Journal of Steroid Biochemistry & Molecular Biology 133 (2013) 129–139 Contents lists available at SciVerse ScienceDirect Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb Identification of microRNAs as a potential novel regulatory mechanism in HSD11B1 expression Yanyan Han, Claudia A. Staab-Weijnitz 1 , Guangming Xiong, Edmund Maser ∗ Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel, Germany article info Article history: Received 22 May 2012 Received in revised form 20 August 2012 Accepted 7 September 2012 Keywords: 11-Hydroxysteroid dehydrogenase type 1 Obesity Diabetes microRNA miR-561 miR-579 abstract 11-Hydroxysteroid dehydrogenase type 1 (11-HSD1, gene HSD11B1) converts glucocorticoid receptor- inert cortisone to receptor-active cortisol. Multiple evidence supports a causal role for 11-HSD1 in the current obesity epidemic. In obese, HSD11B1 expression is increased in adipose tissue but typically decreased in liver, and the underlying tissue-specific mechanisms are largely unknown. In this context, we investigated a potential role of microRNAs (miRNAs). We used several miRNA target prediction tools to identify possible candidates and a publicly available miRNA expression atlas to further select can- didates expressed in hepatocytes. Using a dual luciferase reporter assay, we identified three potential miRNAs, hsa-miR-340, -561 and -579, as potential negative regulators of HSD11B1 expression. Disrup- tion of the corresponding microRNA response elements abolished repression of luciferase activity for hsa-miR-561 and -579, but not for hsa-miR-340. Furthermore, levels of firefly luciferase mRNA were not changed by miR-561 and -579, indicating a mechanism based on translational repression rather than mRNA degradation. Finally, we were able to detect both, miR-561 and -579, in human total liver RNA by reverse-transcription-polymerase chain reaction (RT-PCR). According to the presented results, miR-561 and -579 are likely to be involved in the tissue-specific regulation of HSD11B1 expression. Moreover, literature findings and a pathway enrichment analysis support a potential role of these two miRNAs in glucocorticoid metabolism and signalling and associated diseases. © 2012 Elsevier Ltd. All rights reserved. 1. Introduction 11-Reduced glucocorticoids (cortisol in man, corticosterone in rodents) are secreted from the adrenal gland and fulfil various physiological functions via activation of the glucocorticoid receptor (GR). In nutrient metabolism, for instance, they act on several so- called glucocorticoid target tissues to ultimately mobilise energy reserves in stress situations: they stimulate lipolysis in adipose tis- sue as well as protein degradation and amino acid mobilisation in skeletal muscle, collectively yielding substrates for an increased gluconeogenic rate in the liver. At the same time peripheral glucose consumption is inhibited. Furthermore, glucocorticoids are well- known for their anti-inflammatory properties as they suppress the initiation and promote the resolution of inflammation. Abbreviations: AMO, anti-microRNA oligonucleotide; GR, glucocorticoid recep- tor; 11-HSD1, 11-hydroxysteroid dehydrogenase type 1; miRNA, microRNA; MRE, miRNA response element; RT-PCR, reverse transcription-polymerase chain reaction. ∗ Corresponding author. Tel.: +49 431 597 3540; fax: +49 431 597 3558. E-mail address: maser@toxi.uni-kiel.de (E. Maser). 1 Present address: Institute for Clinical Radiology, Ludwig-Maximilians- University, Pettenkoferstr. 8a, 80336 Munich, Germany. The microsomal enzyme 11-hydroxysteroid dehydrogenase type 1 (11-HSD1, gene name HSD11B1 or SDR26C1 [1]) per- forms intracellular cortisone reduction and thus, leads to the amplification of GR-active cortisol against a typically unfavourable systemic cortisol/cortisone ratio [2–5]. Considering the profound glucocorticoid effects on nutrient metabolism, it is not surpris- ing that deregulation of 11-HSD1 can lead to metabolic disease [6]. Correspondingly, a number of clinical studies have shown that overexpression of 11-HSD1 in adipose tissue correlates with obe- sity [7]. Furthermore, selective overexpression of 11-HSD1 in adipose tissue leads to obesity and all further symptoms of the metabolic syndrome in a mouse model, while 11-HSD1-deficient mice are protected from diet-induced obesity and most associated adverse health effects [8–11]. Clearly, there is strong evidence for an aetiological role of 11-HSD1 in the current obesity epidemic and 11-HSD1 has thus emerged as a major potential drug target in the prevention of the metabolic syndrome [7]. Regulation of HSD11B1 expression is multifactorial and highly tissue-specific [6,12]. For instance, pro-inflammatory cytokines induce HSD11B1 expression in adipocytes, but fail to do so in pri- mary hepatocytes [13,14]. Interestingly, increased 11-HSD1 levels in adipose tissue typically parallel unchanged or decreased 11- HSD1 levels in liver of obese patients and there are indications that 0960-0760/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jsbmb.2012.09.005