Identification of microRNAs as potential novel regulators of HSD11B1 expression
11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1, gene name HSD11B1) is a ubiquitously expressed enzyme that converts glucocorticoid receptor-inert cortisone to receptor-active cortisol. HSD11B1 expression is regulated in a highly tissue-specific manner by immunomodulatory and metabolic regulators. Multiple evidences support a causal role for 11β-HSD1 in the current obesity epidemic. In obese people, HSD11B1 expression is increased in adipose tissue, but typically decreased in liver, and the underlying tissue-specific mechanisms are largely unknown. In this context, a potential role of microRNAs (miRNAs) was investigated. Four different miRNA target prediction tools were used to choose possible candidates and a publicly available miRNA expression atlas to further select candidates expressed in hepatocytes. Using a luciferase reporter assay, where the complete 3’UTR of HSD11B1 mRNA was inserted downstream of the gene for firefly luciferase, three potential miRNAs, hsa-miR-561, -579 and -340 were identified as potential negative regulators of HSD11B1 expression. Moreover, disruption of the corresponding microRNA response elements (MREs) abolished repression of luciferase activity for hsa-miR-561 and -579, but not completely for hsa-miR-340. Therefore, hsa-miR-561 and hsa-miR-579-mediated downregulation of HSD11B1 expression are strictly dependent on the binding of miR-561- and miR-579-MRE in the 3’UTR of HSD11B1 mRNA. Levels of firefly luciferase mRNA were not changed by miR-561 and -579; and levels of endogenous HSD11B1 mRNA were as well unchanged by miR-561 and -579, indicating a mechanism based on translational repression rather than on mRNA degradation. Interestingly, hsa-miR-579 was still performing downregulation of HSD11B1 expression after treatment with glucocorticoids to induce HSD11B1 expression, due to different regulatory mechanisms for HSD11B1 expression by glucocorticoids and miRNAs in a dual luciferase assay system. The function of miR-561 and -579 could be blocked by anti-microRNA oligonucleotides (AMOs). MiR-561 and -579 were amplified by specific stem-loop reverse transcription primers and specific PCR primers from human hepatocytes and HepG2 cells. Although their relative contribution to HSD11B1 expression remains unclear, literature findings and a pathway enrichment analysis of miR-561 and -579 target mRNAs support a role of these miRNAs in glucocorticoid metabolism/signalling and associated diseases.
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