Downregulation of miR-204 expression defines a highly aggressive subset of Group 3/Group 4 medulloblastomas
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Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastoma: WNT, SHH, Group 3 and Group 4. Molecular markers are necessary for accurate risk stratification in the non-WNT subgroups due to the underlying heterogeneity in genetic alterations and overall survival. MiR-204 expression was evaluated in molecularly classified 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC cohort, SickKids, Canada. Low expression of miR-204 in the Group 3 / Group 4 tumors identify a highly aggressive subset of tumors having poor overall survival, in the two independent cohorts of medulloblastomas. Downregulation of miR-204 expression correlates with poor survival within the Group 4 as well indicating it as a valuable risk-stratification marker in the subgroup. Restoration of miR-204 expression in multiple medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. IGF2R was identified as a novel target of miR-204. MiR-204 expression resulted in downregulation of both M6PR and IGF2R that transport lysosomal proteases from the Golgi apparatus to the lysosomes. Consistent with this finding, miR-204 expression resulted in reduction in the levels of the lysosomal proteases in medulloblastoma cells. MiR-204 expression also resulted in inhibition of autophagy that is known to be dependent on the lysosomal degradation pathway and LC3B, a known miR-204 target. Treatment with HDAC inhibitors resulted in upregulation of miR-204 expression in medulloblastoma cells, suggesting therapeutic role for these inhibitors in the treatment of medulloblastomas. In summary, miR-204 is not only a valuable risk stratification marker in the combined cohort of Group 3 / Group 4 medulloblastomas as well as in the Group 4 itself, that has paucity of good prognostication markers, but also has therapeutic potential as indicated by its tumor suppressive effect on medulloblastoma cells.
KeywordsMedulloblastoma MiR-204 Risk stratification Tumor-suppression Autophagy
3′- untranslated region
Analysis of variance
American Type Culture Collection
Dulbecco’s Modified Eagle Medium
Fetal Bovine Serum
Formalin- Fixed, Paraffin-Embedded
Gene Set Enrichment Analysis
Medulloblastoma Advanced Genomics International Consortium
Robust Multi-array Average
Reverse Transcription-Polymerase Chain Reaction
Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
Short Tandem Repeat
Brain tumors are the second most common cancers in children and the leading cause of cancer-related mortality in this age group . Medulloblastoma, a highly malignant tumor of the posterior fossa region of the brain, is the single most common pediatric malignant brain tumor. Genome-wide expression profiling studies have identified four core molecular subgroups of medulloblastomas: WNT, SHH, Group 3 and Group 4 that are not only distinct in their underlying genetic alterations but also differ in clinical characteristics like age, gender related incidence, incidence of metastasis and overall survival rates . WNT subgroup medulloblastomas that are characterized by the activation of the canonical WNT signaling pathway have excellent (> 95%) long term survival . SHH subgroup medulloblastomas having activated Sonic Hedgehog signaling expression profile, have intermediate survival rates with those harboring mutation in the TP53 tumor suppressor gene or amplification of MYCN oncogene having poor survival . The two non-WNT, non-SHH subgroups have some overlap in their expression profiles with a number of transcription factors involved in neural development being overexpressed in both the subgroups . The two subgroups are distinguished based on the preferential expression of proliferation related genes, retina-specific genes in the Group 3 tumors and neuronal differentiation related genes in the Group 4 tumors . Group 3 tumors have the worst survival rates among all the four subgroups while Group 4 tumors have intermediate survival rate. NRL and CRX, the two retina-specific transcription factors have been found to be master regulators of photoreceptor signaling program in the Group 3 medulloblastomas . MYC amplifications are restricted to Group 3 . Structural variants leading to aberrant induction of GFI1/GFI1B oncogenes and MYCN amplifications are found in both Group 3 and Group 4. Pathway analysis of recurrent genetic alterations have found overrepresentation of genes involved in the TGFβ and Notch signaling pathway in Group 3 and chromatin modifiers in Group 4 .
Surgery followed by radiation therapy and chemotherapy is the standard multimodal treatment for medulloblastoma . Long term sequelae of the intense treatment include neurocognitive impairment, endocrine dysfunction, psychiatric, developmental deficits and in some cases secondary malignancies . Accurate risk stratification of medulloblastomas is therefore necessary to spare the children having low risk of recurrence from excessive treatment to the developing brain. On the other hand, survival of high risk medulloblastoma cases can be improved by more aggressive treatment. Considerable heterogeneity exists in each of the three non-WNT subgroups for which molecular markers are necessary so that accurate risk stratification can be done for effective treatment with least side effects .
MicroRNAs are small non-coding molecules that have been shown to regulate a wide array of cell functions, ranging from cell proliferation, differentiation, cell death and stress resistance. Since the first report of miR-15/miR-16 deletion in B cell chronic lymphocytic leukemia, large number of studies have reported microRNA dysregulation in cancer including medulloblastoma [5, 56]. We have earlier reported differential expression profiles of microRNAs in the molecular subgroups of medulloblastomas . Further, we have developed an assay based on the microRNA profile that has 97% accuracy for molecular classification of medulloblastomas and is particularly useful for formalin-fixed, paraffin-embedded (FFPE) tumor tissues . In the present study, miR-204 expression was analyzed in 260 medulloblastomas from an Indian cohort and in 763 medulloblastomas from the MAGIC (Medulloblastoma Advanced Genomics International Consortium) cohort . A subset of Group 3 / Group 4 medulloblastomas having low expression of miR-204 was found to have significantly poor survival. The role of miR-204 expression in medulloblastoma biology was investigated by restoring miR-204 expression in established medulloblastoma cell lines and studying its effect on growth and malignant behavior of medulloblastoma cells.
Materials and methods
Human tissue samples
Medulloblastoma tumor tissues either as fresh frozen or FFPE tissues were obtained after acquiring informed consent from the patients. The study was approved by the Institutional Ethics Committee of the Tata Memorial Centre. The tumor tissues were snap-frozen in liquid nitrogen immediately after surgical resection and stored at − 80 °C. The histopathological diagnosis and grading of the tumor tissues was done as per the World Health Organization 2007 classification of tumors of the Central Nervous System  and only the tumors diagnosed as medulloblastomas were included in the study. Normal human brain tissues were obtained from the Human Brain Tissue Repository at the National Institute of Mental Health and Neurosciences, Bengaluru, India.
Analysis of miR-204 expression
Molecular classification of 260 medulloblastomas from the Indian cohort was carried out using real time RT-PCR (Reverse Transcription-Polymerase Chain Reaction) assay as described before . MiR-204 expression was determined by the Taqman assay. RNU48 was used as a house-keeping small RNA control. Relative Quantity (RQ) was estimated as RQ = 2- (Cttest – Ctcontrol) X 100. In the MAGIC validation cohort, miR-204 expression was analyzed across 763 primary medulloblastoma samples, profiled on the Affymetrix Gene 1.1 ST array as described previously, normalized using the RMA (Robust Multi-array Average) method and, subgrouped / subtyped using similarity network fusion (GSE85217) . Differences across subgroups and subtypes were evaluated using ANOVA (Analysis of variance) in the R statistical environment (v3.4.2). Survival was measured from the time of initial diagnosis to the date of death or last follow up. Survival distribution was estimated according to the Kaplan–Meier method using optimal cut-off selection and log-rank statistics using the survival package (v2.40–1) in the R statistical environment (v3.4.2). P values < 0.01 were considered to be statistically significant.
Human medulloblastoma cell line D283 was obtained from ATCC (American Type Culture Collection), Manassas, VA, USA. Authenticity of the cell lines was confirmed by the Short Tandem Repeat (STR) marker profiling before initiating the experiments. Medulloblastoma cell lines D425, D341 are kind gifts from Dr. Darell Bigner, Duke University Medical Centre, Durham, NC, USA. HD-MB03 cell line is a kind gift from Dr. Till Milde, German Cancer Research Centre, Germany. All the cell lines were checked for the presence of mycoplasma contamination by PCR based assay . The cells were grown in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/ F-12) supplemented with 10% Fetal Bovine Serum (FBS) in a humidified atmosphere of 5% CO2.
Restoration of miR-204 expression in medulloblastoma cells
Genomic region encoding miR-204 was amplified from normal human lymphocyte DNA by PCR and cloned in pTRIPZ lentiviral vector downstream of doxycyline-inducible minimal Cytomegalo virus (CMV) promoter (Additional file 1: Table S1). The medulloblastoma cell lines were transduced with the pTRIPZ-miR-204 lentiviral particles and stable polyclonal populations were selected in the presence of puromycin. The cells transduced with lentiviral particles of empty pTRIPZ vector (Dharmacon, Lafayette, CO, USA) were used as vector control.
Effect of miR-204 expression on proliferation and anchorage-independent growth
Growth of miR-204 expressing cells and control cells was studied by the MTT reduction assay as described before [32, 59]. 2000 cells of the medulloblastoma cell lines were seeded per well of a 96-well micro-titer plate. Cell growth was followed over a period of 10-12 days with replenishment of medium every 3rd day. For studying anchorage-independent growth by soft agar colony formation assay, 2000 cells were seeded in DMEM/F12 medium supplemented with 10% FBS containing 0.3% agarose over a basal layer of 1% agarose in DMEM-F12/10% FBS. The cells were incubated for about 1-2 weeks and the colonies formed were counted.
75,000 cells of D283 / HD-MB03 cell line were seeded in 200 μl of serum-free DMEM / F12 medium in the upper chamber of 8-μm pore size transwell inserts (BD Biosciences, San Hose, CA, USA) coated with Matrigel™, placed in a 24 well micro-titre plate. 750 μl of the medium supplemented with 10% FBS was added to the lower chamber. The cells were allowed to migrate for 56 h to 72 h depending upon the cell line and then labeled with Calcein-AM (Life technologies, Carlsbad, CA, USA), a fluorescent dye, 30 min prior to terminating the invasion. Non-invaded cells from the upper chamber were removed by wiping the upper portion of the insert with a cotton bud. The inserts were photographed using a Zeiss Axiovert 200 M fluorescence microscope. Fluorescence intensity of the Calcein-AM labeled cells on the lower side of the insert was measured using a Mithras LB940 multimode reader (Berthhold Technologies, Bad Wildbad, Germany) using excitation wavelength of 485 nm and emission wavelength of 535 nm.
The experimental protocols were approved by the Institutional Animal ethics committee. Medulloblastoma cells were transduced with lentiviral particles of pCS-CG vector (a gift from Inder Verma, Addgene plasmid #12154 ) expressing firefly luciferase cDNA FL2 (from pCAG-luciferase vector, a gift from Snorri Thorgeirsson, Addgene plasmid #55764 ) under the CMV promoter. 2 X 105 doxycycline-induced cells were injected into the cerebellum of NOD/SCID mice (NOD.CB17-Prkdcscid/NCrCrl, Charles River, USA) through 0.5 mm burr hole in the midline, 2 mm posterior to lambda at 2 mm depth, using small animal stereotaxic frame under anesthesia . Tumor growth was monitored by in vivo bioluminescence imaging using the IVIS Spectrum imaging system (Caliper Lifesciences, Perkin Elmer, MA, USA). Tumor bearing mice were maintained until they succumbed to the tumor or were about to succumb to the tumor as judged by over 40% loss of weight or other clinical symptoms. Upon sacrifice, whole brain was fixed in the neutral buffered formalin and embedded in a paraffin block. Hematoxylin & Eosin stained sections of the paraffin blocks were used for determination of the invasive capacity of the tumor cells without revealing identity of the specimen to the analyst.
Libraries were prepared using the Truseq RNA sample prep kit V2 as per the manufacturer’s protocol (Illumina, San Diego, USA) from the total RNA extracted from the medulloblastoma cells and subjected to 100 nucleotides deep sequencing using the Illumina HiSeq 2500 sequencing system to get a minimum of 10 million reads per library. The reads were aligned to the reference human genome hg19 using the TopHat version 2.0.13 (http://ccb.jhu.edu/software/tophat) with default parameters. Raw counts for the reads aligned to the gene intervals were produced by the python package HTSeq version 0.6.1 (www-huber.embl.de/users/anders/HTSeq) using the default union-counting mode. The data was normalized by variance stabilizing transformation using the DESeq software that takes into account RNA-seq data size of each sample (http://bioconductor.org/packages/release/bioc/html/DESeq.html). Gene Set enrichment analysis of the genes differentially expressed upon miR-204 expression was done using the GSEA (Gene Set Enrichment Analysis) software (software.broadinstitute.org/gsea/index.jsp). Downregulation of expression of known miR-204 target genes upon miR-204 expression in medulloblastoma cell lines was validated by SYBR green real time RT-PCR assay using gene-specific primers (Additional file 1: Table S1).
Anti-LC3B (#2775), anti-p62/SQSTM1 (#8025), anti-Cathepsin D (#2284), anti-Cathepsin B (#31718) and, anti-IGF2R (#14364) antibodies from the Cell signaling technology, Boston, MA, USA.
Anti-GAPDH antibody (SC 47724) from Santa Cruz Biotechnology, Dallas, TX, USA.
Anti-Histone H3 (acetyl K9) antibody (ab10812) from Abcam, Cambridge, UK.
Luciferase reporter assay
Firefly luciferase cDNA was cloned in the pcDNA 3.0 vector (Invitrogen, Carlsbad, CA, USA) downstream of the CMV promoter to generate ‘pLuc’ reporter vector. 3′-UTR regions of the miR-204 target genes were amplified from the genomic DNA of normal human lymphocytes and cloned downstream of the firefly luciferase cDNA in the ‘pLuc’ vector. Putative miR-204 binding sites in the 3′-UTRs were mutated by site-directed mutagenesis using primers having 4 nucleotides corresponding to the binding site altered . Luciferase activity was assessed from the HEK293FT cells transfected with the luciferase reporter plasmid, miR-204 expressing plasmid/vector control pcDNA4 (Invitrogen, Carlsbad, CA, USA), and a plasmid vector expressing EGFP fluorescent protein. Luciferase activity was assessed from the total protein extracted from the transfected HEK293FT cells and was normalized against the EGFP fluorescence measured using the BioTek Cytation Hybrid Multimode Reader, Winooski, VT, USA.
TRPM3/MIR-204 promoter methylation analysis and upregulation of miR-204 expression upon treatment with histone deacetylase inhibitors
Genomic DNA was isolated from the medulloblastoma cell lines using QIAamp DNA mini kit (Qiagen, GmbH, Hilden, Germany) as per the manufacturer’s instructions. Bisulfite conversion of 500 ng of the genomic DNA was performed using EZ DNA Methylation-Gold Kit from Zymo Research, Irvine, CA, USA, as per the manufacturer’s instructions. The 203 bp region covering - 200 to + 3 with respect to the known transcription start site of the TRPM3 gene was PCR amplified using the primers designed to amplify bisulfate converted DNA and sequenced (Additional file 1: Table S1). Medulloblastoma cells were treated with HDAC inhibitors Sodium valproate (6 mM) and Trichostatin A (400 nM) for a period of 16 h. Histone acetylation status was evaluated by separating total protein extracts from the treated cells by SDS-PAGE and probing the Western blot using anti-H3K9 acetylation antibody .
All experiments were performed at least three times and the Student’s t-test was used for evaluating statistical significance of the difference in the test as compared to the control. Error bars indicate standard error of the mean/median.
MiR-204 downregulation identifies a highly aggressive subset of Group 3 / Group 4 medulloblastomas having poor survival
Restoration of miR-204 expression inhibits anchorage-independent growth, and tumorigenicity of medulloblastoma cells
MiR-204 expression inhibits invasion potential of medulloblastoma cells in vitro and in vivo
MiR-204 expression downregulates M6PR, IGF2R genes involved in the lysosomal pathway and inhibits autophagy of medulloblastoma cells
TRPM3/MIR204 promoter methylation analysis and upregulation of miR-204 expression upon treatment with HDAC inhibitors
In a large scale study on 3312 tumors and 1107 non-malignant tissues contributed by 51 different cancer types, miR-204-211 family was found to be the top deleted microRNA family in cancer, suggesting its crucial role as a tumor suppressive miRNA in multiple cancer types . In the present study, miR-204 was found to be differentially expressed in the four core molecular subgroups of medulloblastomas, with almost all SHH and a subset of Group 3/Group 4 tumors showing downregulation of miR-204 expression. Despite the complexity of the heterogeneity and overlap present in the copy number variations, methylation profiles and somatic mutation profiles in the Group 3 / Group 4 medulloblastomas , miR-204 expression levels identify a subset of these tumors having poor survival in the Indian as well as in the large MAGIC cohort. This finding is consistent with lower expression of miR-204 correlating with poor survival in breast cancer , non-small cell lung cancer , and neuroblastoma . Integrated genomic studies have identified novel molecular subtypes within the four core subgroups of medulloblastomas [2, 49]. Among the three Group 3 subtypes, subtype 3γ was found to have the worst 5 year survival rate of 41.9% as compared to that of subtype 3α and subtype 3β at 66.2 and 55.8% respectively . Group 3γ having the worst survival showed the least miR-204 expression among the 3 subtypes of Group 3. The Group 4 subtypes do not show significant difference in their overall survival . MiR-204 expression on the other hand, identified a subset of Group 4 medulloblastomas having significantly poor survival of 59.7% as compared to 80% of the ‘miR-204 high’ subset. Group 3 poor prognostication markers like MYC amplification and isochrome 17q do not have prognostication value in Group 4 . FSTL5 immunopositivity serves as a marker for poor prognostication in both Group 3 and Group 4 medulloblastomas . Adult Group 4 patients have also been reported to have poor survival rates . Loss of chromosome 11 or gain of chromosome 17 identify a small subset of Group 4 patients who have excellent survival . Biology underlying these cytogenetic alterations is however, not understood. Thus, low miR-204 expression serves as a marker of poor prognosis in Group 4 that has paucity of markers for prognostication. Integrated genomic analysis is expensive as well as technically demanding and thus cannot be used in routine clinical practice for risk stratification. MiR-204, a single microRNA on the other hand, can be easily combined with the Nanostring assay that classifies medulloblastomas into the four molecular subgroups . Furthermore, miR-204 due its small size resists degradation during formalin fixation and thus would be a reliable marker even in poor quality FFPE tissues.
Downregulation of miR-204 expression with poor survival is consistent with its tumor-suppressive effect in medulloblastoma cell lines. Restoration of miR-204 expression in multiple established Group 3 medulloblastoma cell lines was found to inhibit their anchorage-independent growth, invasion potential and tumorigenicity. Tumor suppressive effect of miR-204 in the MYC amplified Group 3 cell lines is remarkable since other microRNAs downregulated in medulloblastoma like miR-206 for instance, fail to inhibit tumorigenicity of these cell lines . MiR-204 has been shown to inhibit invasion and tumorigenicity of various cancer cells including glioma, colorectal cancer, endometrial cancer and cervical cancer cells [3, 27, 57, 58]. Thus, the tumor suppressive role of miR-204 in medulloblastoma cells is consistent with its role in other cancers.
MiR-204 has been reported to target a number of genes including RAB22A, FOXC1, EZR, BCL2L2, M6PR, BCL2, MCL1, FOXA1, FOXM1, EPHB2 [22, 42, 50, 52, 57]. Transcriptome sequencing / real time RT-PCR / western blot analysis showed downregulation of RAB22A, M6PR, EZR, EPHB2, upon miR-204 expression in medulloblastoma cells as well. IGF2R was identified and validated as a novel target of miR-204. MiR-204 expression in medulloblastoma cells resulted in downregulation of both M6PR and IGF2R that mediate transport of lysosomal enzymes from the Golgi apparatus to lysosomes . Furthermore, reduction in the levels of lysosomal enzymes Cathepsin B and Cathepsin D upon miR-204 expression in medulloblastoma cells suggests impairment of the lysosomal degradation pathway. Autophagy brings about p62/SQSTM1 mediated degradation of its cargo by lysosomal degradation pathway . MiR-204 is known to target LC3B, a crucial mediator of autophagy . In the present study as well, miR-204 expression in medulloblastoma cells resulted in reduction in the LC3B flux and increase in the levels of p62/SQSTM1 indicating autophagy inhibition. Autophagy has been shown to play role in tumor promotion by sustaining survival in stress, by reducing oxidative stress and, maintaining metabolic homeostasis . Inhibition of tumor growth upon miR-204 expression is consistent with these reports on the role of autophagy in tumor promotion. Autophagy has also been reported to promote invasion by activating Epithelial Mesenchymal Transition of hepatocellular carcinoma cells , by promoting secretion of factors like IL6, MMP2  and by activating the MAP kinase signaling pathway in glioblastoma cells . Consistent with the inhibition of invasion capacity of medulloblastoma cells upon miR-204 expression, downregulation of miR-204 expression was found to be associated with higher incidence of metastasis at diagnosis in Group 3 / Group 4 medulloblastomas. Thus, poor survival of Group 3 / Group 4 medulloblastomas having low miR-204 expression is likely due to their higher invasive capacity and higher malignant potential.
Several microRNAs whose expression is deregulated in medulloblastoma are known to play role in embryonic brain development . MiR-9 and miR-124a that play crucial role in the onset of neurogenesis by targeting transcription factors like SOX9, FOXG1 and MEIS1, are downregulated in medulloblastoma . MiR-9 and miR-199b-5p target HES1, thereby silence Notch signaling pathway at the onset of neuronal differentiation [7, 10]. Low expression of miR-9 and miR-199b-5p has been found to correlate with poor survival in medulloblastoma and their expression in medulloblastoma cell lines promotes growth arrest [7, 10]. MiR-17-92 cluster microRNAs are overexpressed predominantly in the SHH subgroup medulloblastomas . Knock-out of this microRNA cluster brings about reduction in size of cerebellum and inhibits medulloblastoma formation in Ptch knock-out mouse model of SHH subgroup medulloblastomas indicating role of these microRNAs in normal development and tumorigenesis . MiR-204 has been reported to play crucial role in lens and retinal development by targeting MEIS2 transcription factor in Medaka fish . MiR-204 expression has been found to be upregulated during aging in mouse hippocampus and target Ephrin B2 that plays role in axon guidance . MiR-204 has also been reported to control neuronal migration and cortical morphogenesis in mouse embryos presumably by targeting Doublecortin that is known to play role in neuronal migration . Effect of miR-204 on invasive capacity of medulloblastoma cells is consistent with the role of miR-204 in neuronal migration. Thus, miR-204 appears to play role in both normal brain development and tumorigenesis like several other miRNAs that are known to be deregulated in medulloblastoma.
Delineating the molecular mechanism underlying downregulation of miR-204 expression would suggest ways to increase its expression, thereby improving survival rate of medulloblastoma patients. Group 3 medulloblastoma cells treated with HDAC inhibitors showed modest 2 to 4 fold increase in the miR-204 expression levels. Treatment with HDAC inhibitors has been reported to inhibit medulloblastoma cell growth in several studies [20, 29, 43]. Thus, HDAC inhibitors appear to have therapeutic potential in the treatment of medulloblastoma.
In summary, downregulation of miR-204 expression correlates with poor survival in the Group 3 / Group 4 medulloblastomas. Furthermore, within the Group 4 itself, low expression of miR-204 identifies a subset having significantly poor survival, making it a valuable marker for risk stratification in the subgroup that has paucity of prognostication markers. Restoration of miR-204 expression leading to reduction in the invasive capacity and tumorigenic potential of medulloblastoma cells suggests therapeutic potential of miR-204 in the treatment of medulloblastomas. Upregulation of miR-204 expression upon treatment with HDAC inhibitors, although modest, suggests a role of these inhibitors in the treatment of medulloblastomas.
We thank Mr. Anant Sawant for technical assistance and Ms. Nazia Bano, Ms. Amita Wawdekar, clinical trial coordinators. We thank Prof. S. K. Shankar, NIMHANS, Bengaluru for making normal brain tissues available for the study.
We thank Department of Biotechnology, India for the financial support. Dr. V. Ramaswamy is supported by operating grants from the Canadian Institutes for Health Research, the Brain Tumor Foundation of Canada, Meagan’s Walk and the American Brain Tumor Association.
Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable request.
HB, RP and PP have done the major experimental work involving Group 3 medulloblastoma cell lines. AD, SK, RK, KY have contributed to the experimental work that includes MicroRNA profile, molecular classification of medulloblastomas and evaluation of tumorigenicity. SM and Nikhil G have done the transcriptome sequencing and bioinformatics analysis. RJ, ES, TG, AM, AG, NG, PS, GC have contributed by recruitment of medulloblastoma cases, diagnosis and clinical data including treatment given and follow-up. NVS has contributed to the experimental design, implementation, analysis and, interpretation. VR has contributed to the Experimental design, analysis and interpretation. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the Institutional Ethics Committee of the Tata Memorial Centre. Tumor tissues were procured after getting informed consent from the patients. Animal study was approved by the Institutional Animal Ethics Committee.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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