MicroRNAs (miRNAs) are endogenous ∼22 nucleotide (nt) RNAs that play essential gene-regulatory roles in animals and plants by pairing messenger RNAs (mRNAs) of protein-coding genes to direct their posttranscriptional repression by translational inhibition, deadenylation, and mRNA decay.1-3 The human genome is thought to encode over 1,000 miRNAs that regulate the expression of more than 60% of genes. Interestingly, a single miRNA may target multiple genes, potentially providing simultaneous regulation of the genes involved in a physiological pathway. In fact, the complexity in higher organisms is thought to be achieved through sophisticated control and coordinated mechanisms carried out by noncoding RNAs including miRNAs.
MiRNAs have recently emerged as key regulators of lipid metabolism, playing major roles in regulating cholesterol and fatty acid metabolism.4 Among these, miR-122 was the most widely studied miRNA and the first described for its role in regulating total serum cholesterol and hepatic metabolism.5, 6 miR-122 is highly expressed in the liver, and it is estimated to account for ∼75% of all liver miRNAs. Inhibition of miR-122 using antisense oligonucleotides significantly reduces plasma cholesterol levels and reverses hepatic steatosis in mice fed a high-fat diet.6 Similarly, silencing of miR-122 in nonhuman primates results in a significant reduction in plasma cholesterol.7 In addition to miR-122, miR-33a/b have recently been discovered as main regulators of lipid homeostasis. miR-33a/b are miRNAs encoded in intron 16 and 17 of the Srebp-2 and Srebp-1 genes, respectively.8-13 miR-33a/b are cotranscribed with their host genes and target genes involved in cellular cholesterol export, including the adenosine triphosphate binding cassette (ABC) transporters ABCA1 and ABCG1, and fatty acid metabolism, including carnitine O-acetyltransferase (CROT), carnitine palmitoyltransferase 1a (CPT1a), hydroxyacyl-CoA dehydrogenase beta subunit (HADHB), and AMP-activated protein kinase (AMPK). Notably, inhibition of miR-33 in mice and nonhuman primates using a variety of approaches increases hepatic ABCA1 expression and circulating high-density lipoprotein (HDL) levels.11-14 As expected by the significant increase in HDL levels observed in mice treated with anti-miR-33 oligonucleotides, the inhibition of miR-33 expression promotes reverse cholesterol transport and regression of atherosclerosis.15 Overexpression of miR-33 also represses genes involved in the regulation of fatty acid oxidation. Indeed, endogenous inhibition of miR-33 up-regulates CROT, CPT1a, HADHB, and AMPK expression, leading to an increase in β-oxidation.8 Later, Temel, Moore and colleagues confirmed the important role of miR-33 in regulating triglyceride metabolism in nonhuman primates.14
In addition to miR-122 and miR-33, other miRNAs have been shown to play an important role in the posttranscriptional regulation of lipid metabolism, including miR-370, miR-378/378*, miR-335, miR-27, and miR-125a-5p.16-20 miR-27b has been shown to regulate human adipocyte differentiation by directly targeting peroxisome proliferator-activated receptor (PPAR) gamma and C/EBPα, two key regulators of adipogenesis.19 Overexpression of miR-27b represses adipogenic marker gene expression and triglyceride accumulation. Moreover, miR-27b also inhibits PPARα, an important transcription factor that regulates genes encoding lipid-related genes including lipoprotein lipase (LPL) and ABCA1 and ABCG1 transporters.21 miR-27b is a member of the miR-27 microRNA family, encoded in chromosome 9 and clustered with other miRNAs such as miR-23b, miR-3074, and miR-24-1. The molecular mechanism that regulates its expression remains poorly understood. In addition to its role in lipid metabolism (Fig. 1), several reports have pointed out an important role for this miRNA in the cardiovascular system. miR-27b controls venous specification and tip cell fate by regulating the expression of Notch ligand delta-like ligand 4 and sprouty homolog 2.22 Moreover, miR-27a/b also regulates endothelial cell repulsion and angiogenesis by targeting semaphorin 6A and thrombospondin-1 (TSP-1).23, 24 Altogether, these reports suggest that miR-27 may play an important role in regulating lipid metabolism and vascular development. In this issue of HEPATOLOGY, Vickers et al.25 identify miR-27b while studying miRNA regulatory hubs in lipid metabolism using a novel in silico approach. A posttranscriptional “miRNA hub” in lipid metabolism is defined as an miRNA that is predicted to target more lipid metabolism-associated genes than expected by chance. The authors selected a list of 151 lipid-associated genes using three published high-throughput screens. Target sites for three hepatic miRNAs (miR-27b, miR-128, and miR-365) were significantly overrepresented in the 151 known lipid metabolism genes. miR-27b was identified as the strongest such hub in human and mouse liver, with 27 predicted targets. To identify whether or not miR-27b regulates the expression of these predicted target genes, the authors overexpressed miR-27b in the human hepatocarcinoma cell line (Huh7). Huh7 cells transfected with miR-27 mimics showed a significant inhibition of PPARγ, angiopoetin-like 3 (ANGPTL3), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and mitochondrial glycerol-3-phosphate acyltransferase 1 (GPAM). Conversely, endogenous inhibition of miR-27b led to an increase in the expression of these target genes. Altogether, these data strongly suggest that miR-27b regulates lipid metabolism. Another interesting observation is the inverse correlation between the expression of miR-27b and its predicted targets (ANGPTL3 and GPAM), suggesting a potential link between the expression of miR-27b and these genes. Nevertheless, the role of miR-27 in regulating lipid metabolism in vivo remains unclear. Therefore, it would be important to assess whether the inhibition of miR-27b using antisense oligonucleotides influences ANGPTL3 and GPAM expression and hepatic lipid metabolism.
The authors also show that miR-27 is increased in the liver of mice fed a high-fat diet, suggesting that its expression is regulated by lipid content. Similarly, Lin et al.19 found that miR-27a and miR-27b expression were increased in obese mice. Interestingly, the primary transcript of miR-27b (pri-miR-27b) was not affected by dietary lipids in CBL657 mice fed a high-fat diet. This result indicates that miR-27b expression is likely regulated by posttranscriptional processing of pri-miR-27b. Why the pri-miR-27b processing is affected by lipid content and how specific this mechanism is for miR-27 are important questions that remain to be answered. It would also be interesting to assess whether the other 50 miRNAs up-regulated in livers from mice fed a high-fat diet are also up-regulated at the posttranscriptional level.
In addition to miR-27b, miR-27a is a member of the miR-27 miRNA family. Interestingly, miR-27a was also significantly up-regulated in mice fed a high-fat diet. Both miRNAs have the same seed sequence and target similar genes. Therefore, the inverse correlation between the expression of miR-27a/b and their predicted target genes in mice fed a high-fat diet may be due to the combined effect of both miRNAs.
Finally, this article also opens new questions that need to be further explored, including the contribution of miR-27 in regulating lipid metabolism in other relevant cells, such as macrophages and neurons, and how miR-27 therapy may have an effect in models of experimental atherosclerosis and obesity. Moreover, this study elegantly demonstrates the ability of a new in silico approach to identify the functional relevance of miRNAs in regulating gene networks involved in the same physiological pathway. This approach may be used in other studies to identify the relevance of miRNAs in controlling genetic networks.