The online version of this article (doi:10.1186/s13045-014-0096-y) contains supplementary material, which is available to authorized users.
Fotini Tzortzatou-Stathopoulou and Emmanouel Kanavakis contributed equally to this work.
The authors’ declare that they have no competing interests.
MB conceived and designed the study, performed all experiments, evaluated and interpreted data analyses, and drafted the manuscript. GIL performed all data analyses, participated in interpretation of data analyses and in drafting the manuscript, GK assisted in all experiments performance, VM assisted in immunohistochemistry performance, SK performed tumor diagnosis, DKB provided miRNA microarrays raw data for meta-analyses, NP performed all tumor resections, AKol assisted in miRNA microarray performance, AKat treated part of the patients’ cohort, CAS provided the post-mortem specimens, FTS treated the majority of the patients and EK participated in the coordination and supervision of the study. All authors approved the final manuscript.
Although, substantial experimental evidence related to diagnosis and treatment of pediatric central nervous system (CNS) neoplasms have been demonstrated, the understanding of the etiology and pathogenesis of the disease remains scarce. Recent microRNA (miRNA)-based research reveals the involvement of miRNAs in various aspects of CNS development and proposes that they might compose key molecules underlying oncogenesis. The current study evaluated miRNA differential expression detected between pediatric embryonal brain tumors and normal controls to characterize candidate biomarkers related to diagnosis, prognosis and therapy.
Overall, 19 embryonal brain tumors; 15 Medulloblastomas (MBs) and 4 Atypical Teratoid/Rabdoid Tumors (AT/RTs) were studied. As controls, 13 samples were used; The First-Choice Human Brain Reference RNA and 12 samples from deceased children who underwent autopsy and were not present with any brain malignancy. RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed with the mirVANA miRNA isolation kit. The experimental approach included miRNA microarrays covering 1211 miRNAs. Quantitative Real-Time Polymerase Chain Reaction was performed to validate the expression profiles of miR-34a and miR-601 in all 32 samples initially screened with miRNA microarrays and in an additional independent cohort of 30 patients (21MBs and 9 AT/RTs). Moreover, meta-analyses was performed in total 27 embryonal tumor samples; 19 MBs, 8 ATRTs and 121 control samples. Twelve germinomas were also used as an independent validation cohort. All deregulated miRNAs were correlated to patients’ clinical characteristics and pathological measures.
In several cases, there was a positive correlation between individual miRNA expression levels and laboratory or clinical characteristics. Based on that, miR-601 could serve as a putative tumor suppressor gene, whilst miR-34a as an oncogene. In general, miR-34a demonstrated oncogenic roles in all pediatric embryonal CNS neoplasms studied.
Deeper understanding of the aberrant miRNA expression in pediatric embryonal brain tumors might aid in the development of tumor-specific miRNA signatures, which could potentially afford promising biomarkers related to diagnosis, prognosis and patient targeted therapy.
Additional file 1: Table S2.: Common under- and up-regulated miRNAs between tumors following initial and meta-analyses as from Venn diagrams. (XLSX 140 KB)13045_2014_96_MOESM1_ESM.xlsx
Additional file 2: Table S1.: Clinical Raw Data. (XLSX 15 KB)13045_2014_96_MOESM2_ESM.xlsx
Additional file 3: Figure S2.: MicroRNA expression levels and patient age following initial analysis. Kruskal-Wallis analysis of miRNA expression profiles between patient age groups; Group A: < 3 years, Group B: 3–8 years and Group C: 9–18 years. Overall, 11 differentially expressed miRNAs were identified. Among them, 6 miRNAs were overexpressed in Group A as compared to the other groups: miR-1268 (A), miR-3681 (B), miR-3912 (C), miR-601 (D), miR-608 (E) and miR-720 (F). Three miRNAs were up-regulated in Group B as compared to the other groups: miR-3665 (G), miR-519c-3p (H) and miR-891a (I). Finally, two miRNAs were up-regulated as compared to the other groups: miR-2052 (J) and miR-26b (K). (*denotes a p < 0.05 significance and **denotes a p < 0.01 significance). (DOCX 763 KB)13045_2014_96_MOESM3_ESM.docx
Additional file 4: Table S4.: Disease and Drug Association Annotations for DE miRNAs as predicted from enrichment analysis using WebGestalt web tool. (XLSX 48 KB)13045_2014_96_MOESM4_ESM.xlsx
Additional file 5: Figure S3.: MicroRNA expression levels and patient gender following initial analysis. Kruskal-Wallis analysis of miRNA expression profiles between male and female patients within the miRLink dataset (in-house experiments). In total, 3 miRNAs were up-regulated in males miR-26b (A), miR-3162 (B) and miR-1268 (C), while 5 miRNAs were overexpressed in females; miR-720 (D), miR-186* (E), miR-3617 (F), miR-320c (G) and miR-3614-5p (H). (*denotes a p < 0.05 significance between pairs). (DOCX 495 KB)13045_2014_96_MOESM5_ESM.docx
Additional file 6: Table S3.: DE miRNAs log 2 transformed and natural mean expression values and statistical analysis between all clinical associations. (XLSX 22 KB)13045_2014_96_MOESM6_ESM.xlsx
Additional file 7: Figure S4.: MicroRNA expression levels and disease progression following initial analysis. Kruskal-Wallis analysis between DE miRNAs and disease progression, following initial analysis; Relapse (n = 9) or Complete Remission (n = 10) (CR). Overall, 18 miRNAs were differentially expressed; 5 miRNAs were found up-regulated in the group of patients that are in complete remission when compared to the relapsed or the control groups; miR-3681 (A), miR-601 (B), miR-642a (C), miR-136 (D) and miR-26b (E). Additionally, three miRNAs were found up-regulated in relapsed patients when compared to the group of patients that are in Complete Remission (CR) or the control group; mIR-192 (F), miR-320e (G) and miR-34a (H). Finally, ten miRNAs were found overexpressed in the control group when compared to the patients group (relapsed or in Complete Remission (CR)); miR-720 (I), miR-891a (J), miR-522 (K), miR-518c (L), miR-3665 (M), miR-891a (N), miR-382 (O), miR-452 (P), miR-122 (Q), miR-147 (R). (DOCX 891 KB)13045_2014_96_MOESM7_ESM.docx
Additional file 8: Figure S5.: MicroRNA expression levels and survival following initial analysis. Kruskal-Wallis analysis between DE miRNAs and patient outcome; alive (n = 9) or deceased (n = 10). In total, 8 miRNAs were significantly different; five miRNAs weres found up-regulated in alive patients when compared to the group of deceased and control samples. In particular, miR-3681 (A), miR-642a (B), miR-26b (C), miR-136 (D) and miR-320e (E) were increased in alive samples as well as manifested linear regression with respect to expression moving from alive samples to controls. Two miRNAs were found down-regulated in the group of patients that remain alive when compared to the diseased or the control groups. In particular, miR-720 (F) and miR-891a (G) manifested similar linear regression increasing from alive samples to controls. Finally, one miRNA, miR-320c (H), manifested higher expression levels in deceased samples as compared to alive and control samples (* denotes a p < 0.05 significance). (DOCX 549 KB)13045_2014_96_MOESM8_ESM.docx
Additional file 9: Figure S1.: Analysis results of microarray data. Quantile normalization (A), T-test histograms (B), Volcano plot of T-test results (C), False Discovery Rate (FDR) of T-test results (D). (DOCX 1 MB)13045_2014_96_MOESM9_ESM.docx
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- Microrna expression signatures predict patient progression and disease outcome in pediatric embryonal central nervous system neoplasms
George I Lambrou
Diane K Birks
Chara A Spiliopoulou
- BioMed Central
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