Data collection and tumor samples
The Chinese Glioma Genome Atlas (CGGA) RNA sequencing (RNA-seq) dataset (mRNAseq_325) with 325 glioma samples (103 WHO II gliomas, 79 WHO III gliomas, 139 WHO IV gliomas and 4 samples N/A of unknown nature) and corresponding PLCβ1 distribution and survival information, were downloaded from CGGA public database (http://www.cgga.org.cn/). The patients of validation set were divided into high- PLCβ1 and low-PLCβ1 expression group according to the cut-off value of PLCβ1 expression from CGGA dataset. Fifty glioblastoma tumor samples were obtained from the IRCCS Istituto delle Scienze Neurologiche di Bologna, Italy. This study was approved by the AUSL Bologna Ethical Committee (Comitato Etico di Area Vasta Emilia Centro della Regione Emilia-Romagna (CE-AVEC) N° 183/2019/OSS/AUSLBO evaluated on 20/03/2019) and informed consents were obtained from all participants.
DNA mutation and methylation analysis
DNA from fresh/frozen tissues was extracted using the MasterPure™ DNA purification kit. Mutational analysis was performed using a next generation sequencing protocol evaluating the following 4 genes as indicated from WHO and cIMPACT-NOW guidelines : isocitrate dehydrogenase 1 (IDH1) (exon 4), isocitrate dehydrogenase 2 (IDH2) (exon 4), histone H3-3A (exon 1), Telomerase Reverse Transcriptase (TERT) (promoter). Locus-specific amplicon libraries with tagged primers were generated using overhang adapters at 5′ based on Nextera™ sequence at the 5′ for Illumina sequencing; these adapters were recognized by a second round of PCR (eight cycles) to add Illumina P5/P7 adapters and sample-specific indices . Amplicon products were purified with agencourt Ampure XP beads, quantified with the fluorometer Quantus™, pooled and loaded on MiSeq (Illumina). Each next generation sequencings (NGS) experiment was designed to allocate ≥ 1 k reads/region, to obtain a depth of coverage ≥ 1000×. FASTQ files were processed in a Galaxy Project environment , using hg38 human reference genome with BWA (Burrows-wheeler aligner), and visualized on IGV (Integrative genomics viewer). Only mutations with a variant allele frequency (VAF) threshold > 5% and a coverage depth of 10 × in both strands were reported. All mutations were checked in COSMIC database.
DNA methylation: bisulfite treatment of genomic DNA (50–500 ng) was performed using the EZ DNA methylation-lightning kit (Zymo Research Europe, Freiberg, Germany) according to the manufacturer’s protocol. DNA methylation was evaluated using targeted bisulfite NGS for O-6-Methylguanine-DNA Methyltransferase (MGMT). In brief, genomic sequences stored in the Ensembl genome browser (http://www.ensembl.org/index.html) were employed as query sequences to identify putative CpG islands in the promoter and the enhancer regions. MethPrimer (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) designing was applied to identify CpGs and the best primers of choice. Libraries were generated using the same approach for mutation analysis with two PCR steps. FASTQ files were processed in a Galaxy Project environment following a pipeline described previously , in brief after filtering for quality > Q30 and for read lengths (> 80 bp), FASTQ files were then mapped by BWAmeth. BAM files were then in turn processed by MethylDackel using human GRCh38/hg38 as reference genome. The output files assigned the exact methylation level for each investigated CpG position . In parallel, the web tool EPICT-TABSAT was used to confirm DNA methylation level results .
Cell culture and lentiviral transduction
Human glioblastoma astrocytic cell lines U87-MG (HTB-14 ATCC, Old Town Manassas, Virginia, US) and U-251 MG (09063001 Sigma-Aldrich, St Louis, MO, US) were cultured in minimum essential medium eagle (MEM) (Corning, New York, US) with 10% FBS and 1% Penicillin/Streptomycin (Sigma-Aldrich) and Dulbecco’s Modification of Eagle’s Medium (DMEM) (Corning) with 5% FBS and 1% Penicillin/Streptomycin, respectively. Human embryonic kidney HEK 293 T cells (Genecopoeia Inc, US) were cultured in DMEM (Corning) with 10% FBS and 1% Penicillin/Streptomycin. Human Astrocytes HA, isolated from cerebral cortex (Catalog #1800, ScienCell Research Laboratories, California, US), were cultured in Astrocyte Medium (AM, Cat. #1801, ScienCell). All cells were maintained in a humidified incubator at 37 °C with 5% CO2.
HSH096803-LVRH1GP-b, HSH096803-LVRH1GP-c and CSHCTR001-LVRH1GP control vectors (Genecopoeia) were used to construct lentiviruses to silence PLCβ1 and express green fluorescent protein (GFP) as well as lentiviruses coding only for GFP, as control. The Lenti-Pac HIV expression packaging kit (Genecopoeia) was used as packaging system to transfect HEK293T cells according to manufacturer’s protocol. The supernatants containing the viruses were harvested 24–48 h after transfection and filtered through a 0.45 µm cellulose acetate filter. To perform viral transduction, U87-MG and U-251 MG were plated in a 6-well plate at a concentration of 5 × 105 cells/well. Primary HA Astrocytes were seeded at a concentration of 5 × 104/cm2 in 6-well and 12-well plates. The next day virus supernatants together with polybrene 8 µg/ml for U87-MG and 6.4 µg/ml for U-251 MG and HA, were added to the target cells.
U87-MG and U-251 MG cells were treated with 2 µg/ml and 1,5 µg/ml of puromycin (Sigma-Aldrich), respectively, 48 h after transduction and were left in culture for one month to obtain a fully selected clone with stable PLCβ1 silencing. Instead, HA primary astrocytes were treated with 1 µg/ml of puromycin 24 h after transduction and analysed after 96 h.
RNA extraction, reverse transcription, and real-time PCR
RNeasy lipid tissue mini kit (Qiagen, Hilden, Germany) was used to extract total RNA from fresh-frozen clinical tissues following manufacturer’s protocol. Nanodrop spectrophotometer was used to quantify the extracted total RNA. Subsequently, Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and Qubit RNA IQ Assay, were used for quality analysis of the RNA extracted. Only samples with Qubit IQ number of seven or greater, were selected for further experiments. Total RNA extracted from different brain lobes of healthy individuals (Biochain, Newark, CA: R1234062-P, R1234078-P, R1234051-P, R1234066-P) was used as control. In particular, a total of four different pools were used, each containing total RNA from five different healthy donors. RNeasy Mini Kit (Qiagen) was used to extract total RNA from cell lines. Nanodrop spectrophotometer was used to quantify extracted RNA. Using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), 1 µg of total RNA was reverse transcribed into cDNA following the manufacturer’s protocol. Real-time PCR was performed on 10 ng of cDNA per reaction, with QuantStudio1 Real-time PCR system (Thermo Fisher Scientifc) using TaqMan Universal Master Mix II (Thermo Fisher Scientific) and TaqMan probes. GAPDH and 18S were used as housekeeping genes for cell lines and tissue samples, respectively. Data were presented as fold changes relative to the expression levels of control samples in accordance with the 2−ΔΔCt formula. Validated gene probes used are: 18S Hs99999901_s1, GAPDH Hs99999905_m1, PLCβ1 Hs01001930_m1 (Thermo Fisher Scientific).
Protein extraction and Western blot
Cells were lysed with T-PER lysis buffer supplemented with Halt protease and phosphatase inhibitor cocktails (Thermo Fisher Scientifc). Lysed cells were sonicated for 15 s using 40–50% of power. Whole cell lysates were quantified with the Pierce™ Coomassie Plus (Bradford) Assay Reagent (Thermo Fisher Scientific) and 30 µg of total proteins were separated on bolt 4–12% polyacrylamide-0.1% commercial SDS gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membrane. Membranes were washed with PBS-0.1% Tween-20 (PBST) and non-specific binding sites were blocked with blocking buffer (5% w/v non-fat dry milk in PBST) for 1 h at room temperature. Lastly, membranes were incubated with primary antibodies overnight at 4 °C. The antibodies used were diluted in bovine serum albumin (BSA) (Sigma-Aldrich) or milk following manufacturer’s protocols. Membranes were washed again with PBST, then incubated with peroxidase conjugated secondary antibodies (Thermo Fisher Scientifc) diluted 1:1500 (for anti-Mouse) and 1:10’000 (for anti-Rabbit) in PBST for 1 h at room temperature. Westar Antares and Westar Supernova (Cyanagen, Bologna, Italy) were used as chemiluminescence reagents to detect immunoreactive bands. Images were captured with the iBright Imaging System (Thermo Fisher Scientific) and samples were analysed by total protein normalization through the iBright analysis software. The following antibodies were used in Western blotting: anti-PLCβ1 (PA5-78430, Thermo Fisher Scientific), anti-Slug (sc-166476, SantaCruz Biotechnology, Dallas, Texas, US), anti-N-Cadherin (33–3900, Thermo Fisher Scientific), anti-MMP-2 (CST40994, Cell Signaling Technology, Danvers, MA, US), anti-MMP-9 (MA5-32705, Thermo Fisher Scientific), anti- β-Catenin (CST9587, Cell Signaling Technology), anti-Non-phospho (Active) β-Catenin (CST8814, Cell Signaling Technology), anti-C-myc/N-myc (CST13987, Cell Signaling Technology), anti-PPARγ (CST2443, Cell Signaling Technology), anti-phospho-Stat3 Ser727 (CST9134, Cell Signaling Technology), anti-phospho-Stat3 Tyr705 (CST9131, Cell Signaling Technology), anti-p44/42 MAPK (Erk1/2) (CST4695, Cell Signaling Technology), anti-Phospho-p44/42 MAPK (Erk1/2) (CST4376, Cell Signaling Technology).
Cells were fixed in 2% paraformaldehyde at room temperature (RT) for 20 min. After blocking and permeabilizing with 1% BSA and 0,3% Tryton X-100 for 1 h at RT, cells were incubated with primary antibody overnight at 4 °C. Dilutions of primary antibodies were in accordance with the manufacturer’s instructions. Cells were then incubated in the dark at RT for 1 h with corresponding secondary antibodies conjugated to Alexa Fluor plus 555 or Alexa Fluor 488 (Thermo Fisher Scientific). Lastly, nuclei were stained with Hoechst 33,342 (Thermo Fisher Scientifc) and the coverslips were mounted on the slides with Fluoromount-G (Thermo Fisher Scientific). Slides were then examined under a Zeiss AxioImager Z1 fluorescent microscope (Carl Zeiss International, Germany). Different fields were examined at 40× and 63× magnification. The following antibodies were used: anti-PLCβ1 (Thermo Fisher Scientific) diluted 1:100, anti-Ki67 (Cell Signaling Technology) diluted 1:400, anti-Non-phospho (Active) β-Catenin (Cell Signaling Technology) diluted 1:600.
Transwell migration and invasion assays
All the cells were trypsinized and suspended in serum-free medium. 100 µl of the cell suspensions containing 3 × 104 cells/ml (for migration assay) or 6 × 104 cells/ml (for invasion assay), were seeded into the upper chamber of a 24-well transwell (8 µm pore size) (Sarstedt, Nümbrecht, Germany). For the invasion assay, a coating with Geltrex (Thermo Fisher Scientific) was carried out 2 h before the seeding and the plates with coated transwells were left 1 h at 37 °C and 1 h at room temperature before the seeding. For both migration and invasion assays, after the seeding, transwells were inserted into a 24-well plate containing 600 µl of medium supplemented with 10% FBS and incubated at 37 °C in a humidified atmosphere for 18 h to allow the cells to migrate/invade. The next day, non-migrating and non-invading cells on the upper side of the filter were removed with cotton swabs. Migrating and invading cells on the lower side of the filter were fixed for 20 min using 70% EtOH and stained for 15 min with 0.2% crystal violet. The number of migrating and invading cells was manually counted in 4 random and non-repeated fields under an optical microscope (magnification 20×). The average cell numbers of each group were then calculated. Each assay was performed in triplicate.
Wound healing assay
Cells were plated in 6-well plates and the day after reaching 100% confluence a longitudinal scratch was made in the monolayer using a 100 µl sterile micropipette tips. Two independent areas of each lesion were photographed at 0 h and 24 h (for U87-MG) or 48 h (for U-251 MG) using a phase contrast microscope (Motic AE21, Seneco Srl, Milano, Italy) with a digital camera (Visicam 3.0) at ×10 magnification. The gap area was quantified with ImageJ software (National Institutes of Health, Bethesda, MD) and the wound healing effect was calculated as (1-Ax/A0) %, where A0 and Ax represented the empty scratch area at 0 h and 24/48 h, respectively.
Statistical analysis was carried out using Graph Pad Prism 5.0 software (San Diego, CA, US) by applying the unpaired t test for patient’s RNA expression analysis and one-way ANOVA for the other analysis. The differences were considered significant with *p < 0.05, **p < 0.01 and ***p < 0.001.