miRNA-302s may act as oncogenes in human testicular germ cell tumours

Testicular germ cell tumour (TGCT) represents the most common malignancy in young men in large parts of the world, but the aetiology is yet unclear. Multiple TGCT susceptibility loci have been identified, and we have shown that one of these, SPRY4, may act as a TGCT oncogene. Furthermore, many of the loci are in non-coding regions of the genome. miRNAs, a class of non-coding RNAs may play a crucial role in cell proliferation, differentiation, and apoptosis, and alteration in their expression may lead to oncogenesis. Differential expression of miRNAs in TGCT and normal testis has been reported in previous studies. In this study, we used qPCR to analyse, in normal and malignant testis tissue, the expression of the ten miRNAs that we had previously identified by sequencing to be the most upregulated in TGCT. We found high expression of these miRNAs also by qPCR analysis. The levels of miR-302a-3p, miR-302b-3p, and miR-302c-3p were downregulated after treatment of the TGCT cell lines NT2-D1 and 833 K with the chemotherapy drug cisplatin. By using miRNA inhibitor-mediated transient transfection, we inhibited the expression of the three members of miR-302 family (miR-302s). Inhibition of miR-302s resulted in a decreased cell proliferation in NT2-D1 cells, but not in 833 K cells. In both cell lines, inhibition of miR-302s resulted in decreased expression of SPRY4, which we have previously shown to regulate MAPK/ERK and PI3K/Akt signalling pathways in these cells. Inhibition of miR-302b-3p and miR-302c-3p decreased phosphorylation of ERK1/2, whereas inhibition of miR-302a-3p and miR-302b-3p led to decreased expression of the apoptosis inhibitor, survivin. Our findings suggest that miR-302s act as TGCT oncogenes by inducing the expression of SPRY4 and activating MAPK/ERK pathway while inhibiting apoptosis via increased survivin expression.