In this study we have demonstrated that the majority of miRNAs differentially expressed in pediatric malignant GCTs are down-regulated, as has been observed for other types of malignancy (32
). Nevertheless, the most significant differential expression was up-regulation of the miR-371~373 and miR-302 clusters, regardless of histological subtype, tumor site (ovary, testis or extragonadal) or patient age. Over-expression of these miRNAs appears to be specific to malignant GCTs, with no similar findings for other malignancies or diseases to date, save for miR-372~373 over-expression in an isolated case of an exceptionally rare embryonal brain tumor, at much lower levels than in malignant GCTs (33
The miR-371~373 cluster was previously reported to be over-expressed in adult gonadal malignant GCTs, based on qRT-PCR (14
) and RNase protection assay (15
). However, these reports are inconsistent regarding expression of the miR-302 cluster in such tumors. One stated that miR-302a~302d expression was undetectable in many miR-371~373-expressing malignant GCTs, consistent with miR-371~373 over-expression being a selected event in malignant GCT development (15
). In contrast, the other appears to illustrate over-expression of miR-302a~302d in all adult gonadal malignant GCTs, although this was not explicitly commented on (14
). Our re-analysis of the published qRT-PCR data shows that the miR-302 cluster is as significantly over-expressed as miR-371~373 in adult gonadal malignant GCTs compared to non-malignant tissues (teratomas and controls) (), mirroring our observation for pediatric gonadal and extragonadal malignant GCTs. As both miRNA clusters are believed to be ESC-specific pluripotency markers (34
), our findings suggest that expression of the miR-371~373 and miR-302 clusters in malignant GCTs either represents persistence of an embryonic pattern of miRNA expression that is not present in normal tissues and teratomas (the latter having undergone somatic differentiation), or acquired re-expression, regulated by an as yet undetermined mechanism. We observed associations between miR-371~373/miR-302 levels and TF over-expression, warranting future investigations of their functional relationships, which may be complex. For example, NANOG
have binding sites in the miR-302 (35
) and miR-371~373 (40
) cluster promoter regions, while POU5F1
is negatively regulated by miR-145 (41
) which is significantly down-regulated in both pediatric and adult malignant GCTs (Supplementary Table 2A/B
). Our observation of SOX17
over-expression in seminoma and YST, but not in an EC sample, is consistent with previous reports (42
Our Sylamer analysis strongly suggests that over-expression of the miR-371~373 and miR-302 clusters is functionally important in malignant GCTs by globally affecting levels of target mRNAs. We observed significant enrichment of the SCR hexamer GCACTT (complementary to the common miR-372~373 and miR-302a~302d 2-7nt seed AAGUGC) in genes under-expressed in pediatric malignant GCTs, YSTs and EC versus non-malignant controls and in adult YSTs versus testicular controls. Sylamer did not identify over-representation of SCRs corresponding to other over-expressed miRNAs. Nevertheless, such miRNAs may contribute to the clinicopathological heterogeneity of malignant GCTs, by targeting a smaller, more discrete number of mRNAs. For example, each subtype of pediatric malignant GCT showed specific abnormalities of miRNA expression (such as over-expression of miR-182~183 cluster in seminomas, miR-375 in YSTs and miR-515~526 cluster in ECs) and we observed significant differential miRNA expression in intracranial versus extracranial seminomas. Interestingly, however, the striking differences in mRNA expression that we previously observed between pediatric and adult malignant GCTs (2
) were not reflected by similar differences in miRNA expression profiles.
Using GO analysis, we demonstrated that for pediatric malignant GCTs, and their main subtypes YST and seminoma, the down-regulated mRNAs containing the SCR corresponding to the common miR-372~373/miR-302a~302d seed mediate cellular processes important in oncogenesis and malignant progression (signal transduction, cell cycle, development and morphogenesis, etc.), in contrast to the small number of metabolic processes identified for down-regulated mRNAs without the common SCR. Together, these findings indicate the generalized functional significance of miR-372~373 and miR-302a~302d in the biology of malignant GCTs. Interestingly, these miRNA clusters, via their common 2-7nt seed AAGUGC, are known to be essential for regulating G1-S transition and promoting rapid proliferation in embryonic stem cells (45
). Our data further support the use of GO enrichment analysis to identify groups of genes targeted by the same miRNA seed that share a biological function (47
). Of note, considerably weaker signals were obtained from the Sylamer and GO enrichment analysis of the adult mRNA dataset (26
), in which controls were normal adult testis samples only, leading to large numbers of differentially expressed genes being related to male reproduction and spermatogenesis. We obtained a more tractable list of differentially expressed genes by selecting a range of control tissues containing normal germ cells at different developmental stages.
In conclusion, our data indicate that the miR-371~373 and miR-302 clusters are universally over-expressed in malignant GCTs and are of functional significance by down-regulating mRNAs involved in biologically significant pathways. It will now be important to translate our findings clinically. The miRNA expression changes we describe may improve tumor diagnosis and post-treatment monitoring, and enable novel therapeutic approaches that target fundamental abnormalities of malignant GCT cells.