Compilation of CAMs
A list of manually curated 518 CAMs (Supplementary Table 1) was prepared from various sources such as Entrez query ‘CAMs and homo sapiens’, GO term annotations related to cell adhesion and literature was used for analysis in this study.
Differential expression analysis
The gene expression data for GBM was downloaded from TCGA, REMBRANDT, GSE7696, and GSE22866. The differential expression was calculated by subtracting average value of control samples from average of GBM samples. Statistical significance was tested using Wilcox Mann-Whitney test. Genes showing fold change ≤–0.58 or ≥0.58 and significant p-value (p-value≤0.05) were considered as differentially expressed.
Survival Analysis
All CAMs were subjected for univariate cox regression analysis using TCGA Agilent dataset and SPSS software. GraphPad Prism software 5.0 was used for the Kaplan Meier survival analysis.
Copy number variation
Copy number variation of CAMs was analysed using data from cBioPortal (http://www.cbioportal.org/) and calculated the percentage of samples in which a particular CAM amplified or deleted.
Methylation data analysis
The CpGs corresponding to differentially expressed genes were fetched from TCGA DNA Methylation dataset (Illumina Infinium Human DNA methylation 450K array). The data for control samples was taken from GSE79122. For each probe, the differential beta value was calculated by subtracting the average beta value in control from the average beta value in the GBM. In order to identify differentially methylated probes, the difference of >0.3 absolute beta value. Wilcox Mann-Whitney test was used to calculate statistical significance.
MicroRNA
The differentially regulated CAMs were used as an input for miRwalk. The miRNAs which were predicted by minimum, 7 or more algorithms to target the CAMs were taken to further analysis. The miRNAs that were reciprocally regulated with respect to their target CAMs were considered as miRNA and CAM pairs.
Cell lines, normal brain tissues, and plasmids
Glioma cell lines (U373, T98G, U251, U87, LN229, U343, LN18, A172, and U138), immortalized astrocytes (NHA and SVG), and 293T were cultured in DMEM (Sigma-Aldrich, #D5648) supplemented with 10% Fetal Bovine Serum (FBS), Penicillin, and Streptomycin. U251, U87, U373, T98G, and 293T were bought from ECACC. LN229 and NHA were gifted from late Dr. Abhijit Guha (University of Toronto, Canada). Patient tumor derived primary GSC lines MGG8, MGG6, and MGG4 were procured from Dr. Wakimoto H. (Massachusetts General Hospital, Boston, USA) and 1035 was obtained from Dr. Santosh Kesari (University of California, San Diego, USA) and were cultured as neurospheres.
Non-tumorous control brain tissue samples (N1-N5) procured from patients with intractable epilepsy during surgery at National Institute of Mental Health and Sciences (NIMHANS), Bangalore, India. The tissues were snap-frozen in liquid nitrogen and stored at −80 °C and used for RNA isolation. The tissue samples were obtained after informed and taken written consent from all patients prior to use in the study. The study has been approved by the ethics committee of the NIMHANS and Indian Institute of Science (IISc). In the present study control brain tissue samples used for isolation of RNA and to measure PTGFRN transcript levels.
PTGFRN shRNAs TRCN00000057448 to 57452 (Sigma-Aldrich) were used for silencing PTGFRN and pcDNA3.2/V5 mmu-miR-137 (Addgene) was used for overexpression of miR-137.
Lentivirus preparation and transduction
293T cells transfected with shRNA (4 µg) along with the helper plasmids pSPAX and pMD2.G (3:1) using Lipofectamine 2000 (Invitrogen, #11668019) transfection reagent in 60 mm dish. The media was changed after 5 h of transfection and supernatant, which contain virus particles, was collected after 60 h of transfection and the virus was used to infect glioma cells in presence of 8 µg/ml of Polybrene (Sigma, #107689).
cDNA conversion and qPCR
The total RNA was isolated by using Trizol (Sigma-Aldrich, #T9424) method. RNA was converted to cDNA by using cDNA conversion kit (Life Technologies, #4368813). Subsequently, RT-qPCR was performed and fold change was calculated by ΔΔct method.
Western blotting
The RIPA buffer was used to lyse the cells and supernatant was collected. Protein was quantified using Bradford reagent and the required amount of protein was resolved in the SDS-PAGE gel and performed Western blotting. The primary antibodies anti-PTGFRN (ab97567, abcam), anti-phospho-ERK1/2 (#9101, CST), anti-ERK (#9102, CST), anti-phospho-AKT (#9271, CST), anti-AKT (#4691, CST), anti-phospho-p70S6 (#9208, CST), anti-p70S6 (#2708, CST), anti-phospho-4EBP1 (#9456, CST), anti-4EBP1 (#9452, CST) and anti-β-actin (#A3854, Sigma-Aldrich) were used in this study.
Proliferation assay
The cell viability was assessed by trypan blue assay. Briefly, the cells expressing control shRNA or shRNA against PTGFRN were plated in 6 well plate, 104 cells per well. Cell viability checked on every 3rd day using Vi-cell counter (Beckman Coulter, #383722) and normalization was done using the reading of day 1 as control for each condition. Statistical analysis was done using Student’s t-test.
Colony suppression assay
The cells stably expressing shRNA were counted and plated in a 6 well plate in triplicates at the seeding density of 103 cells/well and grown for 2-3 weeks replacing the media every 2-3 days once. Colonies were fixed in chilled methanol overnight followed by staining with crystal violet (0.05% w/v) for 30 min. Colonies were quantified either by manual counting or counting using ImageJ software. The statistical significance was calculated by Student’s t-test.
Soft agar colony formation assay
In the soft agar assay, cells were counted and plated in 104 cells/well in 1.2 ml of 0.4% of low melting agarose (BD Biosciences, #214230) in 6 well plate containing 0.6% agarose base layer. Each condition was plated in triplicates. After 2-3 weeks, images were taken and quantification was performed. Statistical analysis was done using Student’s t-test.
Migration and invasion assays
Trans-well Matrigel invasion assay is an in-vitro method to assess the ability of cells to degrade extracellular matrix proteins in response to a stimulus. Migration of cancer cells was assayed in 24 well Boyden chamber with 8 µm pore size polycarbonate membrane (BD Biosciences, San Diego, USA). For invasion assay the membranes, precoated with matrigel (a mix of extracellular matrix proteins) were used (BD bioscience, Sandiego, USA). The matrigel layer serves as a reconstituted basement membrane which occludes the pores. Cells (5x104) were resuspended in 500 μl serum-free medium and placed in the upper chamber, and the lower chamber was filled with 600 μl medium with 10% FBS (serving as a chemo-attractant). Cells were incubated for 22 hours and after incubation, the cells remaining on the upper surface of the membrane were removed by wiping with a wet cotton bud. The cells on the lower surface of the membrane were fixed in chilled methanol and stained with 0.05% crystal violet and counted under the light microscope. The statistical significance was calculated by student’s t-test.
Cell cycle analysis by Flow cytometry
For analysing the distribution of cells in different phases of cell cycle both the floating and adherent cells were used. The adherent cells were washed with PBS and harvested by trypsinization. The single cell suspension was pelleted down and resuspended in 300 μl of PBS and fixed with chilled 100% ethanol (700 μl) by adding drop by drop while gentle vertexing. Cells were fixed by incubating them in -20°C overnight. Incubation was followed by re-pelleting of cells and complete removal of ethanol by two PBS washes and finally followed by treatment with RNaseA (10 µg/ml) for 2-3 h at 37°C. Cells were stained with propidium iodide (PI), 10 μg/ml and subjected to flow cytometry using FACSVERSE instrument (BD Biosciences) to assay the effects on cell cycle profile.
Annexin V-FITC (Fluorescein isothiocyanate) and PI staining
Annexin V-FITC/PI double staining was utilized to quantify apoptosis in each condition. The apoptotic cells were determined under different conditions by flow cytometry based analyses using Annexin V-FITC Apoptosis kit from Biovision (K-101) following the manufacturer’s instructions. The percentage of healthy population, early/late apoptotic population and necrotic population was determined from each condition and percentage of apoptotic cells was plotted.
GSCs and DGCs
MGG8, MGG6, MGG4, and 1035 GSCs were cultured as neurospheres in Neurobasal medium (Invitrogen, #21103049) supplemented with EGF (20 ng/ml; R&D systems, #236-EG-200), bFGF (20 ng/ml, Peprotech, #100-18B), 0.5x N-2 (Invitrogen, #17502-048), 1x B27 supplement (Invitrogen, #17504-044), 2 µg/ml Heparin (Sigma, #H3149) in low-attachment dishes. For differentiation of GSCs to get DGCs, GSCs were transferred to 10% serum containing DMEM media in regular adherent plates and cultured for 7 days.
Neurosphere assay and Limiting dilution assay
GSC cells were infected with lentivirus expressing either NTshRNA or shRNA against PTGERN or ASTN1. After 48 h of infection, the sphere aggregates formed were dissociated into single cells, counted and plated 104 cells/well in ultra-low attachment 6 well dish and cultured for 7 days. Fresh medium was replenished every 2-3 days. Number of spheres were counted after 7 days of plating and plotted graphs.
For limiting dilution assay, neurospheres were dissociated into single cells and counted. Cells were plated in ultra-low attachment 96 well plates wherein a range of cells (1, 10, 25, 50, 75, and 100 cells/well) were plated into 8 wells for each condition. Fresh medium was replenished every 2-3 days. After 7-10 days, wells forming the spheres were counted in control knockdown and gene knockdown condition. Graph was plotted using ELDA (Extreme Limiting Dilution Analysis) software.