Regulation of adrenal corticosteroidogenesis: the role ofmicroRNAs in the control of aldosterone synthase and11β-hydroxylase expression

R Medicine (General);Q Science (General)

Wood, Stacy ; Davies, Eleanor

University:University of Glasgow

Department:Institute of Cardiovascular and Medical Sciences

关键词: Aldosterone, Cortisol, adrenal, APA, CYP11B1, CYP11B2, microRNA, H295R, 3'UTR;   

Others  :  http://theses.gla.ac.uk/3252/1/2011WoodPhD.pdf

来源: University of Glasgow

【 摘 要 】

Hypertension is a major risk factor for all cardiovascular disease, which is thelargest known cause of global mortality. Essential hypertension-that ishypertension of unknown cause-is thought to have genetic and environmentalrisk factors. The best studied genetic system is that concerning corticosteroidbiosynthesis. In humans, the principal glucocorticoid is cortisol, the mainfunction of which is the control of intermediary metabolism; the majormineralocorticoid is aldosterone, which affects electrolyte and acid-basehomeostasis. These steroid hormones are produced in the adrenal cortex througha series of biosynthetic reactions and under the influence of multiple regulatoryfactors. The final step in cortisol and aldosterone production involves,respectively, the cytochrome p450 enzymes, 11β-hydroxylase and aldosteronesynthase. These are encoded by the CYP11B1 and CYP11B2 genes which have asimilar sequence and are highly polymorphic and lie, in tandem, on humanchromosome 8.Regulation of CYP11B1 and CYP11B2 mRNA abundance and of aldosterone andcortisol production have been extensively investigated. These studies haveidentified that there are several polymorphisms located across the locus whichare associated with an increased aldosterone to renin ratio (ARR; used as anindicator of aldosterone regulation), inefficient 11β-hydroxylation and essentialhypertension. However, to date, no underlying mechanism for these associationshas been established. Regulation of expression by transcription factors has beenwidely 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 abundanceThey are transcribed from endogenous loci, then undergo a series of enzymaticmaturation reactions that result in the production of a single-stranded moleculeof approximately 20 nucleotides. They function by associating with a group ofproteins known as the RNA-induced silencing complex (RISC) and targeting the 3’untranslated region (3’UTR) of specific target mRNAs which they bind withimperfect complementarity. There are approximately 1100 human miRNAs,which have been implicated in the regulation of a range of target mRNAs and inseveral pathologies including cancer and cardiovascular disease. The aim of thisproject was to investigate what role, if any, miRNAs have in the regulation ofCYP11B1 and CYP11B2 expression and in corticosteroid production.The studies in Chapter 3 investigated miRNA regulation of corticosteroidogenesisin the adrenal cell line H295R. miRNA levels were universally reduced bytargeting Dicer mRNA, a key component of the miRNA synthetic pathway, withshort interfering RNA (siRNA). This study identified all of the CYP450 enzymes ofthe corticosteroidogenic pathway (CYP11A1, CYP17A1, CYP21A1, CYP11B1 andCYP11B2) as likely candidates for miR-mediated regulation based on mRNA andsteroid analysis. The study also suggested that StAR, 3βHSDII and 11βHSDII arenot modulated by miRNAs. To determine whether apparent miRNA regulation ofCYP11B1 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 the3’UTR of CYP11B1 and CYP11B2. This chapter therefore provides evidence formiRNA-mediated regulation of corticosteroidogenesis.In Chapter 4, putative miRNA target sites in the CYP11B1 and CYP11B2 3’UTRwere identified using bioinformatic prediction algorithms and the miRNAexpression profile of the normal human adrenal, as determined by microarrayanalysis. Based on miRNA target site prediction and analyses of the 3’UTRsequences (including such parameters as relative length, predicted sequenceconservation and RNA secondary structure), in silico methods indicated thepossibility 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 16adrenal miRNAs predicted to bind putative sites in CYP11B1 and 16 predicted tobind 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 testedindividually in vitro. This was achieved by measuring mRNA expression, steroidproduction and 3’UTR reporter construct activity following artificially inducedincreases or reductions in the levels of specific miRNAs. These studies identifiedsome miRNAs as being false positive predictions, while certain others werevalidated. The miRNA that gave the most striking and consistent results, fortargeting both CYP11B1 and CYP11B2, was miR-24, which significantly decreasedmRNA levels and steroid production. Analysis of adrenal miRNAs predicted onlyto target the CYP11B2 3’UTR confirmed miR-125a-5p and miR-125b as novelregulators, although effects on steroid secretion remain to be assessed. Thestudies in this chapter are the first to report of miRNA-mediated regulation ofCYP11B1 and CYP11B2 expression.Finally, in Chapter 6, the miRNA expression profiles of four aldosterone producingadenoma (APA) samples were generated and compared to those ofnormal 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 expressedbetween the tissue types. This chapter also describes polymorphisms within the3’UTR of the CYP11B1 gene, generated from 26 normotensive patients. No novelSNPs were identified, but three are located in putative miRNA-binding sites.Previously, sequence analysis of the CYP11B2 3’UTR had been used to mapmiRNA binding sites, this identified two miRNA-binding sites which mapped to aknown SNP. Taken together, the studies in this chapter provide a foundation forexploring altered miRNA function and/or expression within the adrenal gland.In summary, the results presented in this thesis support a role for miRNA mediatedregulation of corticosteroidogenesis through actions on CYP11B1 andCYP11B2 expression. It demonstrates that miRNA are present in the adrenalgland, that miRNA-binding sites are present on the 3’UTR of relevant mRNAs,and that miRNAs are capable of post-transcriptional regulation that significantlyalters mRNA abundance and steroid production. My findings describe a novelregulatory mechanism of corticosteroidogenesis. Whether this mechanism isaltered in diseases such as essential hypertension remains to be elucidated. Ifso, miRNAs could, in the longer term, be used as targets for novel therapies oras biomarkers to classify more precisely specific pathologies.