Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506 ~ 514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-Associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.
Bibliographical noteFunding Information:
Clinical samples. A total of 15 frozen tumors from 14 TGCT patients (TGCT1–14) and 5 histopathologically verified NT (NT1-5) were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute, USA. Other investigators may have received specimens from the same subjects.
Acknowledgements. We thank Drs Leendert HJ Looijenga and Peter Andrews for TGCT cell lines; Dr John Higgins for tissue microarrays; the Cooperative Human Tissue Network for frozen tissue samples; the Stanford Sequencing Facility for sRNA-sequencing; and Dr Andrew Fire and the members of the sRNA group for their helps and suggestions. This work was supported by Swedish Research Council; Cancer Research Funds of Radiumhemmet; Swedish Cancer Society; Åke Wiberg’s Foundation; and Karolinska Institutet and Stockholm County Council. DM Özata was supported by the Karolinska Institutet PhD program (KID) and J Liu was supported by a China Scholarship Council training grant.