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Title: Downregulation of miRNA expression in malignant germ cell tumours : mechanism and functional significance
Author: Ferraresso, Marta
ISNI:       0000 0004 7660 8426
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Germ cell tumours (GCTs) are clinically and pathologically heterogeneous neoplasms that arise at gonadal (testicular/ovarian) and extra-gonadal sites. The chemotherapy burden for patients with malignant germ cell tumours (mGCTs) that require treatment results in substantial longterm side-effects, and, furthermore, poor-risk patients have < 50% survival. Consequently, identifying common molecular changes and novel therapeutic targets in mGCTs is of major clinical importance. MicroRNAs are short, non-protein coding RNAs that regulate gene expression. We previously showed that miR-99a-5p/-100-5p and miR-125b-5p are among the most frequently underexpressed microRNAs in mGCTs, regardless of anatomical site, histological type or patient age. The present study investigates the upstream causes and downstream consequences of such under-expression. The mature form of miR-125b-5p is the product of two genomic loci, which form a cluster with either miR-99a-5p (on chromosome 21q) or miR-100-5p (on chromosome 11q). MiR-99a-5p/- 100-5p share identical 'seed' regions (at nucleotide positions 2-7), which determine their mRNA targets. Cross-reactivity experiment revealed that both miR-99a-5p and miR-100-5p probes were highly cross-reactive to each other's target (from 91% to 95%), indicating functional overlap. Linear regression analysis of qRT-PCR data reveals a strong positive correlation between miR-99a-5p/-100-5p and miR-125b-5p levels (R2 =0.989) in mGCTs, strongly suggesting co-regulation. Primary microRNA transcripts (pri-miR-99a/-100 and pri-miR-125b), and other genes that colocalise to these miRNA clusters (e.g. BLID on chromosome 11), were quantified by RT-qPCR in four representative cell lines - TCam2, 1411H, 2102Ep, and GCT44 - which were derived from a range of common histological types of mGCTs. A significant down-regulation (p < 0.0001) of all primary transcripts was observed, suggesting transcriptional repression of the entire cluster regions. Treatment of the cell lines with 5'-azacytidine resulted in significant upregulation of all three miRNAs (p < 0.002), as well as BLID (p < 0.02). The methylation status of potential CpG islands at the region of interest on chromosome 11 and chromosome 21 was therefore investigated by Pyrosequencing. Significant hyper methylation was found in 2102Ep, 1411H and GCT44 cell lines, suggesting that the miR-99a-5p/-100-5p and miR-125b-5p clusters are likely transcriptionally silenced by DNA methylation. To assess the functional relevance of these microRNAs in GCT progression, co-transfection of microRNA mimics (8.3 nM miR-99a-5p/-100-5p + 8.3 nM miR-125b-5p) was performed. A significant decrease in cell growth was seen in 1411H (p < 0.01) and TCam2 (p < 0.03) cells. To identify the mimics' downstream mRNA targets, HumanHT-12 v4 Expression Bead Chip (Illumina) mRNA arrays were used and data analysed using Sylamer. This analysis showed that mimic-treated cells were enriched in downregulated genes involved in pro-proliferative mechanisms. Among those, further functional characterisation focussed in particular on TRIM71, FGFR3, E2F7 and LIN28A. Moreover, restoring miR-99a-5p/-100-5p and miR-125b-5p in TCam2 cells also resulted in G0-G1 accumulation, consistent with a cell cycle effect. These data support a functionally important role for miR99a-5p/-100-5p and miR-125b-5p in GCT progression. They also raise the possibility of a therapeutic replenishment approach for treating these, and potentially other, tumours.
Supervisor: Coleman, Nicholas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: microRNA ; germ cell tumour ; methylation ; cell cycle regulator