Hypertension is a major risk factor for all cardiovascular disease, which is the largest known cause of global mortality. Essential hypertension-that is hypertension of unknown cause-is thought to have genetic and environmental risk factors. The best studied genetic system is that concerning corticosteroid biosynthesis. In humans, the principal glucocorticoid is cortisol, the main function of which is the control of intermediary metabolism; the major mineralocorticoid is aldosterone, which affects electrolyte and acid-base homeostasis. These steroid hormones are produced in the adrenal cortex through a series of biosynthetic reactions and under the influence of multiple regulatory factors. The final step in cortisol and aldosterone production involves, respectively, the cytochrome p450 enzymes, 11β-hydroxylase and aldosterone synthase. These are encoded by the CYP11B1 and CYP11B2 genes which have a similar sequence and are highly polymorphic and lie, in tandem, on human chromosome 8. Regulation of CYP11B1 and CYP11B2 mRNA abundance and of aldosterone and cortisol production have been extensively investigated. These studies have identified that there are several polymorphisms located across the locus which are associated with an increased aldosterone to renin ratio (ARR; used as an indicator of aldosterone regulation), inefficient 11β-hydroxylation and essential hypertension. However, to date, no underlying mechanism for these associations has been established. Regulation of expression by transcription factors has been widely studied but, in this thesis, it is the role of a novel regulator, microRNA (miRNA) that is central. miRNAs are short, non-coding RNAs which negatively regulate mRNA abundance They are transcribed from endogenous loci, then undergo a series of enzymatic maturation reactions that result in the production of a single-stranded molecule of approximately 20 nucleotides. They function by associating with a group of proteins known as the RNA-induced silencing complex (RISC) and targeting the 3’ untranslated region (3’UTR) of specific target mRNAs which they bind with imperfect complementarity. There are approximately 1100 human miRNAs, which have been implicated in the regulation of a range of target mRNAs and in several pathologies including cancer and cardiovascular disease. The aim of this project was to investigate what role, if any, miRNAs have in the regulation of CYP11B1 and CYP11B2 expression and in corticosteroid production. The studies in Chapter 3 investigated miRNA regulation of corticosteroidogenesis in the adrenal cell line H295R. miRNA levels were universally reduced by targeting Dicer mRNA, a key component of the miRNA synthetic pathway, with short interfering RNA (siRNA). This study identified all of the CYP450 enzymes of the corticosteroidogenic pathway (CYP11A1, CYP17A1, CYP21A1, CYP11B1 and CYP11B2) as likely candidates for miR-mediated regulation based on mRNA and steroid analysis. The study also suggested that StAR, 3βHSDII and 11βHSDII are not modulated by miRNAs. To determine whether apparent miRNA regulation of CYP11B1 and CYP11B2 expression occurs by direct action at their 3’UTRs, reporter constructs were generated and tested. Under both basal and stimulated (AngII) conditions, these studies support a regulatory mechanism involving the 3’UTR of CYP11B1 and CYP11B2. This chapter therefore provides evidence for miRNA-mediated regulation of corticosteroidogenesis. In Chapter 4, putative miRNA target sites in the CYP11B1 and CYP11B2 3’UTR were identified using bioinformatic prediction algorithms and the miRNA expression profile of the normal human adrenal, as determined by microarray analysis. Based on miRNA target site prediction and analyses of the 3’UTR sequences (including such parameters as relative length, predicted sequence conservation and RNA secondary structure), in silico methods indicated the possibility that miRNAs can target CYP11B1 and CYP11B2 mRNA. Furthermore, the expression of 107 miRNAs in the normal adrenal gland was confirmed. Cross-referencing of microarray expression and bioinformatic data identified 16 adrenal miRNAs predicted to bind putative sites in CYP11B1 and 16 predicted to bind CYP11B2; 12 of these miRNAs were common to both genes. These formed the basis of the miRNA target validation studies in Chapter 5. Sixteen adrenal miRNAs identified by bioinformatic analysis were tested individually in vitro. This was achieved by measuring mRNA expression, steroid production and 3’UTR reporter construct activity following artificially induced increases or reductions in the levels of specific miRNAs. These studies identified some miRNAs as being false positive predictions, while certain others were validated. The miRNA that gave the most striking and consistent results, for targeting both CYP11B1 and CYP11B2, was miR-24, which significantly decreased mRNA levels and steroid production. Analysis of adrenal miRNAs predicted only to target the CYP11B2 3’UTR confirmed miR-125a-5p and miR-125b as novel regulators, although effects on steroid secretion remain to be assessed. The studies in this chapter are the first to report of miRNA-mediated regulation of CYP11B1 and CYP11B2 expression. Finally, in Chapter 6, the miRNA expression profiles of four aldosterone producing adenoma (APA) samples were generated and compared to those of normal adrenal gland. Analysis identified 67 miRNAs expressed within the APAs; 54 were also present in the normal tissue. The levels of several miRNAs, including miR-24 and miR-125a-5p, were shown to be differentially expressed between the tissue types. This chapter also describes polymorphisms within the 3’UTR of the CYP11B1 gene, generated from 26 normotensive patients. No novel SNPs were identified, but three are located in putative miRNA-binding sites. Previously, sequence analysis of the CYP11B2 3’UTR had been used to map miRNA binding sites, this identified two miRNA-binding sites which mapped to a known SNP. Taken together, the studies in this chapter provide a foundation for exploring altered miRNA function and/or expression within the adrenal gland. In summary, the results presented in this thesis support a role for miRNA mediated regulation of corticosteroidogenesis through actions on CYP11B1 and CYP11B2 expression. It demonstrates that miRNA are present in the adrenal gland, that miRNA-binding sites are present on the 3’UTR of relevant mRNAs, and that miRNAs are capable of post-transcriptional regulation that significantly alters mRNA abundance and steroid production. My findings describe a novel regulatory mechanism of corticosteroidogenesis. Whether this mechanism is altered in diseases such as essential hypertension remains to be elucidated. If so, miRNAs could, in the longer term, be used as targets for novel therapies or as biomarkers to classify more precisely specific pathologies.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:643080 |
| Date | January 2012 |
| Creators | Wood, Stacy |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/3252/ |
Page generated in 0.0192 seconds