PO-052 Significance of miR-100–5 P/99A-5P and MIR-125B-5P under-expression in malignant germ cell tumours

Abstract

IntroductionGerm-cell-tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. They occur across a wide age-range, from early childhood to late adulthood. Poor-risk patients have <50% survival and the chemotherapy burden results in substantial long-term side-effects. Identifying common molecular changes and therapeutic targets in GCTs is of major clinical importance.Material and methodsFour representative Malignant Germ Cell Tumour (mGCT) cell lines (TCam2, 1411 hour, GCT44, 2102Ep), were co-transfected using 16 nM mimics (8.3 nM miR-99a-5p/100–5p+8.3 nM miR-125b-5p) in order to assess the functional relevance of the miRNAs in cancer progression. Transfected cells were then analysed using Illumina mRNA arrays to assess the genes mostly affected by the treatment.Results and discussionsWe previously showed that miR-100–5 p/99a-5p/125b-5p are among the most frequently under-expressed microRNAs in malignant GCTs.The mature form of miR-125b-5p is the product of two genomic loci, which cluster with either miR-99a-5p on chromosome21, or miR-100–5 p on chromosome11. MiR-100–5 p/99a-5p share identical seed regions, which determine their mRNA targets, indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-100–5 p/99a-5p and miR-125b-5p (R2=0.9) in malignant GCTs, suggesting co-regulation.After mimics transfection, a significant decrease in cell growth was seen in 1411 hour (p<0.01) and TCam2 (p<0.03) at day 7. We used Illumina mRNA arrays to analyse the genes that were mostly affected by mimic treatment at days 2 and 7 and observed an enrichment in genes involved in cell proliferation/apoptosis, in particular TRIM71, FGFR3, E2F7 and LIN28A. Restoring miR-100–5 p/99a-5p/125b-5p in TCam2 cells also resulted in G0-G1 accumulation from day 3.Previous work in our lab demonstrated that LIN28A knockdown resulted in de-repression of let-7 (a class of microRNAs that are highly downregulated in GCTs). Its downregulation led to de-repression of let-7 implying a contribution of miR-125b-5p in this regulatory pathway.ConclusionThese data support a significant role of miR99a-100–5 p/125b-5p in GCT progression. We discovered that replenishment of these miRNAs can also reduce downregulation of other miRNAs, opening the possibility that multiple benefit can be achieved with a therapeutic replenishment approach for treating these and potentially other tumours.