2.1 Introduction

Proper maintenance of cellular and plasma cholesterol levels is critical for proper metabolic function and as such is regulated through tightly controlled mechanisms at both the transcriptional and posttranscriptional level. Cardiometabolic diseases, including atherosclerosis, a prominent cause of human morbidity and mortality in western societies (Glass and Witztum 2001; Lusis 2000), are caused in large part by dysregulation of cholesterol and lipid homeostasis. Although many environmental and genetic factors are known to contribute to atherogenesis, elevated levels of low-density lipoprotein cholesterol (LDL-C) are the primary risk factors for atherosclerosis and are sufficient to drive the progression of this disease. For this reason, the pathways governing plasma LDL-C levels have been extensively studied, and their modulation has led to effective therapies for the treatment of atherosclerosis.

The primary treatment option for patients with hypercholesterolemia is statins, a class of drugs which competitively inhibit 3-hydroxy-3methylgutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthesis pathway (Steinberg 2008; Steinberg et al. 2008). Reduced intracellular cholesterol synthesis in the liver, in response to statins, leads to activation of the sterol regulatory element-binding protein (SREBP), which increases the expression of the LDL receptor (LDLR). This in turn promotes LDL-C uptake from the blood and reduces levels of pro-atherogenic lipoproteins in circulation (Brown and Goldstein 1976). Although statin therapies have proven to be effective at reducing cholesterol levels and limiting cardiovascular-related deaths, some patients have poor tolerance for statin therapies, and the majority of patients still experience adverse coronary events despite treatment with statins (Steinberg 2008; Steinberg et al. 2008). As a result, developing novel approaches for lowering cholesterol that can be used alone or in combination with statins has become a major goal for cardiovascular research. Among these, approaches that modulate noncoding RNAs, including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs), have generated a great deal of interest due to their ability to regulate key pathways in cholesterol metabolism and their dysregulation in different disease states (Grundy 2008; Grundy et al. 2004). In this review, we highlight recent work demonstrating the importance of miRNAs and lncRNAs in regulation of cholesterol metabolism and discuss the potential of noncoding RNA-based therapeutic approaches for the treatment of atherosclerosis.

2.2 miRNAs Regulating HDL-C Metabolism and Reverse Cholesterol Transport

In addition to lowering levels of circulating pro-atherogenic lipids, much interest has recently been focused upon developing strategies to promote the removal of cholesterol from arterial macrophages, as a means to limit plaque progression and promote plaque regression. This process, known as reverse cholesterol transport, involves efflux of cholesterol esters from arterial macrophages by the lipid transporters ATP-binding cassette transporter A1 (ABCA1) and ABCG1 onto circulating high-density lipoprotein cholesterol (HDL-C) molecules (Brooks-Wilson et al. 1999; Gelissen et al. 2006; Oram and Vaughan 2000). These cholesterol esters are then transported to the liver where they can be converted into bile acids and removed from the body (Brooks-Wilson et al. 1999; Gelissen et al. 2006; Oram and Vaughan 2000). In recent years, numerous miRNAs have been shown to control numerous aspects of the reverse cholesterol transport pathway, including HDL biogenesis and uptake, cellular cholesterol efflux, and bile acid synthesis and secretion (Fig. 2.1). These include miR-10b, miR-27b, miR-33, miR-96, miR-125a, miR-128a, miR-144, miR-148a, miR-185, miR-223, miR-302a, and miR-455 (De Aguiar Vallim et al. 2013; Goedeke et al. 2015a, b; Ramirez et al. 2013b; Vickers et al. 2014; Wagschal et al. 2015; Wang et al. 2012, 2013). While all of these miRNAs have the potential to be important regulators of reverse cholesterol transport and atherogenesis, only a few have thus far been shown to have a significant impact on atherosclerotic plaque progression. Two independent groups have identified miR-144 as an important regulator of ABCA1 expression in both monocyte/macrophages and the liver (De Aguiar Vallim et al. 2013; Ramirez et al. 2013b). These studies further demonstrate that overexpression of miR-144 decreases circulating HDL-C, while inhibition of miR-144 was found to increase circulating HDL-C. Importantly, further work has since demonstrated that administering miR-144 mimics to ApoE −/− mice on a pro-atherogenic diet reduces hepatic ABCA1 expression and plasma HDL-C levels leading to increased atherosclerotic plaque formation (Hu et al. 2014). More recently, miR-302a has also been found to control the expression of ABCA1, thereby influencing HDL-C levels. Treatment with inhibitors of miR-302a in vivo was demonstrated to increase hepatic ABCA1 and plasma HDL-C leading to reduce atherosclerosis (Meiler et al. 2015). While most of these miRNAs have not been extensively studied for their role in regulating atherosclerosis, a great deal of work has been done exploring the role of the miR-33 family of miRNAs in regulation of this disease.

Fig. 2.1
figure 1

miRNA regulation of HDL-C metabolism. Schematic overview of miRNAs involved in the regulation of HDL-C metabolism. Blue boxes highlight miRNAs, which regulate genes that control HDL-C. ABC indicates ATP-binding cassette, SR-BI scavenger receptor B1, FXR farnesoid X receptor, and LCAT lecithin–cholesterol acyltransferase (Figure was created using the Servier Medical Art illustration resources (http://www.servier.com))

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2.2.1 miR-33a/miR-33b

The miR-33 family consists of two members, miR-33a and miR-33b. These are intronic miRNAs, which are encoded within the SREBP2 and SREBP1 genes, respectively (Najafi-Shoushtari et al. 2010; Rayner et al. 2010; Marquart et al. 2010; Gerin et al. 2010; Horie et al. 2010). The SREBP1 transcription factor is regulated in response to factors such as insulin and LXR ligands and is primarily responsible for the induction of genes involved in fatty acid synthesis (Horton et al. 2002). SREBP2, on the other hand, is regulated by changes in sterol levels and is the primary factor involved in the induction of genes regulating cholesterol biosynthesis and uptake, thereby allowing cells to carefully regulate their intracellular cholesterol levels (Horton et al. 2002). miR-33a and miR-33b have been demonstrated to be co-transcribed along with their host genes, so conditions that modulate the expression of the SREBP genes result in concomitant changes in miR-33 levels (Rayner et al. 2010; Najafi-Shoushtari et al. 2010; Marquart et al. 2010). While the SREBP transcription factors have different target gene specificities, the miR-33 isotypes share the same seed sequence and are therefore believed to target the same set of genes. However, it is not clear whether slight differences in the mature sequence of these miRNAs may alter their affinity for certain targets.

The important role of miR-33 in regulating HDL-C metabolism was first revealed in studies showing that miR-33 could target the cholesterol transporters ABCA1 and ABCG1, both in hepatocytes and macrophages (Najafi-Shoushtari et al. 2010; Rayner et al. 2010; Marquart et al. 2010; Gerin et al. 2010; Horie et al. 2010). Overexpression of miR-33 reduced the expression of ABCA1 and ABCG1 in the liver of mice and decreased plasma HDL-C (Marquart et al. 2010; Najafi-Shoushtari et al. 2010; Rayner et al. 2010; Gerin et al. 2010). Conversely, antisense oligonucleotides (ASOs) that reduced miR-33 level increased the expression of ABCA1 and ABCG1 and raised plasma HDL-C (Rayner et al. 2010; Najafi-Shoushtari et al. 2010; Marquart et al. 2010). Similarly, genetic loss of miR-33 in mice was reported to increase liver ABCA1 expression and plasma HDL-C (Horie et al. 2010). Importantly, inhibition of miR-33 in nonhuman primates was also demonstrated to significantly increase plasma HDL-C (Rayner et al. 2011a; Rottiers et al. 2013). Additionally, miR-33 was found to regulate hepatic bile acid synthesis through targeting of CYP7a1 (Li et al. Hepatology 2013) and secretion via modulation of ABCB11 and ATP8B1 (Allen et al. 2012). Due to its key role in regulating the removal of cholesterol from macrophages, HDL-C biogenesis in the liver, and bile acid metabolism, miR-33 was proposed to be an important regulator of reverse cholesterol transport and the progression of atherosclerosis (Allen et al. 2012; Rayner et al. 2011b). Collectively, these data indicated that ASOs against miR-33 might be a promising therapeutic option to raise HDL-C and treat patients with atherosclerosis.

2.2.2 miR-33a/miR-33b and Atherosclerosis

In response to these initial findings, numerous studies have been performed assessing the impact of miR-33 antagonism on atherosclerosis in mice. The first study of this sort demonstrated that short-term (4-week) administration of miR-33 inhibitors to Ldlr −/− mice with established plaques was capable of raising HDL-C levels and promoting atherosclerotic plaque regression (Rayner et al. 2011b). However, another study found that treatment with anti-miR-33 ASOs did not sustain elevated plasma HDL-C levels during a 12-week atherosclerosis progression study, raised plasma triglycerides, and did not have any effect on plaque development (Marquart et al. 2013). Three additional atherosclerosis progression studies using Ldlr −/− (Rotllan et al. 2013; Ouimet et al. 2015) and ApoE −/− mice (Karunakaran et al. 2015b) reported a decrease in plaque burden in animals treated with miR-33 inhibitors. Surprisingly, although the expression of ABCA1 in the liver and/or macrophages was elevated in mice treated with miR-33 ASOs, HDL-C remained unchanged in these progression studies (Rotllan et al. 2013; Ouimet et al. 2015; Karunakaran et al. 2015b). Together, these findings suggest that the anti-atherosclerotic effects of the ASOs may be the result of direct effects on the plaque rather than alterations in circulating lipids. Consistent with this theory, anti-miR-33 ASOs were found to be efficiently taken up by macrophages in the aortic root of Ldlr −/− mice, which could promote ABCA1-/ABCG1-mediated cholesterol efflux. Additionally, it has been reported that anti-miR-33 ASOs decreased inflammatory burden and promoted the recruitment of M2-like macrophages (Rayner et al. 2011b; Ouimet et al. 2015) and Treg cells (Ouimet et al. 2015). Furthermore, bone marrow transplant experiments in which ApoE −/− mice were reconstituted with ApoE −/− × miR-33 −/− bone marrow demonstrated that loss of miR-33 in hematopoietic cells caused reduced lipid accumulation in atherosclerotic plaques but did not affect HDL-C levels or total plaque size (Horie et al. 2012). However, whole-body loss of miR-33 in ApoE −/− mice did increase plasma HDL-C and reduce total plaque size (Horie et al. 2012). While the low levels of HDL-C and impaired macrophage cholesterol efflux of ApoE −/− mice make this a poor model in which to study effects on reverse cholesterol transport, these findings do indicate that both the liver and macrophages are likely involved in mediating the effects of miR-33 on atherosclerotic plaque formation, although the specific contributions are still not entirely clear.

In addition to its role in regulating lipid metabolism, further studies have showed that miR-33 can also target a number of genes involved in other metabolic processes including fatty acid β-oxidation, insulin signaling, and mitochondrial function (Karunakaran et al. 2015b; Gerin et al. 2010; Davalos et al. 2011; Ramirez et al. 2013a). These findings highlight the complex role of miR-33 in regulating metabolic function and indicate that it may have more diverse functions than have previously been elucidated. Consistent with this, miR-33 −/− mice on a high-fat diet were found to gain more weight than control animals, leading to more rapid development of hepatic steatosis and insulin resistance (Horie et al. 2013). Similarly, some studies have indicated that prolonged anti-miR-33 therapy may cause unwanted effects such as hypertriglyceridemia (Marquart et al. 2013; Allen et al. 2014; Goedeke et al. 2014) and hepatic steatosis (Goedeke et al. 2014). In contrast, similar long-term miR-33 inhibitor studies showed no change (Rotllan et al. 2013; Ouimet et al. 2015; Horie et al. 2012; Rottiers et al. 2013) or even a decrease (Karunakaran et al. 2015a; Rayner et al. 2011a) in plasma triacylglycerides (TAGs) in mice and nonhuman primates.

Overall, these studies indicate that anti-miR-33 therapies may be a promising approach for developing novel therapies for atheroprotection. However, the multitude of miR-33 targets involved in many different metabolic functions, and the potential for adverse effects of long-term miR-33 inhibition warrant further exploration. Additionally, because there are two isoforms of miR-33 present in humans, but mice and other rodents do not express miR-33b, little is known about the role of this miRNA in regulating atherosclerotic plaque development and overall metabolic function. This is especially important because the host genes for miR-33a and miR-33b (SREBP2 and SREBP1) are regulated by different nutritional and hormonal stimuli and miR-33b is more likely to be altered under conditions of metabolic dysfunction such as diabetes and obesity. Recently, a miR-33b knock-in model was reported (Horie et al. 2014), which should allow further exploration of the role of miR-33b in atherosclerosis and other metabolic diseases.

2.3 miRNAs and LDL-C Metabolism

While the role of miRNAs in regulating HDL-C metabolism has been extensively studied, the importance of miRNAs in controlling LDL-C remains to be fully elucidated. Nevertheless, a number of studies have reported the importance of miRNAs in controlling circulating LDL-C by regulating cholesterol biosynthesis, very-low-density lipoprotein (VLDL) secretion, and LDLR activity (Fig. 2.2).

Fig. 2.2
figure 2

miRNA regulation of LDL-C metabolism. Schematic overview of miRNAs involved in the regulation of LDL-C metabolism. Blue boxes highlight miRNAs, which regulate genes that control LDL-C. LDLR indicates low-density lipoprotein receptor, MTTP microsomal triglyceride transfer protein, LPL lipoprotein lipase, and NSF N-ethylmaleimide-sensitive factor (Figure was created using the Servier Medical Art illustration resources (http://www.servier.com))

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2.3.1 miRNAs That Regulate Cholesterol Biosynthesis and VLDL Secretion

2.3.1.1 miR-122

The first miRNA identified as a major regulator of cholesterol metabolism was miR-122 (Elmen et al. 2008; Esau et al. 2006; Krutzfeldt et al. 2005). This miRNA is the most abundant miRNA in the liver, accounting for at least 80% of the total miRNA content. Importantly, pharmacological inhibition of miR-122 in mice and nonhuman primates using ASOs or genetic ablation of miR-122 in mice results in a marked reduction of plasma cholesterol levels (Elmen et al. 2008; Esau et al. 2006; Krutzfeldt et al. 2005). Despite these intriguing findings, the molecular mechanisms by which miR-122 controls lipoprotein metabolism remain largely unknown. It is thought that the reduced circulating cholesterol observed in mice treated with miR-122 antagonists is mediated by a combination of different metabolic processes including inhibition of cholesterol and fatty acid biosynthesis, enhanced fatty acid oxidation, and decreased VLDL secretion (Elmen et al. 2008; Esau et al. 2006; Krutzfeldt et al. 2005). However, the direct miR-122 target genes that influence lipid metabolism remain largely unknown. While these findings suggest that the use of miR-122 inhibitors might represent a useful approach to treat dyslipidemia, two recent studies have shown that miR-122 deficiency in mice results in hepatic steatosis, fibrosis, and hepatocellular carcinoma, raising concerns about the potential therapeutic value of miR-122 inhibitors.

2.3.1.2 miR-30c in Lipid Metabolism and Atherosclerosis

In addition to miR-122, miR-30c levels also regulate circulating lipids by regulating the expression of microsomal triglyceride transfer protein (MTTP), an enzyme that lipidates Apo-B and triglycerides and is required for the secretion of plasma lipoproteins that contain Apo-B (Irani et al. 2016; Soh et al. 2013). Moreover, miR-30c inhibits hepatic lipid synthesis. As a result, overexpression of miR-30c using lentiviral constructs or miRNA mimics significantly reduces plasma cholesterol levels and attenuates the progression of atherosclerosis (Irani et al. 2016; Soh et al. 2013). Interestingly the inhibition of MTTP expression and VLDL secretion does not influence the accumulation of lipids in the liver, suggesting that miR-30c therapies might be an interesting approach for treating patients with homozygous hypercholesterolemia.

2.3.1.3 miR-33a/miR-33b

Besides the abovementioned role of miR-33a/miR-33b in regulating HDL-C metabolism and the progression and regression of atherosclerosis, several reports have shown that miR-33 might also control VLDL metabolism (Allen et al. 2014; Goedeke et al. 2014). In this regard, Baldán and colleagues found that chronic inhibition of miR-33 in rodents enhances hepatic VLDL-C. The authors showed that targeting miR-33 in vivo increases the expression of N-ethylmaleimide-sensitive factor (NSF), an ATPase enzyme involved in intracellular trafficking and membrane fusion (Allen et al. 2014). These findings correlate with another study that shows a marked increase in circulating TAG (Goedeke et al. 2014). In agreement with these observations, genetic ablation of miR-33 in mice results in obesity, insulin resistance, hepatosteatosis, and dyslipidemia (Horie et al. 2013). Despite these results, other similar studies in mice and nonhuman primates showed no differences or even a decrease in circulating TAG (Rayner et al. 2011a). The factors that might explain these discrepancies need further investigation.

2.3.1.4 miR-96/miR-182/miR-183

miR-96/miR-182/miR-183 was identified as the most upregulated miRNA in mice fed a chow diet supplemented with a mixture of lovastatin plus ezetimibe (to deplete hepatic cholesterol and enhance cholesterol biosynthesis) compared to mice fed a chow diet supplemented with cholesterol (to increase hepatic cholesterol content and inhibit cholesterol synthesis) (Jeon et al. 2013). The miR-96/miR-182/miR-183 locus is regulated by SREBP and regulates the expression of key proteins that control the maturation and activation of SREBP. miR-96 and miR-182 inhibit the expression of INSIG and FBXW7, proteins that control the retention of the SREBP/SCAP complex in the ER and the degradation nuclear SREBP, respectively (Jeon et al. 2013). As a consequence, overexpression of all three miRNAs enhances cholesterol and fatty acid synthesis in HeLa cells. Further experiments in vivo will be important to define the specific contribution of these miRNAs in regulating hepatic lipid homeostasis and lipoprotein metabolism.

2.3.2 miRNAs That Regulate LDLR Expression and LDL-C Clearance

Several studies have recently identified miRNAs that regulate LDL-C metabolism via posttranscriptional regulation of LDLR. Notably, miR-27a/miR-27b, miR-128-1, miR-130b, miR-148a, miR-185, miR-199a, and miR-301 were shown to directly target the 3’UTR of LDLR and modulate LDLR expression in human and mouse hepatic cells (Alvarez et al. 2015; Goedeke et al. 2015b; Jiang et al. 2015; Wagschal et al. 2015). Of these miRNAs, only miR-128-1, miR-148a, and miR-185 significantly altered plasma LDL-C in vivo (Goedeke et al. 2015a; Jiang et al. 2015; Wagschal et al. 2015; Yang et al. 2014). Thus, we will only discuss in greater detail how these miRNAs regulate hepatic lipid metabolism and circulating LDL-C.

2.3.2.1 miR-148a

miR-148a has been recently identified by two independent studies as an important regulator of hepatic LDLR expression and lipoprotein metabolism in a number of mouse models (Goedeke et al. 2015a; Wagschal et al. 2015). In the first study, Goedeke and colleagues developed a high-throughput genome-wide screening assay to systematically identify miRNAs that regulate LDLR activity in human hepatic cells (Goedeke et al. 2015a). From this screen, the authors identified and characterized miR-148a as a negative regulator of LDLR expression and activity. miR-148a is highly expressed in the liver, and its expression is regulated by dietary lipids and SREBP1. Of note, pharmacological inhibition of miR-148a using ASOs lowered plasma LDL-C levels in two different mouse models of hypercholesterolemia (Goedeke et al. 2015a; Wagschal et al. 2015). Surprisingly, targeting miR-148a in vivo also increases hepatic ABCA1 expression and circulating HDL-C. Further experiments demonstrated that ABCA1 is also a miR-148a target gene. Collectively these studies underscore the therapeutic potential of modulating miR-148a expression to treat dyslipidemias (high plasma LDL-C and low circulating HDL-C).

Several labs independently identified SNPs (rs4722551, rs4719841, and rs6951827) in the promoter region of miR-148a associated with altered plasma TC, LDL-C, and TAG levels (Do et al. 2013; Global Lipids Genetics Consortium et al. 2013; Huan et al. 2015). In particular, a miR-eQTL analysis performed in human livers revealed a strong correlation between SNP status and miR-148a expression (Wagschal et al. 2015). Although the exact mechanism by which these SNPs contribute to altered plasma lipids remains unknown, it could be possible that these genetic variations might influence the regulation of miR-148a expression via SREBP1. However, whether these SNPs affect SREBP1-induced transcription requires further investigation. The role of miR-148a in regulating lipid metabolism is likely to be more complex and not only mediated by its targeting on LDLR and ABCA1. In particular, miR-148a was demonstrated to directly target the 3’UTR of other genes involved in lipid metabolism, including PGC1a, AMPK, and INSIG1 (Wagschal et al. 2015; Goedeke et al. 2015a). Taken together, these findings highlight the importance of miR-148a in regulating lipid metabolism in mice and humans and underscore the therapeutic potential of modulating miR-148a expression to treat dyslipidemias.

2.3.2.2 miR-128-1

Besides miR-148a, Wagschal and colleagues also found a strong association of a number of SNPs in the miR-128-1 gene locus and altered plasma lipid levels (Wagschal et al. 2015). miR-128a regulates the expression of numerous genes associated with multiple metabolic pathways. miR-128a directly targets the 3’UTR of the LDLR and ABCA1, and its inhibition in mice results in a significant decrease in circulating cholesterol and TAG. In addition to its role in controlling lipoprotein metabolism, targeting miR-128-1 in vivo also improved glucose tolerance and insulin sensitivity. Mechanistically, the authors found that miR-128-1 regulates the expression of the insulin receptor (INSR) and insulin receptor substrate 1 (IRS-1) and downstream phosphorylation levels of Akt. Finally, it was also found that miR-128-1 controls the expression of genes associated with fatty acid synthesis, including the fatty acid synthase (FASN) and SIRT1, an NAD+-dependent energy sensor and deacetylase that can directly deacetylate and inactivate SREBP1 and thus modulate SREBP1-dependent lipogenesis. Altogether, these observations suggest that miR-128a expression might influence plasma lipid levels by regulating the expression of genes associated with lipid and glucose metabolism. Further studies are necessary to define the specific contribution of miR-128a target genes in different tissues and their impact on regulating lipid and glucose metabolism.

2.3.2.3 miR-185

Several reports have recently shown that miR-185 regulates cholesterol metabolism in vitro and in vivo by regulating the expression of LDLR and SRBI, a hepatic HDL-C receptor that facilitates the uptake of cholesteryl esters from HDL in the liver (Jiang et al. 2015; Wang et al. 2013; Yang et al. 2014). Interestingly, miR-185 also targets KH-type splicing regulatory protein (KSRP), a RNA-binding protein that negatively regulates the expression of the human LDLR (Jiang et al. 2015). These findings suggest that inhibiting miR-185 in vivo might attenuate atherosclerosis by reducing circulating LDL-C and promoting reverse cholesterol transport. Indeed, a recent study demonstrated that inhibition of miR-185 in the atheroprone ApoE −/− mice significantly reduced plasma cholesterol levels and attenuated the progression of atherosclerosis (Jiang et al. 2015).

2.4 LncRNAs and Cholesterol Metabolism

While the contribution of miRNAs in regulating lipoprotein metabolism has been deeply studied in the last few years, the role of lncRNAs in controlling cholesterol homeostasis has just started to emerge. Recent studies have demonstrated the important contribution of lncRNAs as key regulators of cholesterol metabolism (Fig. 2.3).

Fig. 2.3
figure 3

LncRNA regulation of cholesterol metabolism. Schematic overview of lncRNAs involved in the regulation of hepatic cholesterol homeostasis and lipoprotein metabolism. Blue boxes highlight lncRNAs, which regulate genes that control cholesterol metabolism. FXR indicates farnesoid X receptor, LeXis liver-expressed LXR-induced sequence, LncLSTR liver-specific triglyceride regulator, ApoA1-AS ApoA1 antisense, LPL lipoprotein lipase, and TDP-43 TAR DNA-binding protein 43 (Figure was created using the Servier Medical Art illustration resources (http://www.servier.com))

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2.4.1 LeXis

Tontonoz’s group has recently identified liver-expressed LXR-induced sequence (LeXis), a liver noncoding RNA that is markedly induced in response to LXR agonists and high-fat diet (Sallam et al. 2016). Hepatic LeXis overexpression reduces circulating cholesterol, attenuates cholesterol biosynthesis, and inhibits the expression of cholesterol biosynthetic genes. Conversely, genetic ablation of LeXis or acute pharmacological inhibition using ASOs enhances the hepatic expression of genes associated with cholesterol biosynthesis, leading to a significant accumulation of cholesterol in the liver. Mechanistically, the authors found that LeXis interacts with and influences the binding of RALY to DNA. The authors hypothesize that RALY might cooperate with SREBP2 to control the expression of cholesterol biosynthetic genes. However, further studies are needed to support this hypothesis. Another important question that remains to be addressed is the study of the contribution of LeXis in regulating cholesterol metabolism in humans. Importantly, mouse and human genomic comparison revealed a moderate conservation on the LeXis genetic locus in a region adjacent to the human ABCA1 gene. Further experiments are warranted to determine whether the putative lncRNA annotated in this region (TCONS_00016452) regulates cholesterol metabolism in humans.

2.4.2 Lnc-HC and ApoA1-AS

Other lncRNAs that have been associated to the regulation of cholesterol metabolism include Lnc-HC and ApoA1-AS (Halley et al. 2014; Lan et al. 2016). Lnc-HC is highly expressed in the liver and interacts with hnRNPA2B1 forming a RNA-protein complex, which can then bind to the target mRNAs, Cyp7a1 and Abca1 (Lan et al. 2016). Inhibition of Lnc-HC increased Cyp7a1 and Abca1 expression in hepatocytes, thus promoting cholesterol catabolism. Lnc-HC is conserved in humans and rodents and appears to be highly expressed in the liver and fat (Lan et al. 2016). These observations suggest that Lnc-HC might play a role in regulating lipid metabolism. However, additional studies in vivo are needed to define the role of Lnc-HC in regulating hepatic lipid homeostasis and lipoprotein metabolism. Another interesting finding is the identification of an antisense LncRNA (ApoA1-AS) encoded in the apolipoprotein gene cluster that contains four different transcripts including ApoA1, ApoA4, ApoA5, and ApoC3. ApoA1-AS controls the expression of the apolipoprotein gene cluster epigenetically by recruiting histone-modifying enzymes (Halley et al. 2014). Targeting ApoA1-AS using ASOs increases ApoA1 expression in both monkey and human cells and enhances hepatic RNA and protein expression in African green monkeys. While these results are of interest, it is still not known whether the increase in circulating ApoA1 influences plasma lipid levels and/or lipoprotein metabolism.

2.4.3 LncLSTR

Liver-specific triglyceride regulator (LncLSTR) was identified using an unbiased screen aimed to determine lncRNAs highly expressed in the liver (Li et al. 2015). Of note, specific inhibition of LncLSTR leads to a marked reduction in circulating TAG. Mechanistically, LncLSTR depletion increases apoC2 levels, an activator of the lipoprotein lipase (LPL), thus enhancing VLDL and chylomicron catabolism, leading to an increase in plasma TAG clearance (Li et al. 2015). Hepatic LncLSTR expression is regulated by FXR and forms a complex with TDP-43, thus regulating Cyp8b1 expression, a critical enzyme involved in bile acid synthesis.

2.5 Concluding Remarks

Work in recent years has clearly established both miRNAs and lncRNAs as important regulators of lipid metabolism. Since the human genome encodes thousands of lncRNAs, it is expected that in the near future, other lncRNAs will also be identified as major regulators of lipid metabolism. Despite this exciting future, the analysis and the identification of functional lncRNAs will be challenging because of the modest conservation of these RNA molecules between species. Additionally, a more complete understanding of the mechanisms by which these lncRNAs excerpt their effects and additional studies directly assessing the impact of the lncRNAs on atherosclerotic plaque formation will be needed to properly assess the therapeutic potential of lncRNA-based therapeutic approaches.

On the other hand, a large amount of work has been done demonstrating that miRNAs are capable of targeting key factors regulating lipid metabolism and can have an important impact on the development and progression of atherosclerosis in animal models. As such, miRNA-based therapies may provide useful complimentary approaches for the treatment of atherosclerosis in humans. However, the complicated role of many miRNAs in targeting numerous different genes in different tissues and under different physiologic conditions raises concerns that these therapeutic approaches may result in unintended and possibly detrimental outcomes. These risks are highlighted by the adverse outcomes apparent in some experiments examining inhibition/ablation of miR-33 and miR-122. As such, additional studies including tissue-specific knockouts and careful target gene assessment will be important to understand the full impact of miRNA alterations prior to developing treatment strategies for human patients.