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’, and gene ontology (GO) term annotations related to cell adhesion was used for the analysis in this study.
Differential expression analysis
The gene expression data for GBM was downloaded from TCGA, REMBRANDT, GSE7696, and GSE22866 datasets. The differential expression was calculated by subtracting the average value of control samples from the average of GBM samples. Statistical significance was tested using the Wilcoxon-Mann-Whitney test. Genes showing fold change ≤ − 0.58 or ≥ 0.58 and significant p-value (p-value≤0.05; t-test: Wilcoxon-Mann-Whitney test and additional Benjamini/Hochberg FDR correction was applied) were considered as differentially expressed.
For PTGFRN protein level validation, we utilized the immunohistochemical data derived from the Human Protein Atlas (www.proteinatlas.org). The antibody used for this analysis is from Sigma-Aldrich (Cat# HPA017074; Rabbit polyclonal antibody). The IHC staining intensity was scored on a scale of 0-3 (0-no staining, 1+ -weak staining, 2+ -moderate staining, 3+-strong staining) with 1+ and above was considered as positive. The cells showing percent tumor cell positivity values >25, <25%, and none were considered.
All CAMs were subjected to 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 analyzed using data from cBioPortal (http://www.cbioportal.org/) and calculated the percentage of samples in which a particular CAM was amplified or deleted.
Methylation data analysis
The list of CpGs corresponding to differentially expressed genes was fetched from the TCGA DNA Methylation dataset (Illumina Infinium Human DNA methylation 450 K array). The CpG probes present in all parts of the gene were considered for this analysis. The data for control samples were taken from GSE79122. For each probe, the differential beta value (methylation value) was calculated by subtracting the average beta value of the control from the average beta value of the GBM. A difference of >0.3 absolute beta value was applied to identify differentially methylated probes. The Wilcoxon-Mann-Whitney test was used to calculate statistical significance.
The differentially regulated CAMs were used as an input for miRwalk. The miRNAs which were predicted by a minimum of seven or more algorithms to target the CAMs were taken for 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 (U373, T98G, U251, U87, LN229, U343, LN18, A172, and U138), Immortalized human astrocytes (IHA and SVG), and 293T cell lines 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 IHA were gifted by 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) were procured from patients with intractable epilepsy during surgery at the National Institute of Mental Health and Sciences (NIMHANS), Bangalore, India. The tissue samples were obtained with written consent from all patients before using them in the current 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 were used for the isolation of RNA and to measure PTGFRN transcript levels.
PTGFRN shRNAs TRCN00000057448 to TRCN00000057452 (Sigma-Aldrich) were used for silencing PTGFRN, and pcDNA3.2/V5-mmu-miR-137 (Addgene) was used for overexpression of miR-137. The luciferase reporter construct pmiR-GLO-3’UTR of PTGFRN was a kind gift from Dr. Markus Stoffel (Institute of Molecular Health Sciences, ETH Zurich, Switzerland).
Lentivirus preparation and transduction
293T cells were transfected with shRNA (4 μg) along with the helper plasmids pSPAX and pMD2.G (3:1) using Lipofectamine 2000 (Invitrogen, #11668019) transfection reagent in a 60 mm dish. The media was changed after 5 hours of transfection, and supernatant, which contains virus particles, was collected after 60 h of transfection. The virus suspension was used to infect glioma cells in the 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 the ΔΔct method.
The RIPA buffer was used to lyse the cells, and the supernatant was collected. Protein was quantified using Bradford’s reagent, and the required amount of protein was resolved in the SDS-PAGE gel, and Western blotting was performed. 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.
The cell viability was assessed by trypan blue assay. Briefly, the cells expressing either control non-targeting shRNA (shNT) or shRNA against PTGFRN (shPTGFRN) were plated in a six-well plate with 104 cells per well. Cell viability was checked every 3rd day using the Vi-cell counter (#383722, Beckman Coulter), and normalization was done using the reading of day-1 for each condition. Statistical analysis was done using the Student’s t-test.
Colony suppression assay
The cells stably expressing shRNAs were counted and plated in a six-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. Colonies were fixed using chilled methanol overnight, followed by staining with crystal violet (0.05% w/v) for 30 min. Colonies were quantified by counting using ImageJ software. The statistical significance was calculated by the Student’s t-test.
Soft agar colony formation assay
In the soft agar assay, cells were counted, and 104 cells/well in 1.2 ml of 0.4% low melting agarose (#214230, BD Biosciences) was plated in a six-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 the 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 pre-coated with matrigel (a mix of extracellular matrix proteins) were used (BD Biosciences, Sandiego, USA). The matrigel layer serves as a reconstituted basement membrane that occludes the pores. Cells (5 × 104) 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 using chilled methanol and stained with 0.05% crystal violet and counted under the light microscope. The statistical significance was calculated by the Student’s t-test.
Cell cycle analysis by flow cytometry
For analyzing the percentage of cells in different phases of the 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 vortexing. Cells were fixed by incubating them at −20 °C overnight. Incubation was followed by re-pelleting cells and complete removal of ethanol by two PBS washes followed by treatment with RNase A (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 FACS-VERSE instrument (BD Biosciences) to assay the effects on cell cycle profile. The statistical significance was calculated by the Student’s t-test.
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 using flow cytometry-based analyses using Annexin V-FITC Apoptosis kit from Bio-vision (K-101) following the manufacturer’s instructions. The percentage of the healthy population, early/late apoptotic population, and necrotic population were determined from each condition, and the percentage of apoptotic cells was plotted. The statistical significance was calculated by the Student’s t-test.
TaqMan advanced miRNA assay for measuring miR-137
To measure the miR-137 levels in IHA and GBM cell lines, we extracted the total small RNA from cell lines using Trizol lysis followed by column-based RNA purification method (miRNeasy Mini kit, #1038703; Qiagen). To synthesize cDNA from small RNA, TaqMan advanced miRNA cDNA synthesis kit was used (#A25576; Thermo Fisher Scientific, USA). To quantify the specific mature miR-137, TaqMan advanced miRNA assay (has-miR-137; Assay ID: 477904_mir; Thermo Fisher Scientific, USA) was performed using RT-qPCR method and miR-191 was used as an internal control.
Luciferase reporter assay
293T cells were co-transfected with 1000 ng of pcDNA3.2/V5 vector or pcDNA3.2/V5-miR-137 overexpression plasmid along with 100 ng of pmiR-GLO-3’UTR of PTGFRN luciferase reporter plasmid in a 12 well-plate. The pmiR-GLO-3’UTR of PTGFRN construct contained ~1 kb insert DNA of 3’UTR of mouse PTGFRN and which covers one binding site for miR-137 in the middle of the insert . After 48 hours of transfection, cells were harvested and assayed for luciferase activity using dual luciferase assay system (Promega). Firefly luciferase activity was normalized with Renilla luciferase activity, an internal control.
GSCs and DGCs
MGG8, MGG6, MGG4, and 1035 GSCs were cultured as neurospheres in Neurobasal medium (#21103049, Invitrogen) supplemented with EGF (20 ng/ml; #236-EG-200, R&D systems), bFGF (20 ng/ml; #100-18B, Peprotech), 0.5x N-2 (#17502-048, Invitrogen), 1x B27 supplement (#17504-044, Invitrogen), 2 μg/ml Heparin (#H3149, Sigma) in ulta-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. The statistical significance was calculated by the Student’s t-test.
Neurosphere assay and limiting dilution assay
GSCs were infected with lentivirus expressing either shNT or shPTGFRN. After 48 h of infection, the sphere aggregates formed were dissociated into single cells, counted, and plated at a density of 104 cells/well in an ultra-low attachment six-well plates and cultured for 7 days. Fresh medium was replenished every 2-3 days. The number of spheres was counted after 7 days of plating, and graphs were plotted. The statistical significance was calculated by the Student’s t-test.
For limiting dilution assay, neurospheres were dissociated into single cells and counted. Cells were plated in an ultra-low attachment 96 well plates wherein a range of cells (1, 10, 25, 50, 75, and 100 cells/well) were plated into eight 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 conditions. A graph was plotted using ELDA (Extreme Limiting Dilution Analysis) software.