Thematic Review Series: Functional Regulation of Lipid Homeostasis by microRNA: Complexity of microRNA function and the role of isomiRs in lipid homeostasis

MicroRNAs (miRNAs) are key posttranscriptional regulators of biological pathways that govern lipid metabolic phenotypes. Recent advances in high-throughput small RNA sequencing technology have revealed the complex and dynamic repertoire of miRNAs. Specifically, it has been demonstrated that a single genomic locus can give rise to multiple, functionally distinct miRNA isoforms (isomiR). There are several mechanisms by which isomiRs can be generated, including processing heterogeneity and posttranscriptional modifications, such as RNA editing, exonuclease-mediated nucleotide trimming, and/or nontemplated nucleotide addition (NTA). NTAs are dominant at the 3′-end of a miRNA, are most commonly uridylation or adenlyation events, and are catalyzed by one or more of several nucleotidyl transferase enzymes. 3′ NTAs can affect miRNA stability and/or activity and are physiologically regulated, whereas modifications to the 5′-ends of miRNAs likely alter miRNA targeting activity. Recent evidence also suggests that the biogenesis of specific miRNAs, or small RNAs that act as miRNAs, can occur through unconventional mechanisms that circumvent key canonical miRNA processing steps. The unveiling of miRNA diversity has significantly added to our view of the complexity of miRNA function. In this review we present the current understanding of the biological relevance of isomiRs and their potential role in regulating lipid metabolism

Inhibition of miR-29 has a significant lipid-lowering benefit through suppression of lipogenic programs in liver

MicroRNAs (miRNAs) are important regulators and potential therapeutic targets of metabolic disease. In this study we show by in vivo administration of locked nucleic acid (LNA) inhibitors that suppression of endogenous miR-29 lowers plasma cholesterol levels by ~40%, commensurate with the effect of statins, and reduces fatty acid content in the liver by ~20%. Whole transcriptome sequencing of the liver reveals 883 genes dysregulated (612 down, 271 up) by inhibition of miR-29. The set of 612 down-regulated genes are most significantly over-represented in lipid synthesis pathways. Among the up-regulated genes are the anti-lipogenic deacetylase sirtuin 1 (Sirt1) and the anti-lipogenic transcription factor aryl hydrocarbon receptor (Ahr), the latter of which we demonstrate is a direct target of miR-29. In vitro radiolabeled acetate incorporation assays confirm that pharmacologic inhibition of miR-29 significantly reduces de novo cholesterol and fatty acid synthesis. Our findings indicate that miR-29 controls hepatic lipogenic programs, likely in part through regulation of Ahr and Sirt1, and therefore may represent a candidate therapeutic target for metabolic disorders such as dyslipidemia

Transcriptomic Analysis of Chronic Hepatitis B and C and Liver Cancer Reveals MicroRNA-Mediated Control of Cholesterol Synthesis Programs

ABSTRACT Chronic hepatitis B (CHB), chronic hepatitis C (CHC), and associated hepatocellular carcinoma (HCC) are characterized by cholesterol imbalance and dyslipidemia; however, the key regulatory drivers of these phenotypes are incompletely understood. Using gene expression microarrays and high-throughput sequencing of small RNAs, we performed integrative analysis of microRNA (miRNA) and gene expression in nonmalignant and matched cancer tissue samples from human subjects with CHB or CHC and HCC. We also carried out follow-up functional studies of specific miRNAs in a cell-based system. These studies led to four major findings. First, pathways affecting cholesterol homeostasis were among the most significantly overrepresented among genes dysregulated in chronic viral hepatitis and especially in tumor tissue. Second, for each disease state, specific miRNA signatures that included miRNAs not previously associated with chronic viral hepatitis, such as miR-1307 in CHC, were identified. Notably, a few miRNAs, including miR-27 and miR-224, were components of the miRNA signatures of all four disease states: CHB, CHC, CHB-associated HCC, and CHC-associated HCC. Third, using a statistical simulation method (miRHub) applied to the gene expression data, we identified candidate master miRNA regulators of pathways controlling cholesterol homeostasis in chronic viral hepatitis and HCC, including miR-21, miR-27, and miR-33. Last, we validated in human hepatoma cells that both miR-21 and miR-27 significantly repress cholesterol synthesis and that miR-27 does so in part through regulation of the gene that codes for the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase ( HMGCR ). IMPORTANCE Hepatitis B virus (HBV) and hepatitis C virus (HCV) are phylogenetically unrelated hepatotropic viruses that persistently infect hundreds of millions of people world-wide, often leading to chronic liver disease and hepatocellular carcinoma (HCC). Chronic hepatitis B (CHB), chronic hepatitis C (CHC), and associated HCC often lead to cholesterol imbalance and dyslipidemia. However, the regulatory mechanisms underlying the dysregulation of lipid pathways in these disease states are incompletely understood. MicroRNAs (miRNAs) have emerged as critical modulators of lipid homeostasis. Here we use a blend of genomic, molecular, and biochemical strategies to identify key miRNAs that drive the lipid phenotypes of chronic viral hepatitis and HCC. These findings provide a panoramic view of the miRNA landscape in chronic viral hepatitis, which could contribute to the development of novel and more-effective miRNA-based therapeutic strategies

MicroRNA-27b is a regulatory hub in lipid metabolism and is altered in dyslipidemia

Cellular and plasma lipid levels are tightly controlled by complex gene regulatory mechanisms. Elevated plasma lipid content, or hyperlipidemia, is a significant risk factor for cardiovascular morbidity and mortality. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and have emerged as important modulators of lipid homeostasis, but the extent of their role has not been systematically investigated. In this study, we performed high-throughput small RNA sequencing and detected approximately 150 miRNAs in mouse liver. We then employed an unbiased, in silico strategy to identify miRNA regulatory hubs in lipid metabolism, and miR-27b was identified as the strongest such hub in human and mouse liver. In addition, hepatic miR-27b levels were determined to be sensitive to plasma hyperlipidemia, as evidenced by its ~3-fold up-regulation in the liver of mice on a high-fat diet (42% calories from fat). Further, we showed in a human hepatocyte cell line (Huh7) that miR-27b regulates the expression (mRNA and protein) of several key lipid-metabolism genes, including Angptl3 and Gpam. Finally, we demonstrated that hepatic miR-27b and its target genes are inversely altered in a mouse model of dyslipidemia and atherosclerosis

MicroRNA-223 coordinates cholesterol homeostasis

Results from this study represent a breakthrough in our understanding of posttranscriptional control of cholesterol metabolism and how microRNAs (miRNAs) are at the heart of cholesterol regulatory circuitry and homeostasis. Although cells are adept at maintaining proper cholesterol levels, it was unknown how cells posttranscriptionally coordinate cholesterol uptake, efflux, and synthesis. MicroRNA-223 (miR-223) transcription and expression are maintained by cholesterol, and, as a feedback network, miR-223 inhibits cholesterol biosynthesis and uptake and increases cholesterol efflux. This study clearly demonstrates the extensive role that miRNAs play in coordinating metabolic adaptation to disease and general homeostasis. This work highlights a unique regulatory control point for cholesterol homeostasis and illustrates how important the study of miRNAs is to the greater understanding of dyslipidemia and cardiovascular disease

HDL-transferred microRNA-223 regulates ICAM-1 expression in endothelial cells

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223−/− mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL’s anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells

Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium

Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field

Intercellular transport of MicroRNAs

Extracellular microRNAs (miRNA) are present in most biological fluids, relatively stable, and hold great potential for disease biomarkers and novel therapeutics. Circulating miRNAs are transported by membrane-derived vesicles (exosomes and microparticles), lipoproteins, and other ribonucleoprotein complexes. Evidence suggests that miRNAs are selectively exported from cells with distinct signatures that have been found to be altered in many pathophysiologies, including cardiovascular disease. Protected from plasma ribonucleases by their carriers, functional miRNAs are delivered to recipient cells by various routes. Transferred miRNAs use cellular machinery to reduce target gene expression and alter cellular phenotype. Similar to soluble factors, miRNAs mediate cell-to-cell communication linking disparate cell types, diverse biological mechanisms, and homeostatic pathways. Although significant advances have been made, miRNA intercellular communication is full of complexities and many questions remain. This review brings into focus what is currently known and outstanding in a novel field of study with applicability to cardiovascular disease