Targeting oncogenic microRNAs in malignant germ cell tumours.
University of Cambridge
Doctor of Philosophy (PhD)
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Bailey, S. (2021). Targeting oncogenic microRNAs in malignant germ cell tumours. (Doctoral thesis). https://doi.org/10.17863/CAM.69865
Malignant germ cell tumours (mGCTs) are a heterogenous group of tumours that occur primarily in the testes and ovaries but also in other anatomical sites, and affect people of all ages. It was established by the Coleman group through microRNA profiling in 2010 that all mGCTs, regardless of tumour site, patient age or histological subtype, display common microRNA profiles that segregate them from non-malignant controls. In particular, two clusters of miRNAs, miR-371~3 and miR-302/367 were found to be markedly over-expressed in mGCTs. MicroRNAs are short, non-protein-coding RNAs that regulate gene expression through binding of the microRNA seed region to the 3’untranslated region (3’UTR) of messenger RNAs, primarily resulting in mRNA degradation. Six of the eight main microRNAs from the two over-expressed clusters share an identical seed region ‘AAGUGC’, that has been found to down-regulate a range of functionally significant genes. This common seed region is associated with cisplatin resistance in GCT cells and has dose-dependent effects, with higher levels found in GCT subtypes with inferior outcomes. My hypothesis was that targeting these over-expressed microRNAs would confirm their functional significance, identify the pathways in which they function in mGCTs, and identify them as potential therapeutic targets. Multiple strategies were employed in the attempt to target these microRNAs, including gene deletion through the CRISPR/Cas9 system, targeting the primary transcript (pri-miRNA) of each cluster using locked nucleic acid (LNA)-based gapmeRs and targeting the mature miRNAs with peptide nucleic acid and LNA-based inhibitors. Targeting the mature microRNAs using LNA-based inhibitors resulted in a significant and reproducible negative growth phenotype. Gene expression profiling was performed, and although there was no de-repression of individual mRNAs, Sylamer analysis revealed significant enrichment of mRNAs containing the seed complementary region amongst upregulated genes. Pathway analysis of the upregulated mRNA targets demonstrated that these targets were primarily involved in cell cycle regulation. This was functionally validated through cell cycle analysis using flow cytometry. This demonstrated that inhibiting the key, over-expressed microRNAs with LNA-based inhibitors results in cell cycle arrest of mGCT cell lines.
germ cell tumour, AAGUGC, miR-302
Action Medical Research (GN224)
Embargo Lift Date
This record's DOI: https://doi.org/10.17863/CAM.69865
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