Cells, plasmids, and siRNAs
Human cell lines were purchased from ATCC and FuHeng, and maintained with 1% penicillin and streptomycin (Gibco Life Technologies) at 37 °C with 5% CO2. DAOY cells (ATCC Cat# HTB-186, RRID:CVCL 1155) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco), 1 × glutamine (Gibco), and 1 mM sodium pyruvate (Gibco). D283-Med cells (ATCC Cat# HTB-185, RRID:CVCL_1155) were cultured in MEM medium supplemented with 10% FBS. Full-length human PRKAA1 cDNA was generated by PCR and cloned into the pRK5 vector (RRID:Addgene_32693). siRNAs specific for the human PRKAA1, GLI1 and CCL5 were purchased from GenePharma (Shanghai, China).
Medulloblastoma tissues obtained from Children’s hospital of Nanjing Medical University (Nanjing, China) were carried out to detect the phosphorylation of AMPKα by immunohistochemistry. Mouse heterograft tumor tissues were detected Ki67 to measure the growth of heterograft tumors. And standard procedures for immunohistochemical staining were followed with rabbit two-step detection kit (ZSGB-BIO). The primary antibodies were rabbit anti-phospho-AMPKα (Cell Signaling Technology; 1:100), rabbit anti-Ki67 (Abcam; 1:100).
Transfected cells or grinded tumor tissues were lysed in modified RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% vol/vol NP-40, 1% n-Dodecyl β-D-maltoside, 0.25% wt/vol sodium deoxycholate, 1 mM DTT, and 1 × Roche complete Protease Inhibitor Cocktail) for 1 h at 4 ℃. The lysate was clarified by centrifugation for 20 min at 14,000 × g. The protein concentration was determined using a bicinchoninic acid assay and equal amounts of total protein from each of the samples was supplemented with 5 × SDS loading buffer, incubated at 95 ℃ for 5 min, subjected to SDS-PAGE, followed by western blot analysis. The following antibodies were used: rabbit anti-β-actin (Affinity; 1:5000), rabbit anti- AMPKα (Cell Signaling Technology; 1:1000), rabbit anti-Phospho-AMPKα (Cell Signaling Technology; 1:1000), mouse anti-E-cadherin (Cell Signaling Technology; 1:1000), rabbit anti-N-cadherin (Elabscience; 1:1000), rabbit anti-MMP3 (Proteintech; 1:1000), rabbit anti-GLI1 (Cell Signaling Technology; 1:1000), rabbit anti-GLI2 (NOVAS; 1:1000), goat anti-GLI3 (R&D; 1:1000), mouse anti-IκBα (Cell Signaling Technology; 1:1000), rabbit anti-NF-κB p65 (Cell Signaling Technology; 1:1000) and rabbit anti-Phospho-NF-κB p65 (Cell Signaling Technology; 1:1000).
Cell Count Kit-8 (CCK-8) assays
Treated DAOY and D283-Med cells were separately seeded in 96-well plates at a density of 0.4 × 104 cells/well and 1 × 104 cells/well. After adhesion, the cell viability was obtained by the CCK-8 kit for 72 h. The following formulae were used for calculations:
Cell viability (%) = ([OD (experiment) – OD (blank)])/ ([OD (control) – OD (blank)]) × 100%
EdU incorporation assays
Transfected cells were seeded at 5 × 104 cells/well in 24-well plates containing round coverslips and maintained in medium overnight. Cell proliferation was further evaluated through measuring the incorporation of EdU with EdU Cell Proliferation Assay Kit (RiboBio). Images were captured by using a fluorescence microscope, and the proliferating cells in five different fields were counted.
Colony formation assays
For the colony formation assay, transfected cells were plated in triplicate at 200 cells per well in 6-well plates and cultured for 2 weeks. The cells were fixed with 4% paraformaldehyde for 15 min at 4 ℃, and then stained with 0.1% crystal violet for 30 min, colonies with more than 50 cells were counted.
Flow cytometric analysis for cell cycle
Cells were inoculated in 6-well plates and transfected with siRNA for 48 h, then these cells were collected and fixed with pre-cooled 75% ethanol. Then cell cycle distribution was determined by the flow cytometer (BD Biosciences).
Tumorsphere formation assays were measured by in vitro limiting dilution assay, as previously reported [31, 32]. Briefly, decreasing numbers of cells per well (50, 20, 10, 5 and 1) were plated into Ultra-Low Attachment 96-wells plates (Costar®) in Neural basal medium (Gibco) supplemented with 100 × penicillin and streptomycin (Gibco Life Technologies), 100 × Glutamine (Gibco), 100 × D-( +)-Glucose solution (Sigma), 100 × Insulin-Transferrin-Selenium (Gibco), 50 × B27 Supplement (Gibco), N-Acetyl Cysteine (16ug/ml), EGF (20 ng/ml) and FGF (20 ng/ml). Spheres with a diameter equal or higher than 40 μm were deemed tumourspheres. The presence and number of tumorspheres in each well were recorded seven days after plating. Extreme limiting dilution analysis was performed using the software available at [31, 33].
CRISPR-Cas9 genome editing
Genome editing was achieved using the CRISPR-Cas9 technique in DAOYs. Briefly, a single guide RNA (sgRNA) targeted the eighth and tenth exon of human PRKAA1 was designed and cloned into pX330 vector (RRID: Addgene_101733). The sgRNA sequences are:
5′- CACCGAACTATATGATGGATCCTC -3′;
5′- CACCGCAACTATCGATCTTGCCAA -3′.
Cells were co-transfected with CRISPR/Cas9 plasmids and empty plasmids containing puromycin resistance genes. 48 h after transfection, transfected cells were selected using Puromycin (3 μg/ml) for 7 days, followed by another 4 days without selection for expansion. Then the survived DAOYs were seeded at 1 cell per well in 96-well plates for single cell colonies formation and screened by genotyping and Western Blot.
In vivo xenograft tumor model
6 weeks female BALB/c nude mice were purchased from Nanjing Medical University Experimental Animal Center. Nude mice were subcutaneously inoculated with 5 × 106 PRKAA1+/+ or PRKAA1−/− DAOY cells in double side axillae. The tumor sizes were measured every week. The tumor volume was calculated as follows: volume (mm3) = length (mm) × width (mm)2/2. 8 weeks after xenograft inoculation, mice were euthanized and their tumor nodules were excised, photographed, weighed and harvested.
Intracranial brain tumor xenografts
NOD-SCID mice were anesthetized, and an incision was made to expose the skull. A hole was created in the calvarium above the right cerebellar hemisphere, where 2 mm lateral (right) to the sagittal suture, and 2 mm posterior of the lambdoid suture, using a microdrill. Orthotopic MB xenografts were performed by injection of PRKAA1+/+ DAOY-Luc and PRKAA1−/− DAOY-Luc cells severally, that had been previously infected with lentiviral particles to stably express luciferase gene, into the cerebellum of five NOD-SCID mice. Bioluminescence imaging was performed weekly using IVIS Spectrum (Perkin Elmer). Tumor growth was evaluated by quantifying the bioluminescence signals using the integrated fluxes of photons within each area of interest using the Living Images Software Package 4.5 (Perkin Elmer). All mice were euthanized when the mice showed symptoms of metastasis.
Movement of living cells
Cells were seeded in 60-mm plate at 30% density for 12 h and then treated with 2 μg/ml mitomycin C for 24 h. After, cells were cultured with fresh culture medium and recorded at one image every 5 min with Celldiscoverer 7 automatic live cell imaging system (ZEISS) for 20 h. The cells motion trajectory image and data were obtained using ImageJ (RRID:SCR_003070).
Transfected cells were seeded on glass coverslips for 24 h and fixed with 4% PFA for 20 min at 4℃, then the cells were permeabilized with 0.5% TritonX-100 for 10 min at room temperature. Fluorescently labeled phalloidin working solution was added a well and incubated at room temperature for 30 min for staining and stained with DAPI. The cell cytoskeleton images were acquired with laser scanning confocal microscope.
In vitro migration and invasion assays
The 24-well plate with 8 µm pore polycarbonate membrane inserts (Millipore) was used to analyze the migratory abilities of tumor cells and then the membrane was coated with 60μL diluted Matrigel (1:30; Corning) to detect cells’ invasion. After adding 600μL 10% FBS medium into the lower chambers, 2.5 × 104 transfected cells in 300μL serum-free medium were seeded into the insert for incubation at 37 °C in 5% (v/v) CO2 incubator for 16 h. Then, the cells migrating to the lower surface of the membrane insert were stained with the crystal violet (Beyotime) and quantified by counting five randomly chosen microscopic fields.
Total RNA was isolated with RNAiso Plus reagent (TaKaRa) from DAOY cells transfected with siNC or siPRKAA1 for 48 h, then purified to meet the following requirements were used in subsequent experiments: RNA integrity number (RIN) > 7.0 and a 28S:18S ratio > 1.8. The triplicate samples of both assays were constructed an independent library, and sequenced on an Illumina Novaseq 6000 sequencer by CapitalBio Technology (Beijing, China). The sequencing raw data was uploaded to the GEO dataset (GSE218076).
Reverse transcription (RT) and real-time PCR
Total RNAs were isolated from cultured cells and reverse transcribed using HiScript II Q RT SuperMix (Vazyme). Quantitative real-time PCR (qPCR) was carried out using AceQ qPCR SYBR Green Master Mix (Vazyme). Each measurement was repeated three times, and each sample was analyzed in triplicate with hypoxanthine phosphoribosyl transferase (HPRT) as an internal control. The qPCR primers are listed:
Human PRKAA1: Forward: 5′- CAACTATCGATCTTGCCAAAGG -3′
Reverse: 5′- AACAGGAGAAGAGTCAAGTGAG -3′
Human CCL3: Forward: 5′- AGGACACGGGCAGCAGACAG -3′
Reverse: 5′- GGACAGCAAGGGCAGCAGTG -3′
Human CCL5: Forward: 5′- CAGCAGTCGTCCACAGGTCAAG -3′
Reverse: 5′- TTTCTTCTCTGGGTTGGCACACAC -3′
Human CXCL10: Forward: 5′- CTCTCTCTAGAACTGTACGCTG -3′
Reverse: 5′- ATTCAGACATCTCTTCTCACCC -3′
Human VCAM1: Forward: 5′- CAGGCTGGAGATAGACTTACTG -3′
Reverse: 5′- CCTCAATGACAGGAGTAAAGGT -3′
Combination index with simultaneous treatment of Vismodegib and TPCA-1
To understand the Combination Index (CI), cells were treated with Vismodegib (GDC-0449) alone, or TPCA-1 alone, or a combination of both in the following concentrations (0, 1/4 IC50, 1/2 IC50, IC50, 2 IC50, and 4 IC50). To test for synergy, and then the CCK8 data was validated using CompuSyn software based on Chou-Talalay quantitative method. The dose–effect curve was simulated and CI was calculated at a constant ratio of 1:2.
Analysis of published datasets
Human medulloblastoma expression dataset from GSE85217 and GSE124814 was used. Correlations between different gene were determined by Spearman correlation analysis. Overall survival curves were analyzed with Kaplan–Meier plotter (https://hgserver1.amc.nl/cgi-bin/r2/main.cgi). The best cutoff value was auto selected in the analysis. The hazard ratio with 95% confidence interval and log rank P value were calculate, and significance was set at P < 0.05.
Statistical analyses were performed with GraphPad Prism 8.0 (RRID:SCR_002798). Each measurement was repeated at least three times. Comparisons between indicated groups were performed using independent-samples t-test. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, and ***P < 0.001. n.s. not significant.