher) following the manufacturer's instructions. cDNA was synthesized using the M-MLV Reverse Transcriptase Kit (Cwbio, Beijing, China). RT-qPCR analyses were conducted to quantify mRNA expression levels using Real SYBR Mixture (Cwbio) on a Lightcycler 480 II instrument (Roche Applied Science, USA). GAPDH severed as an internal control. The specific oligonucleotide primer sequences are listed in Supplementary Table 1.
Western blot analysis
Cells were lysed using RIPA lysis buffer (Cell Signal Technology, Beverly, MA), and the protein concentration was measured by bicinchoninic acid (BCA) assay (Cwbio). For E2F7 analysis, p53 OE and p53 KO cells were treated with or without Dox at concentrations of 0.5-1 µM for 24 hours. Cell lysates were subjected to standard Western blot assay with the specific antibodies to detect the target proteins. The primary antibodies used were anti-E2F7 (1:1000; cat# NBP1-80266; NOVUS, USA), anti-p53 (1:2000; cat# MAB11254; Abnova, China), anti-phospho-p53 (Ser46) (1:2000; cat# 2521S; CST, USA) and GAPDH (1:5000; cat# 60004-1-Ig; Proteintech, China). Finally, the blots were visualized using the ECL substrate solution (Vazyme). Results were quantified using Image J software.
Luciferase assay
To evaluate p53 transcriptional activity, the luciferase assay was conducted using the Dual-Luciferase Reporter Assay Kit (Promega, Madison, WI) according to the manufacturer's protocol. Briefly, S and R cells were seeded in 24-well plates and transfected with pGL4.38[luc2P/p53 RE/Hygro] (p53 RE-pGL4.38, Promega, Madison, WI) to evaluate p53 transcriptional activity. After 24 hours of transfection, the cells were lysed, and the firefly and renilla luciferase activities were measured. The pRL-TKRenilla luciferase plasmid (Promega) served as a DNA transfection control through all experiments.
ChIP and ChIP RT-qPCR Assays
The ChIP assay was performed using the Hyperactive pG-MNase CUT&RUN Assay Kit followed by RT-qPCR (Vazyme) according to the manufacturer's instructions. Briefly, 2×107 cells and anti-p53 antibody (5 µg; cat# ab1101; Abcam, UK) were used for each ChIP. The mouse IgG was applied as negative control and spike-in DNA was used for homogenization correction. Through a protein G-fused MNase nuclease, the target protein was precisely targeted with antibody guidance, and the DNA was subsequently fragmented near the target site. After reversing cross-links and purifying the DNA, the samples were used for quantitative RT-qPCR with specific primers targeting the promoters (Supplementary Table 1).
Animal experiments
Four-week-old male nude mice were obtained from the Changzhou Cavens Experimental Animal Co., Ltd (Jiangsu, China) and held under specific pathogen-free conditions, and all mice were assigned randomly. All methods were carried out in accordance with relevant guidelines and regulations. For the intracranial tumor model, cells expressing the firefly luciferase gene were infected with lentivirus over-expressing E2F7 or control lentivirus. A total of 2.5×105 luciferase-labeled cells over-expressing E2F7 or control cellswere injected into the right hemisphere of individual nude mice brains. After 14 days of tumor growth, the mice were randomly divided into two groups. The tumor-bearing mice were treated with TMZ (20 mg/kg) or vehicle every 2 days starting from the 14th day post-implantation for a total of 7 doses. Tumor progression was monitored by in vivo bioluminescence imaging and quantified by Living Image J Software. At the end of the experiments, the animals were deeply anesthetized and euthanized with an intraperitoneal injection of pentobarbital. All animal care and handling procedures were performed in accordance with the National Institutes of Health's Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Review Board of Nanjing Medical University. All animal experiments were performed by two technicians blinded to the treatment condition of the mice. No mice were excluded from the analysis.
Statistical analysis
All experiments were independently repeated at least three times, and the data are expressed as mean ± standard deviation (SD). The difference between 2 independent samples or among multiple groups was determined by the student t-test or one-way ANOVA followed by a Student-Newman-Keuls multiple comparison test (SNK test) respectively. Survival analyses were performed using the Kaplan-Meier method with the log-rank test. The correlation of gene expression was evaluated using Spearman’s rank correlation analysis. Statistical significance was set at p < 0.05. SPSS 20.0 package (IBM, USA) and GraphPad Prism 8.0 software (GraphPad, USA) were used for all statistical analyses and data graphing respectively.