Author: Kanchana Veronika Bandara
Bandara, Kanchana Veronika, 2014 Hypoxic regulation of microRNA biogenesis, Flinders University, School of Medicine
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Hypoxia is a key feature of many cancers and the presence of hypoxia is associated with more aggressive and metastatic tumours. MicroRNAs are 17-22 nucleotides, non-coding, single stranded RNA that are important regulators of gene expression. Functional studies show that microRNAs are involved in regulating many cellular processes including developmental timing, cell differentiation, cell proliferation and cell death. The expression levels of many microRNAs are deregulated in human disease conditions including cancer. In addition to deregulation of specific microRNAs in cancer, it has emerged that most tumour cell lines and cancers are characterised by global reductions in microRNA expression when compared to adjacent normal tissue. Cancers are commonly characterised by hypoxia and also by global reductions in the levels of mature microRNAs. This thesis examined the hypothesis that hypoxia mediates the global reduction of microRNAs through repressive effects on microRNA biogenesis proteins. Cancer cell lines were exposed to hypoxia and manipulations of hypoxia inducible factor (HIF) and HIF hydroxylase activity. The effects of hypoxia on the mRNA and protein levels of enzymes involved in microRNA biogenesis (DICER, DROSHA, TARPB2, DCGR8, XPO5) were determined by RT PCR and immunoblotting. The effect of hypoxia on microRNA biogenesis and function was determined with microarrays, RT PCR, activity assays and reporter assays. In two breast cancer lines (MCF7 and SKBR3), a colorectal cancer cell line (HT29) and a non-cancer cell line (HUVEC) there were significant reductions of DICER mRNA and protein levels after exposure to hypoxia. This effect was independent of HIF but dependent on the HIF hydroxylase PHD2 and was partly mediated by feedback effects by microRNAs. Furthermore, several other proteins with critical roles in microRNA biogenesis such as DROSHA, DGCR8, TARBP2 and XPO5 also showed significant and co-ordinated repression under hypoxic conditions. The significant and consistent reduction in the levels of proteins with central roles in microRNA biogenesis under hypoxia did not have a substantial effect on the expression levels of mature microRNAs over the time course of these experiments. Even though hypoxia exerted only modest effects on the production of mature microRNAs, a significant influence of hypoxia on the function of exogenously introduced precursor microRNA was observed. These observations provide further and important interfaces between oxygen availability and gene expression and a potential mechanistic explanation for the reduced levels of microRNAs observed in some cancers. They provide further support for the existence of feedback mechanisms in the regulation of the microRNA biogenesis pathway and the relative stability of microRNAs.
Subject: Medicine thesis
Thesis type: Doctor of Philosophy
School: School of Medicine
Supervisor: Professor Jonathan Gleadle