Clin Chem Lab Med 2017; 55(5): 667–686

Review

Véronique Desgagné, Luigi Bouchard and Renée Guérin* microRNAs in lipoprotein and lipid metabolism: from biological function to clinical application

DOI 10.1515/cclm-2016-0575 Received June 29, 2016; accepted October 31, 2016; previously Introduction ­published online December 17, 2016 Since their first discovery in worms in 1993 [1], microRNAs Abstract: microRNAs (miRNAs) are short (~ 22 nucleo‑ (miRNAs) have been described as important regulators tides), non‑coding, single-stranded RNA molecules that of several biological processes from cellular differentia‑ regulate the expression of target genes by partial sequence- tion and development to metabolic function regulation, specific base-pairing to the targeted mRNA 3′UTR, block‑ and their deregulation in diseased states has repeatedly ing its translation, and promoting its degradation or its been reported. The role of miRNAs as central regulators sequestration into processing bodies. miRNAs are impor‑ of lipoprotein metabolism and cholesterol homeosta‑ tant regulators of several physiological processes includ‑ sis is now well established in the literature (reviewed in ing developmental and metabolic functions, but their [2–5]). However, the role of lipoprotein-bounded miRNAs concentration in circulation has also been reported to be as endocrine regulators has recently emerged. Although altered in many pathological conditions such as familial studies on lipoprotein-transported miRNAs suggest dis‑ hypercholesterolemia, cardiovascular diseases, obesity, tinct miRNA profiles in the two major lipoproteins found in type 2 diabetes, and cancers. In this review, we focus on circulation, the data support that miRNAs have a stronger the role of miRNAs in lipoprotein and lipid metabolism, role in high-density lipoprotein (HDL) than in low-density with special attention to the well-characterized miR-33a/b, lipoprotein (LDL) in regulating their metabolic functions and on the huge potential of miRNAs for clinical applica‑ [6–8]. The discovery of miRNAs transported by lipopro‑ tion as biomarkers and therapeutics in the context of car‑ teins thus opens up the way for two main clinical appli‑ diometabolic diseases. cations: biomarkers of diseases and targeted therapies. In this review, we describe the basics of miRNA biology Keywords: blood miRNA; cholesterol; high-density lipo‑ and focus on the important role of miRNAs in lipoprotein protein (HDL). and lipid metabolism. We also describe where potential clinical applications currently stand and discuss future research perspectives and their challenges.

miRNA biogenesis and biological *Corresponding author: Renée Guérin, PhD, CSPQ, FCACB, Département de biochimie, Université de Sherbrooke, Sherbrooke, function Québec, Canada; and Département de biologie médicale, CIUSSS du Saguenay–Lac-St-Jean, Hôpital de Chicoutimi, Saguenay, Québec, miRNAs are evolutionarily-conserved short [~ 22 nucleo‑ Canada, Phone: (418) 541-1000-3403, tides (nt)], non‑coding, single-stranded RNAs involved in E-mail: [email protected] the post-transcriptional regulation of potentially > 60% of Véronique Desgagné: Département de biochimie, Université de Sherbrooke, Sherbrooke, Québec, Canada; and Laboratoire the human genes [9]. The miRNAs biogenesis pathway is ECOGENE-21, CIUSSS du Saguenay–Lac-St-Jean, Hôpital de detailed in Figure 1. Briefly, miRNAs are encoded in the Chicoutimi, Saguenay, Québec, Canada genome and are transcribed, mostly by the RNA polymer‑ Luigi Bouchard: Département de biochimie, Université de ase II, as a ~ 1000-nt capped and polyadenylated primary Sherbrooke, Sherbrooke, Québec, Canada; Laboratoire ECOGENE-21, miRNA (pri-miRNA) from their own promoter (intergenic) CIUSSS du Saguenay–Lac-St-Jean, Hôpital de Chicoutimi, Saguenay, Québec, Canada; and Département de biologie médicale, CIUSSS du or from co-expression with their host protein-coding gene Saguenay–Lac-St-Jean, Hôpital de Chicoutimi, Saguenay, Québec, (intragenic) [10]. The pri-miRNA is then cleaved by the Canada microprocessor complex (Drosha/DGCR8) to generate a 668 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism

Cytoplasm miRNA gene Nucleus Intergenic Intragenic

Mirtron Pol II Spliceosome debranching

Drosha Dicer RISC DGCR8 Exportin 5-Ran-GTP TRBP RISC m G- -AAAAA miRNA Mature Pri-miRNA Pre-miRNA duplex miRNA

7 m G- -AAAAA Target mRNA

Sequestrationintoprocessing Repression of protein bodies or degradation of mRNA translation RISC RISC

7 7 m G- -AAAAA m G- -AAAAA Ribosome

Figure 1: Canonical miRNA biogenesis pathway. miRNA genes (intragenic or intergenic) are transcribed, mostly by the RNA polymerase II (Pol II), as a primary miRNA (pri-miRNA) [10]. The pri-miRNA is cleaved the microprocessor complex (Drosha/DGCR8) to generate a hairpin-structured precursor miRNA (pre-miRNA) which is then exported to the cytoplasm by the Exportin5-RanGTP system [10]. The pre-miRNA is subjected to a second cleavage by the Dicer/TRBP complex, releasing a ~ 22 nt miRNA duplex (miRNA-3p/miRNA-5p), which is then loaded onto an Argonaute protein (AGO1-4, in humans) to form the pre-RNA-induced silencing complex (RISC) [10]. Based on relative thermodynamic stability of the ends of the two miRNA strands, one strand (guide) is selected to form the mature RISC complex, while the other strand (passenger) is quickly removed and degraded [10]. Non-canonical miRNA biogenesis pathways [e.g. bypassing the processing by either Drosha (e.g. mirtrons) or Dicer (e.g. miR-451)] have also been characterized for a few specific miRNAs (reviewed in [10]). Through (mostly partial) base-pairing to the 3′ UTR of its target mRNA, miRNA regulates the expression of target genes by repressing mRNA translation and promoting mRNA sequestration into processing bodies as well as mRNA destabilization and degradation (mRNA decay, accelerated by mRNA deadenylation and decapping) [10].

~ 70-nt hairpin-structured precursor miRNA (pre-miRNA), synthesis through their (mostly partial) base-pairing to which is exported to the cytoplasm by the Exportin5- the 3′ untranslated region (UTR) of their mRNA target, RanGTP system [10]. The pre-miRNA is subjected to a which is mainly dependent of the complementarity at second cleavage by the Dicer/TRBP complex, releasing nucleotides 2–8 of the miRNA (i.e. miRNA seed region; at a ~ 22 nt miRNA duplex (miRNA-3p/miRNA-5p) [10]. The the 5′-end of the miRNA), although the nucleotides 13–16 miRNA duplex is subsequently loaded onto an Argo‑ also contribute to mRNA targeting specificity (i.e. miRNA naute protein (AGO1-4, in humans), by an ATP-dependent 3′ supplementary site) [12]. This interaction consequently mechanism involving the heat shock cognate 70 (HSC70)– inhibits mRNA translation and promotes mRNA seques‑ heat shock protein 90 (HSP90) chaperone complex, to tration into processing bodies as well as its degradation form the pre-RNA-induced silencing complex (RISC) [10]. (mRNA destabilization and decay, accelerated by mRNA Based on the relative thermodynamic stability of the two deadenylation and decapping; Figure 1), the latter pro‑ miRNA strands, the one with the weakest binding at its 5′ ducing greater repressive effect at steady state [10, 13–15]. end is more likely to become the strand (guide) selected These miRNA-mediated gene-silencing mechanisms have to form the mature RISC complex, while the other strand been reviewed in [15] and [16]. However, it has been shown (passenger) is quickly removed and degraded [10, 11]. This that miRNAs could bind functional sites in the 5′UTR as selection, however, is flexible depending on cell types, well as in the protein-coding region of mRNAs, promoting developmental stages and disease states [11]. This means gene translation in certain circumstances [17–19]. While a that either the miRNA-3p or the miRNA-5p may be selected single miRNA may target several (often, but not always, to form the mature RISC complex (reviewed in [11]). functionally related) genes, a single mRNA may harbor The biological function of miRNAs is usually, but many binding sites for the same or different miRNAs [20]. not exclusively, attributed to the repression of protein Moreover, two miRNA-binding sites closely located on a Desgagné et al.: microRNAs in lipoprotein and lipid metabolism 669 mRNA could act synergistically to post-transcriptionally urine, as well as feces [38–42], demonstrating their remark‑ regulate gene expression [21]. able stability and supporting their great potential as non- Although the central role of miRNAs is to negatively invasive biomarkers. In plasma, miRNAs are transported regulate protein synthesis, other functions of miRNAs are and protected against RNAse-dependent degradation­ by emerging (reviewed in [22] and [23]). Particularly, miRNAs various carriers, such as vesicles (exosomes, microvesi‑ have been found to regulate the stability of nuclear tran‑ cles, apoptotic bodies), RNA-binding proteins [AGO2 and scripts (e.g. mRNAs, long non-coding RNAs (lncRNAs) nucleophosmin 1 (NPM1)], and lipoproteins [HDL, inter‑ or pri-miRNAs), induce epigenetic alterations at specific mediate density lipoprotein (IDL) and LDL] [37, 43]. The gene promoters, and likely modulate alternative splicing next section focuses on the role of miRNAs in lipoprotein events in the nuclear compartment [22–26]. Moreover, metabolism and function regulation. two studies have reported few specific miRNAs as ligands for toll-like receptor (TLRs; TLR7 in mice and TLR8 in humans) activation [27, 28]. Overall, miRNAs are impor‑ tant gene expression regulators that, cooperatively and at Role of miRNAs in lipoprotein and various steps of the gene expression process, orchestrate the fine-tuning of gene expression at diverse development lipid metabolism stages, for different cell types and disease states [20]. miRNAs as regulators of lipid metabolism and function miRNA expression regulation and Cholesterol is an essential molecule for animal cell physi‑ stability ology. It is involved in the regulation of the plasma mem‑ brane fluidity and integrity, as well as in membrane protein The steady-state concentration of mature miRNAs in cells functionality (e.g. cell signaling, intracellular transport). depends on their tightly regulated transcription, matura‑ It is the precursor for steroid hormones, bile acids, and tion, and decay (reviewed in [10] and [29]). It has been pro‑ vitamin D biosynthesis [44, 45]. As altered cholesterol posed that approximately ≈ 100 copies of a mature miRNA levels have been repeatedly associated with cardiometa‑ per cell is required for biologically relevant repression of bolic diseases (e.g. atherosclerosis), a strict regulation of gene translation [30, 31]. The regulation of the miRNA con‑ the lipoprotein metabolism (Figure 2; reviewed in [62]) is centration therefore plays an important part in the control essential to maintain cholesterol homeostasis and thus of the miRNA regulatory function. Briefly, miRNA tran‑ health. Since their discovery [1], miRNAs have been pro‑ scription is regulated by specific transcription factors and posed as important regulators of metabolism homeostasis epigenetic marks (e.g. DNA methylation, histone modifi‑ in humans [63–65]. The number of miRNAs with validated cations) [13]. Like protein-coding genes, certain miRNAs target genes involved in lipid and lipoprotein metabolism display a tissue-specific expression pattern [32], support‑ has increased significantly in the last decade (Figure 2 and ing their roles in cellular differentiation and develop‑ Table 1). So far, the best characterized miRNA that regu‑ ment [33]. Moreover, the miRNA-processing efficiency is lates lipid and lipoprotein metabolism is miR-33a/b. regulated through various post-translational modifica‑ tions of the maturation machinery (Drosha, DGCR8, Dicer, TRBP, AGO) [10, 13]. Also, the miRNA itself is subjected to miR-33a/b post-transcriptional modifications such as RNA editing, methylation, uridylation and adenylation [13]. These The miR-33 family member miR-33a is evolutionarily con‑ modifications, in addition with protein complex forma‑ served across animal species, whereas miR-33b is con‑ tion (e.g. AGO) and exposure to nucleases [e.g. 5′–3′ exori‑ served only in certain mammals [81, 127]. In humans, bonuclease 2 (XRN-2)], modulate the miRNA stability and miR-33a and miR-33b are encoded within intronic regions decay [29]. of the sterol regulatory element-binding transcription miRNAs show high stability (> 24 h) in cells and factor (SREBF) genes SREBF2 and SREBF1, respectively plasma, with miRNA persistence varying from one miRNA [82]. SREBFs encode key membrane-bound transcription to another [34–37]. Interestingly, miRNAs have also been factors involved in the activation of numerous genes impli‑ found in virtually all biological fluids, including cerebro‑ cated in biosynthesis and uptake of cholesterol and in the spinal fluid, tears, saliva, breast milk, amniotic fluid, synthesis of fatty acids [e.g. 3-hydroxy-3-methyglutaryl-CoA 670 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism

Peripheral cell

Intestine Circulation LPL* Liver B48 MTP* B48 B48 LRP1 B48 E E E LDLR* A5 A5 * Syn-1 Exogenous pathway CM A5 mRNA CMR CMR miR-27a/b miR-30c CM miR-128 miR-130b miR-148a LPL* B100 miR-185 B100 miR-301b MTP* B100 E LDLR* E Size VLDL Density Syn-1 VLDL IDL LPL*, HL B100 LDLR* Endogenous pathway E LDL

LCAT A1 SR-BI* A1

* A1 mRNA ecto-F1-ATPase/P2Y miR-33a/b miR-145 HDLd HDL 13 miR-26 miR-148a miR-27a/b miR-223 ABCA1*

miR-19b miR-301b A1 miR-10b miR-302a ABCA1 * miR-101 miR-378 ABCG1 * miR-106b miR-455 miR-128 miR-613 Macrophage Reverse transpor t miR-130b miR-758 miR-144

Figure 2: Lipoprotein metabolism. Mature lipoproteins are spherical structures composed (roughly) of phospholipids (in white), cholesterol (in yellow), triglycerides (in red) and apolipoproteins, which allow the transport of lipids in the bloodstream [44, 46]. Lipoproteins present diverse sizes (7–1200 nm of diameter) and can be classified based on their respective density or electrophoretic mobility [47]. The exogenous lipoprotein pathway, which comprises the apoB48-containing lipoproteins, consists in dietary lipid transport from the intestine to energy-demanding tissues such as muscle, adipose tissue and the liver [46, 48–53]. The endogenous lipoprotein pathway, which comprises the apoB100-containing lipoproteins, allows the transport of lipids from the liver to peripheral tissues [46, 49–55]. The reverse cholesterol pathway consists in the efflux of excess cholesterol from extrahepatic tissues to HDLs and its transport to the liver for metabolization and excretion into the bile in the intestine [56–60]. Importantly, circulating lipoproteins undergo constant remodeling by interacting with various partners (e.g. LCAT, CETP, PLTP, HL, LPL), which implicates cargo exchanges between different lipoproteins (reviewed in [57] and [61]). All these pathways are well described with more details in the references mentioned above. Several miRNAs with validated target genes (*) involved in these lipoprotein metabolism pathways (in blue) have been identified so far (detailed in Table 1). miR-33a/b and miR-223 (in bold) have been extensively described in this review. A1, apolipoprotein-A1; A5, apolipoprotein-A5; ABCA1, ATP-binding cassette subfamily A member 1; ABCG1, ATP- binding cassette subfamily G member 1; B48, apolipoprotein-B48; B100, Apolipoprotein-B100; CETP, cholesterol ester transfer protein; CM, chylomicron; CMR, chylomicron remnant; E, apolipoprotein-E; HDL, high-density lipoprotein HDLd, nascent discoidal HDL; HL, hepatic lipase; IDL, intermediate density lipoprotein; LCAT, lecithin/cholesterol acyltransferase; LDL, low-density lipoprotein LDLR, low-density lipoprotein receptor; LPL, lipoprotein lipase; LRP1, low-density lipoprotein receptor-related protein 1; MTP, microsomal triglyceride transfer protein; PLTP, phospholipid transfer protein; SR-BI, scavenger receptor class B type I; Syn-1, syndecan-1 receptor; TG, triglyceride; VLDL, very low-density lipoprotein.

reductase (HMGCR), low-density lipoprotein receptor (SREBP1), repressing fatty acid oxidation, and increasing (LDLR), fatty acid synthase (FASN), stearoyl-CoA desatu‑ intracellular cholesterol levels (miR-33b) [83, 128, 130]. rase (SCD), and acetyl-CoA carboxylase alpha (ACACA)] Several studies performed using miRNA mimics/inhibi‑ [128, 129]. Under low intracellular cholesterol levels, tors in cultured cells and animal models have suggested an MIR33A is transcribed concomitantly with SREBF2 important role of miR-33a/b in HDL and lipid metabolism and increases the cholesterol biosynthesis and uptake regulation (Table 1; reviewed in [130] and [2]). miR-33a/b (SREBP2) as well as reduces cholesterol efflux and excre‑ have been shown to regulate cholesterol efflux by tar‑ tion (miR-33a) [83, 128, 130]. Alternatively, MIR33B is co- geting the cholesterol transporter gene ABCA1 in human transcribed with SREBF1 under insulin and liver x receptor hepatocytes and human and mice macrophages, and to (LXRs) activation [5], promoting fatty acid synthesis regulate ABCG1 in mice macrophages [84, 127]. miR-33a/b Desgagné et al.: microRNAs in lipoprotein and lipid metabolism 671

Table 1: miRNAs implicated in the regulation of lipid and lipoprotein metabolism. miRNA Regulation of miRNA Target Effect/pathway involved References expression genes miR-1 Not known LXRα Transcriptional regulation of lipid metabolism genes [66] miR-9 CREB ACAT1 Intracellular cholesterol homeostasis [67] miR-10b Protocatechuic acid (gut ABCA1 HDL biogenesis and cholesterol efflux (reverse cholesterol [68, 69] microbiota metabolite), TWIST ABCG1 transport) PPAR-α Transcriptional regulation of lipid metabolism genes miR-19b Not known ABCA1 Cholesterol efflux (reverse cholesterol transport) [70] miR-21 Dietary lipids HMGCR Cholesterol biosynthesis [71] miR-26 LXR ABCA1 Cholesterol efflux (reverse cholesterol transport) [72] ARL7 miR-27a/b Hyperlipidemia, C/EBPα ABCA1 Cholesterol trafficking and efflux (reverse cholesterol [73–77] OSBPL6 transport) ACAT1 Intracellular cholesterol homeostasis SR-BI HDL-Cholesterol uptake LDLR ApoB100-containing lipoproteins (VLDL, IDL, LDL) LDLRAP1 clearance RXRα Transcriptional regulation of lipid metabolism genes PPARγ GPAM Triglyceride-rich lipoprotein metabolism NDST1 ANGPTL3 LPL miR-29 Insulin resistance, FOXA2 HMGCR Fatty acids and cholesterol metabolism [78, 79] HMGCS2 AHR SIRT1 FOXO3 miR-30c C/EBPα MTP ApoB100-containing lipoprotein assembly and secretion [80] LPGAT1 miR-33a/b Sterols, LXR, insulin ABCA1 HDL biogenesis, cholesterol trafficking and efflux (reverse [77, 81–91] ABCG1a cholesterol transport) NCP1 OSBPL6 CROT Fatty acid oxidation CPT1A HADHB CYP7A1 Bile acid synthesis and secretion ABCB11 ATP8B1 IRS2 Insulin signaling G6PC PCK1 Glucose metabolism PDK4 Mitochondrial biogenesis and ATP production SLC25A25 PPARGC1A NSF VLDL vesicular trafficking/global apolipoprotein secretion SRC3 Transcriptional regulation of lipid metabolism genes RIP140 NFYC SREBP1 SIRT6 Post-translational regulation of metabolism-associated genes miR-34a Sterols, FFAs, FXR/SHP/p53 SIRT1 Post-translational regulation of metabolism-associated [92, 93] HNF4α genes, hepatic lipid storage and secretion miR-96 SREBP2 SR-BI HDL-Cholesterol uptake [94, 95] INSIG-2 Regulation of SREBPs activity 672 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism

Table 1 (continued) miRNA Regulation of miRNA Target Effect/pathway involved References expression genes miR-101 IL-6, TNFα ABCA1 Cholesterol efflux (reverse cholesterol transport) [96] miR-106b Not known ABCA1 Cholesterol efflux (reverse cholesterol transport) [97] miR-107 Dietary lipids FASN Triglyceride biosynthesis and storage [98, 99] CLOCK Circadian rhythm, metabolic homeostasis miR-122 HNF-4α, REV-ERBα, Circadian AGPAT1 TG biosynthesis and lipid storage [100–102] rhythm CIDEC PPARβ/δ Transcriptional regulation of lipid metabolism genes miR-125a Trophic hormones, cAMP, oxLDL SR-BI HDL-Cholesterol uptake, steroidogenesis [103, 104] ORP9 Lipid uptake (macrophage) miR-128 SREBP2 ABCA1 HDL biogenesis and cholesterol efflux (reverse cholesterol [105, 106] ABCG1 transport) RXRα Transcriptional regulation of lipid metabolism genes SREBP2 LDLR ApoB100-containing lipoproteins (VLDL, IDL, LDL) clearance INSR, IRS1 Insulin signaling SIRT1 Post-translational regulation of metabolism-associated genes miR-130b Dietary lipids, TNFα, NFκB ABCA1 Cholesterol efflux (reverse cholesterol transport) [105, 107–109] LDLR ApoB100-containing lipoproteins (VLDL, IDL, LDL) clearance PPARγ Transcriptional regulation of lipid metabolism genes, PPARGC1A adipogenesis miR-144 Sterols, LXR, FXR ABCA1 HDL biogenesis and cholesterol efflux (reverse cholesterol [110, 111] transport) miR-145 PPARγ ABCA1 HDL biogenesis and cholesterol efflux (reverse cholesterol [112–114] transport) miR-148a Dietary lipids, SREBP1c ABCA1 HDL biogenesis and cholesterol efflux (reverse cholesterol [105, 115] transport) LDLR ApoB100-containing lipoproteins (VLDL, IDL, LDL) clearance SIK1 Post-translational regulation of lipogenesis genes CPT1A Fatty acid oxidation miR-181a TGF-β, Wnt/β-catenin, STAT3 IDH1 Regulation of lipid biosynthesis and β-oxidation [116] miR-182 SREBP2 FBXW7 Regulation of SREBPs activity [95] miR-185 Sterols, Dietary lipids, SREBP1c SR-BI HDL-Cholesterol uptake [94, 117, 118] SREBP2 Transcriptional regulation of lipid metabolism genes LDLR ApoB100-containing lipoproteins (VLDL, IDL, LDL) clearance miR-206 Not known LXRα Transcriptional regulation of lipid metabolism genes [66] miR-223 Sterols, C/EBPα SR-BI HDL-Cholesterol uptake [94, 119] SP3 HDL biogenesis and cholesterol efflux (reverse cholesterol transport) HMGCS1 Cholesterol biosynthesis SC4MOL miR-301b Not known ABCA1 Cholesterol efflux (reverse cholesterol transport) [105] LDLR ApoB100-containing lipoprotein (VLDL, IDL, LDL) clearance miR-302a Sterols ABCA1 HDL biogenesis and cholesterol efflux (reverse cholesterol [120] transport) miR-378 Coenzyme Q10, Dietary lipids ABCG1 Cholesterol efflux (reverse cholesterol transport) [121–123] p110α Insulin signaling CRAT Energy metabolism homeostasis MED13 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism 673

Table 1 (continued) miRNA Regulation of miRNA Target Effect/pathway involved References expression genes miR-455 Trophic hormones, cAMP SR-BI HDL-Cholesterol uptake, steroidogenesis [103] miR-613 SREBP1c, Insulin, PPARγ ABCA1 Cholesterol efflux (reverse cholesterol transport) [124, 125] LXRα Transcriptional regulation of lipid metabolism genes miR-758 Sterols ABCA1 HDL biogenesis and cholesterol efflux (reverse cholesterol [126] transport) aThe miR-33a/b binding site in the 3′UTR of ABCG1 gene is present in rodents, but is not conserved in humans. have been found to target other genes associated with cho‑ in terms of both structure and functionality (reviewed lesterol metabolism, including the Niemann-Pick disease in [132]). Indeed, HDLs are composed of a large variety type C1 (NPC1) [84], cholesterol 7α-hydroxylase (CYP7A1), of proteins (> 80), lipid species (> 200), and miRNAs [6, ATPase aminophospholipid transporter class 1 type 8B 133]. The pioneering work of Vickers et al. first suggested member 1 (ATP8B1), and ATP-binding cassette subfamily that HDLs transport and deliver functional miRNAs to B member 11 (ABCB11) genes [85, 86]. In addition to their recipient cells with gene regulation capabilities [6]. role in cholesterol metabolism homeostasis, miR-33a/b Interestingly, the HDL-carried miRNA profile was consid‑ have been found to regulate the cellular fatty acid metabo‑ ered biologically distinct from the one observed in puri‑ lism by repressing many genes contributing to fatty acid fied LDLs and exosomes, these latter being more closely oxidation in hepatocytes [e.g. carnitine palmitoyltrans‑ related to each other [6]. One of the most abundant ferase 1A (CPT1a), carnitine O-octanoyltransferase(CROT)] and best characterized miRNAs carried by HDLs is miR- [83, 87] and to regulate insulin signaling by targeting the 223-3p [6, 7, 134]. HDL-transported miR-223-3p concentra‑ insulin receptor substrate 2 (IRS2) and sirtuin 6 (SIRT6) tions have been reported to be significantly increased genes [83]. Finally, the in vivo administration of miR-33a/b in participants with homozygous familial hypercholes‑ inhibitor oligonucleotides in mice and non-human pri‑ terolemia (FH) [135], and in athero-prone mice [Apolipo‑ mates has sustainably raised plasma HDL-C levels and protein E (ApoE)−/− or LDLR−/− mouse models on high-fat decreased very low-density lipoprotein (VLDL)-associated diet (HFD)] (Table 2) [6]. Moreover, hypercholesterolemia triglyceride levels [81, 82, 84, 88, 89], two factors associ‑ has also been associated with increased hepatic cho‑ ated with the metabolic syndrome [131]. Overall, miR- lesterol and miR-223-3p levels in ApoE−/− mice on HFD, 33a/b are important regulators of metabolic homeostasis, suggesting that liver miR-223-3p expression is associ‑ including cholesterol, lipid and glucose/insulin metabo‑ ated with cholesterol levels in vivo [119]. Accordingly, lism and represent a potential novel therapeutic target in miR‑223‑3p concentration in cultured human hepato‑ atherosclerosis and the metabolic syndrome [88, 89]. cytes and mice macrophages was found positively asso‑ ciated with intracellular cholesterol concentrations, and the MIR223 promoter activity was demonstrated to be cholesterol-sensitive (downregulated in low-cholesterol miRNAs carried by lipoproteins state) [119]. Importantly, miR-223-3p was found to target several genes involved in the cholesterol metabolism. High-density lipoprotein At high intracellular cholesterol levels, miR-223-3p is upregulated and represses cholesterol biosynthesis [tar‑ HDL-carried miRNA concentration is altered in diseased geting the 3-hydroxy-3-methylglutaryl-CoA synthase 1 state (HMGCS1) and methylsterol monooxygenase 1 (SC4MOL) mRNAs] as well as HDL-C uptake [targeting the scaven‑ HDLs exert their anti-atherogenic action primarily ger receptor class B type I (SR-BI) mRNA], and indirectly through their important role in promoting the reverse enhances ABCA1-mediated cholesterol efflux to HDLs cholesterol transport [56], but they also mediate several [targeting the transcription factor SP3 (SP3) mRNA] [119]. other less well-characterized cardioprotective functions, Under low intracellular cholesterol levels, miR-223-3p such as anti-inflammatory, antioxidant, anti-thrombotic, expression is downregulated and its targets repres‑ anti-apoptotic, and insulin-secretagogue actions [132]. sion is relieved; increasing cholesterol biosynthesis and HDLs comprise an heterogeneous population of particles uptake [119]. Consequently, miR-223-3p appears as an 674 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism

Table 2: Lipoprotein-transported miRNAs altered in cardiovascular disease compared to healthy controls. miRNA Disease Effect References

HDL let-7f-5p Familial hypercholesterolemia + 12.3-folds [6]a miR-24-3p Familial hypercholesterolemia + 65.3-folds [6]a miR-30c-5p Acute coronary syndrome − 1.7-folds [7]b miR-92a-3p Acute coronary syndrome − 3.0-folds [7]b c Stable angina + 2.3-folds (HDL2) [8]

+ 3.8-folds (HDL3) c Unstable angina + 2.3-folds (HDL2) [8]

+ 10.0-folds (HDL3) c Myocardial infarction + 2.8-folds (HDL2) [8]

+ 7.5-folds (HDL3) miR-106a-5p Familial hypercholesterolemia + 7.9-folds [6]a c miR-122-5p Stable angina + 7.5-folds (HDL3) [8] c Unstable angina + 8.0-folds (HDL3) [8] miR-135a-3p Familial hypercholesterolemia − 2.4-folds [6]a miR-138-1-3p Familial hypercholesterolemia − 2.0-folds [6]a miR-146a-5p Acute coronary syndrome − 1.3-folds [7]b c Unstable angina + 4.7-folds (HDL3) [8] c Myocardial infarction + 4.7-folds (HDL3) [8] miR-188-5p Familial hypercholesterolemia − 2.7-folds [6]a miR-191-5p Familial hypercholesterolemia + 4.7-folds [6]a miR-218-5p Familial hypercholesterolemia + 7.8-folds [6]a miR-222-3p Familial hypercholesterolemia + 8.2-folds [6]a miR-223-3p Familial hypercholesterolemia + 3780.6-folds [6]a miR-323-3p Familial hypercholesterolemia − 2.5-folds [6]a miR-342-3p Familial hypercholesterolemia + 30.4-folds [6]a miR-412-5p Familial hypercholesterolemia + 9.5-folds [6]a c miR-486-5p Stable angina + 1.8-folds (HDL2) [8] c Unstable angina + 3.6-folds (HDL2) [8] c Myocardial infarction + 5.0-folds (HDL2) [8] miR-509-3p Familial hypercholesterolemia − 3.7-folds [6]a miR-520c-3p Familial hypercholesterolemia − 5.4-folds [6]a miR-572 Familial hypercholesterolemia − 2.5-folds [6]a miR-573 Familial hypercholesterolemia − 3.6-folds [6]a miR-625-3p Familial hypercholesterolemia − 11.0-folds [6]a miR-632 Familial hypercholesterolemia − 3.3-folds [6]a miR-877-5p Familial hypercholesterolemia − 2.3-folds [6]a LDL None yet IDL None yet

M, men; W, women. amiRNA differently concentrated in HDLs from patients with familial hypercholesterolemia (n = 5; 3 M/2 W) compared to healthy subjects (n = 6; 6 M/0 W). bmiRNAs showing different concentration in HDLs from patients with acute coronary syndrome (n = 10; 6 M/4 W) or coronary artery disease (CAD; n = 10; 6 M/4 W) compared to healthy subjects (n = 10; 6 M/4 W). No statistically significant dif- ference was observed for LDL-carried miRNAs (n = 5 for each group). cmiRNA differently concentrated in lipoproteins from 95 patients with CAD as compared to 16 healthy subjects (3 M/13 W). Patients with CAD were separated into three groups: stable angina (n = 30; 20 M/10 W), unstable angina (n = 39; 28 M/11 W) and myocardial infarction (n = 26; 22 M/4 W). miRNA analyses were performed on pooled samples (three pools per group).

important mediator of intracellular and systemic choles‑ Wagner et al. were the first to assess the contribu‑ terol homeostasis. Other HDL-carried miRNAs have been tion of HDL-carried miRNAs to the total plasmatic miRNA associated with hypercholesterolemia, inflammation, pool [7]. The percentages of HDL-bound miRNAs related and acute coronary syndrome (ACS) when compared to to those detected in plasma associated to other carriers healthy subjects (Table 2) [6, 7]. were found to be relatively low (< 1% to ~ 8%) in healthy Desgagné et al.: microRNAs in lipoprotein and lipid metabolism 675 subjects, with HDL-carried miR-223-3p contributing HDL-carried miRNA concentration is affected by the diet the most to the plasmatic pool of circulating miRNAs (~ 8%) [7]. A contribution level varying from one miRNA More recently, studies from our and other groups tested to another suggests that over the relatively large variety whether HDL-transported miRNA concentrations were of plasmatic miRNAs carried by HDLs, a specific subset sensitive to diet-induced metabolic changes. Tabet et al. of plasmatic miRNAs might be enriched in HDLs. These showed a mild decrease in HDL-carried miR-223-3p con‑ miRNAs would be of particular biological relevance, centration following a 12-week diet rich in proteins and potentially contributing to HDL functionality (e.g. miR- that this change in HDL-miR-223-3p level was associated 223-3p, which is abundantly transported in HDLs and with a diet-induced weight loss in overweight and obese have regulatory functions in cholesterol metabolism [119] men (Table 3) [137]. Our group reported an increased con‑ and in inflammation [136]). centration of HDL-carried miR-223-3p and miR-135a-3p, Very recently, Niculescu et al. showed that miR- which was associated with an overall worsening of the car‑ 486-5p and miR-92a-3p were upregulated in the serum of diovascular disease (CVD) risk profile (e.g. lowered HDL-C patients with CAD [including stable angina (SA), unstable and increased high-sensitivity C-reactive protein (hsCRP) angina (UA) and myocardial infarction (MI)] compared and TG concentrations; Table 3) following a 4-week dou‑ to healthy subjects (Table 2), and that these differ‑ ble-blinded, randomized, crossover, controlled dietary ences were also found significant in the HDL2 and HDL3 intervention rich in trans fatty acids (TFA) from industrial subpopulations[8]. Most importantly, the authors con‑ (iTFA) or ruminant (rTFA) sources [134]. cluded that these differences in HDL-carried miR-486-5p and miR-92a-3p concentrations may discriminate between stable and vulnerable CAD patients [8]. Interestingly, miRNA transfer to HDLs miR-92a-3p has been identified as one of the most con‑ centrated miRNAs in HDLs by Wagner et al. [7]. However, The HDL-carried miRNA profile is distinct but also shares miR-92a-3p was not preferentially associated to HDLs similarities with that of plasma, LDLs, and (to a lesser relatively to other plasmatic carriers (~ 2% contribution extent) exosomes [6, 7]. This was expected considering to the plasmatic miRNA pool) [7]. This miRNA might well that HDLs are an integral part of plasma (like other car‑ remain an excellent biomarker of CAD, but our enthusi‑ riers), that a complex interplay exists between different asm is diminished regarding its specific role in the regula‑ lipoprotein pathways, and that circulating miRNAs (inde‑ tion of the many HDL functions. pendently of the carrier) may be of common cellular origin

Table 3: Lipoprotein-carried miRNAs associated with diet-induced changes in cardiometabolic risk markers. miRNA Cardiometabolic risk marker Diet Correlation direction References

HDL miR-135a-3p LDL-triglyceride Rich in rTFA (3.7% energy) + [134]b b HDL2-cholesterol Rich in iTFA (3.7% energy) − [134] HDL-ApoA1 Rich in iTFA (3.7% energy) − [134]b Total triglyceride Rich in iTFA (3.7% energy) + [134]b miR-223-3p Weight High in proteins (30% energy) + [137]a C-reactive protein Rich in rTFA (3.7% energy) + [134]b HDL-cholesterol Rich in iTFA (3.7% energy) − [134]b Total ApoA1 Rich in iTFA (3.7% energy) − [134]b HDL-ApoA1 Rich in iTFA (3.7% energy − [134]b

ApoA1, apolipoprotein A1; iTFA, trans fatty acids from industrial source; rTFA, trans fatty acids from ruminants. aObese and overweight men followed either a diet rich in proteins (HP; 30% daily energy; n = 20) or a control diet with normal protein amount (NP; 20% daily energy; n = 27), for 12 weeks. Relatively to pre-diet baseline, mean HDL-carried miR-223-3p concentration was lowered (~ –1.3-folds) following the HP diet only, and this change in HDL-miR-223-3p level was associated with diet-induced weight loss. b Following a double-blind, randomized, crossover, controlled design, nine healthy men were fed each of the three isoenergetic diets for 4 weeks each, with a washout period of ≥ 3 weeks between diets: (1) rich in iTFA (3.7% daily energy); (2) rich in rTFA (3.7% daily energy); (3) low in TFAs (control; 0.8% daily energy). The variations in HDL-carried miR-223-3p and miR-135a-3p concentration following a diet rich in TFAs compared to the control diet were strongly correlated with diet-induced changes in cardiometabolic risk markers. 676 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism

(e.g. hepatocytes, macrophages, endothelial cells). Never‑ that Huh7 cells treated with HDLs from FH patients theless, certain miRNAs (e.g. miR-223-3p) were more impor‑ (abnormally abundant in miR-105-5p) showed increased tantly transported by HDLs in plasma [7], suggesting that intracellular miR-105-5p levels, whereas no change was a subset of miRNAs might potentially be more specifically observed when cells were incubated with HDLs from (actively) loaded in HDLs rather than in other carriers. healthy patients [6]. Interestingly, 60% of the concomi‑ This process could be supported by either cell type-speci‑ tantly downregulated mRNAs in Huh7 cells treated with ficity of membrane transporters and miRNA expression or HDLs from FH subjects harbored a putative target site of post-transcriptional modifications of miRNAs that would miR-105-5p [6]. This work proposed for the first time that increase their affinity for HDLs. endogenous HDL-carried miRNAs may be transferred to The exact mechanism by which miRNAs are trans‑ recipient cells with functional capabilities, although the ferred to HDLs and delivered to recipient cells is still possible contribution of other constituents of HDLs to the poorly described [138]. An obvious transporter is ABCA1, observed transcriptomic changes in cultures hepatocytes since it mediates the cholesterol and phospholipid could not be excluded. efflux to lipid-poor ApoA1 [84], but its overexpression In a more recent publication, the same group ele‑ in cultured mice macrophage resulted only in a modest gantly demonstrated that native HDL efficiently trans‑ increase in miR-223-3p levels (1.8-folds) in reconstituted fers miR-223-3p to human coronary artery endothelial HDLs (rHDL) [6]. Interestingly, sphingomyelin (SM) binds cells (HCAEC), into which miR-223-3p was not naturally RNA and prevents its degradation by RNAses [139, 140]. transcribed nor processed [136]. Importantly, Tabet et al. Moreover, a few groups have reported that small RNAs showed that change in intracellular miR-223-3p levels could bind to phospholipid membranes using vesicular following incubation with HDLs was transcription-inde‑ models [141–144]. Whether the plasma membrane SM, pendent in cells pretreated with actinomycin D, suggest‑ enriched into lipid rafts, could also bind miRNA and ing that this change in miR-223-3p levels might likely be contribute to its export to HDLs has not been published due to HDL miRNA transfer to the cell [136]. Moreover, they so far. However, the chemical inhibition of the neutral demonstrated that HDL-transferred miR‑223-3p efficiently sphingomyelinase 2 (nSMase2; the rate-limiting enzyme repressed ICAM-1 using luciferase assays [136]. This sug‑ in ceramide biosynthesis from SM) was shown to reduce gested for the first time that HDL-carried miR-223-3p likely the cellular miRNA export by exosomes [145–147]. Using contribute to the atheroprotective HDL anti-inflammatory inhibition of the nSMase2 in cultured mice macrophage, action in endothelial cells, by limiting monocyte adhe‑ Vickers et al. reported that the miR-223-3p export to rHDLs sion to endothelial cells [136]. This is concordant with the was significantly increased (35-folds), suggesting distinct previously identified anti-inflammatory function of miR- and possibly opposing mechanisms for miRNA export to 223-3p in macrophages [149]. HDLs and exosomes through the SM-ceramide pathway Another group has measured the HDL-carried miRNA [6]. This pathway may nevertheless constitute an impor‑ transfer to human umbilical venous endothelial cells tant regulatory mechanism that will have to be further (HUVEC), vascular smooth muscle cells (SMC) and periph‑ explored. eral blood mononuclear cells (PBMC) [7]. The authors first showed that HDL-introduced exogenous Caenorhabditis elegans (cel)-miR-39 can be transferred to HUVECs, but in Delivery of functional HDL-carried miRNAs to recipient low levels only (< 10 copies/cell) [7]. This group did not cells show a significant miRNA transfer from native HDLs to HUVECs, SMCs and PBMCs, but reported a slight decrease Circulating miRNAs have been suggested to play a role in in intracellular miR-223-3p, miR-92a-3p and miR-126-3p endocrine-like intercellular communication [6, 148]. The levels, especially in the PBMCs [7]. This was observed first evidence of HDL-carried miRNA biological function‑ after a relatively short incubation with native HDLs from ality was provided in 2011 by Vickers et al. [6]. As a proof healthy subjects [7]. Interestingly, the intracellular miRNA of concept, the authors showed that native HDLs loaded levels were decreased in PBMCs after incubation with with miR‑375 or miR-223-3p miRNA mimics efficiently deliv‑ HDLs from healthy subjects, whereas they were increased ered these miRNAs to cultured human hepatocytes, with after incubation with HDLs from CAD and ACS patients a concomitant decrease in mRNA levels of two putative [7]. This differential cellular response to HDLs from target genes of miR‑223‑3p [6]. Moreover, the miRNA trans‑ healthy and diseased subjects may reflect physiological fer to recipient cells was demonstrated to be mostly SR-BI- changes in HDL functions [150]. The analysis of intra­ dependent [6]. Importantly, Vickers’ group demonstrated cellular pri-miRNA and pre-miRNA levels revealed that Desgagné et al.: microRNAs in lipoprotein and lipid metabolism 677 the observed changes in PBMCs might be due to transcrip‑ plaque and thus vascular inflammation [152]. Noteworthy, tional changes in miR-126-3p, but not miR-223-3p and miR- the miR-155-5p concentration in macrophages was found 92a-3p [7], raising the interesting hypothesis that PBMCs to be increased in presence of native and mildly oxidized may export miRNAs to HDLs. The direction of the miRNA LDLs, whereas profoundly oxidized LDLs lowered the miR- exchange between HDL and cells (import or export) could 155-5p concentration in macrophages [152]. Although the possibly be influenced by cell culture conditions and con‑ functional potential of LDL-carried miRNAs has not been tribute to explain, at least partially, the observed discrep‑ investigated yet, this observation may suggest a possible ancies between these studies. transfer of LDL-carried miR-155-5p to macrophages or, Although the transfer (and possibly exchange) of alternatively, a change in cellular miR-155-5p expression functional HDL-carried miRNAs to recipient cells has been induced by LDLs, which is likely affected by the degree demonstrated in different cultured cells [6, 7, 119], the of LDL oxidation [7]. Taken together, these pioneering HDL’s capacity to deliver (exchange) functional miRNAs studies suggest that LDL- and IDL-carried miRNA profiles in vivo has not been investigated so far. This matter will be may be altered in the diseased state, and more particularly of major importance in the future. in CAD (Table 2), and support the assessment of the role of these lipoproteins miRNAs profiles in larger studies [7, 8].

Low-density lipoprotein and other ­ApoB-containing lipoproteins Biomarkers As for HDLs, IDL and LDL particles were also found to transport miRNAs in circulation [6–8]. Despite the recog‑ miRNA as potential biomarkers of CVDs nized implication of LDLs in atherosclerosis development and the strong association between high LDL-C levels and Due to their gene regulatory function in diverse biological increased risk of CVDs [4], the LDLs’ miRNA cargo has processes and metabolic functions [63–65, 155], as well as been less studied than that of HDLs. Initial work from their high stability in biological fluids [34–37], circulat‑ Vickers et al. allowed the identification of several miRNAs ing miRNAs have been pointed out as an exciting avenue particularly abundant in LDLs including, among others, for the identification of new, non-invasive biomarkers miR-223-3p, miR-150-5p, miR-19b-3p, miR-24-3p, miR-92a-3p of diseases. Compared with the usual protein biomark‑ and miR-146a-5p [6]. From the nine miRNAs quantified by ers, miRNAs could provide early disease markers (there Wagner’s group, miR-223-3p was also reported as the most usually needs to be tissue damage for protein biomark‑ concentrated in LDLs from healthy subjects [7]. Moreover, ers to be liberated) or greater specificity. Indeed, miRNAs miR-92a-3p has been repeatedly identified as one of the can be detected quantitatively by the real-time quantita‑ most abundant miRNA carried by LDLs and IDLs [6–8]. tive polymerase chain reaction (RT-qPCR) technique with Although most miRNAs found by Wagner et al. in high sensitivity, specificity and multiplexing potential LDLs and IDLs from healthy subjects were more abundant [43, 156]. However, RT-qPCR remains a relatively time- in HDLs [7, 8], which may partially explain the greater consuming technique compared to many analyses cur‑ enthusiasm toward the HDL-carried miRNA transport as rently used in clinical laboratories, and no consensus on compared to LDLs, Wagner’s group identified one miRNA, a data normalization method is currently established (see miR-155-5p, that was more concentrated in LDLs than in Kappel et al. in this special edition for details on miRNA HDLs [7]. miR-155-5p is implicated in the regulation of detection) [156, 157]. Also, the amount of miRNA in easily various physiological processes including hematopoie‑ accessible biological matrixes (e.g. plasma, urine) is gen‑ sis, immunity, inflammation and adipogenesis. Its over‑ erally small, making a reliable miRNA quantification and expression has been previously associated with several results reproducibility more difficult [156, 157]. Some inflammation-related pathologies such as cancers, ath‑ miRNAs are modulated in more than one pathology, lim‑ erosclerosis, CVD and obesity [151–154]. miR-155-5p plays iting the specificity and thus the applicability of these a central role in the regulation of macrophage polariza‑ miRNAs as biomarkers [156]. tion toward the M1 pro-inflammatory phenotype, and Despite these challenges, several studies have inves‑ was found to play a role in the atherogenic programming tigated the potential usefulness of miRNAs based on of macrophages by indirectly upregulating the mono‑ reported differences in miRNA signature in health and cyte chemotactic protein 1 (MCP1), a chemokine known disease [43, 156, 157]. To date, most of the literature in the to promote monocyte recruitment to the atherosclerotic field focuses on cancers and CVDs [158–160]. Numerous 678 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism reviews describe miRNAs as potential biomarkers for related cluster with CVD is much more abundant and gen‑ CVDs, individually or in a panel signature, for single or erally agrees with Niculescu’s study [173, 174]. Very inter‑ mixed use (diagnostic, prognostic, and staging) [159, estingly, miR-92a-3p has been identified as an atheromiR 161]. Some studies have compared these miRNAs to the in a large scale screen in human umbilical vein endothe‑ current reference biomarkers such as troponin (I and T) lial cells (HUVECs) searching for upregulated miRNAs in [159, 161]. Interestingly, Zampetaki et al. challenged the response to the presence of oxidized LDL and low shear use of miRNAs as predicators of cardiovascular events stress [175]. This could help in refining the CAD status and showed that circulating miR-126, miR-197 and miR-223 prediction (see Zeller et al. in this special edition for more are strongly associated with the risk of future MI, and that details on miRNAs in CVD). adding the circulating levels of these three miRNAs to the The use of circulating miRNAs in a non-invasive usual Framingham risk score (FRS) improves by ~ 15% the screening, diagnostic or prognostic of disease is an excit‑ risk classification [162]. This improvement is better than ing and awaited clinical application of miRNAs. Based on that observed when adding the hsCRP levels to the risk the current knowledge, plasmatic and lipoprotein-associ‑ score calculation [163], supporting the high potential of ated miRNAs (individually or as a set of related miRNAs) circulating miRNAs. have great potential to become clinically used biomark‑ ers in the future. However, larger well-designed studies with standardized methodologies (e.g. sampling, miRNA Lipoprotein-bound miRNAs as a biomarker extraction and quantification techniques, data normaliza‑ tion method) are needed to achieve this important goal. Lipoproteins are widely used as biomarkers in the evalua‑ tion of the CVD risk. Although these biomarkers are mainly based on lipoprotein-cholesterol levels, more recent studies suggested that the lipoprotein characteristics (e.g. Therapy composition, functionality) were more strongly associ‑ ated to the CVD risk than solely its cholesterol content Nowadays, statins are one of the most prescribed classes [164–168]. The discovery that miRNAs are transported by of drugs to treat dyslipidemia and for the prevention and lipoproteins opens up our minds to new possibilities [6]. management of CVDs [176]. Statins decrease the de novo Although many plasmatic miRNAs have been identified cholesterol synthesis through HMGCR inhibition, and as potential CVD biomarkers, most of them have not been lower the cholesterol levels in plasma through indirect correlated yet with established CVD biomarkers [161]. An upregulation of LDLR via SREBP2 activation (in response interesting hypothesis would be that the carrier-specific to decreasing cellular cholesterol levels) [176–179]. Up to miRNA signature is a more accurate biomarker of a disease now, statins have been the only lipid lowering drug con‑ state than that of plasma. The association of the specific ferring a significant (~ 25%) reduction of the risk of CVD HDL-carried miRNA profile with various diseases and [176–178]. Others lipid lowering drugs, such as fibrates metabolic status (Tables 2 and 3), as well as these miRNAs’ [180, 181], niacin [178] and ezetimibe [178], are also biological relevance illustrate the potential of HDL-trans‑ available, but their effects on CVD risk prevention are ported miRNAs as biomarkers (and therapeutics; see next limited (alone or in combination with statins). Numerous section). No study has yet evaluated the clinical benefits others molecules are currently in development targeting of HDL-associated miRNAs over that of standard biomark‑ various lipoprotein biosynthesis steps, including inhibi‑ ers. As reported above, Niculescu et al. have showed that tors of ApoB biosynthesis, MTP activity, and ApoC-III among the six miRNAs upregulated in plasma from CAD synthesis, as well as the very promising proprotein con‑ compared to healthy subjects, miR-486-5p and miR-92a-3p vertase subtilisin/kexin type 9 (PCSK9) inhibitors [178, could discriminate between CAD stages in regard to their 182, 183]. In contrast, the HDL-raising therapies devel‑ levels in HDLs [8]. MiR-486-5p has been identified in oped over the past years, including over-expression of various cancer studies [169], and its serum concentration APOA1 gene, infusion of reconstituted HDL particles or has been reported negatively correlated with systemic ven‑ recombinant lecithin-cholesterol acyltransferase (LCAT) tricular contractility following a cardiac bypass surgery and CETP inhibitors [178, 184], have failed to prevent [170]. More importantly, miR-486-5p has been associated CVD occurrence despite a huge increase (up to 180%) with the angiogenesis process and was recently shown in HDL levels [177, 178, 185–187]. This clearly shows that to regulate the cholesterol efflux from macrophages [171, the HDL particle is much more complex than previously 172]. Literature about the association of miR-92a-3p and its thought. Accordingly, our understanding of different Desgagné et al.: microRNAs in lipoprotein and lipid metabolism 679

HDL functions and the molecular mechanisms involved still a challenge, but one that needs to be addressed in needs to be refined. order to limit the undesirable side-effects of the treat‑ To this end, novel classes of molecules are currently ment [32]. Pharmacodynamics is also difficult to assess in development, targeting HDLs’ metabolism, parti‑ since miRNAs regulate numerous genes and pathways cle structure and function [178, 185]. One such class is and the summation of these effects requires sophisticated miRNAs, which have emerged as potential candidates analytical models. Anti-miR and miR-mimetic treatments because they are important regulators of lipid metabolism may induce toxicity related to the chemical structure of and can target several mRNAs implicated in a given the molecule and also toxicity related to the hybridiza‑ pathway. As for conventional drugs, the development of tion potential of the molecule to other undesirable target a miRNA-based drug is a challenging multi-step process mRNAs (off-target) or tissue. (recently reviewed in [188] and [189]). It begins with the Even though a good number of miRNAs have been profiling of miRNAs in health and disease states [188]. described in the literature in various pathologies and Either downregulated or upregulated miRNAs (i.e. biologi‑ are candidates to become potential miRNA-based treat‑ cally relevant in disease development or progression) are ments, only a few are now under serious development then selected and tested in vitro and in vivo for gain and [188, 189]. Two miRNA mimics (miR-34 and let-7) targeting loss of function [188]. Since miRNA levels may be either oncogenes implicated in the development of solid tumors increased or decreased, two classes of miRNA-based treat‑ are presently in preclinical development [195]. miRNA ments have been described [188–190]. miRNA antagonists, antagonists are also of interest, with candidates under also known as antagomiR or anti-miR, are antisense oli‑ development: three miRNAs (miR-195 et miR-208/499) in gonucleotides which complementary bind to the targeted preclinical targeting CVD and one miRNA (miR-122) in mature miRNA, causing its loss of function by creating phase II clinical trial targeting hepatitis C viral infection a duplex unable to bind its target mRNA and inhibit its [196, 197]. Importantly, a miR-33 antagonist is currently translation [189]. Since free-synthetic and exogenous under preclinical development [129, 189]. As reported miRNAs are rapidly degraded in biological fluids, these above, miR-33 is central in cholesterol homeostasis, and antagomiRs need to be structurally stabilized in order its inhibition raises HDL-C levels, increases the choles‑ to increase their half-life and cellular uptake [189]. The terol reverse transport from macrophages and is athero‑ antagomiR structural stabilization can be achieved by the protective. Other miRNAs are also good candidates for incorporation of 2′-O-methyl modified oligonucleotides as the treatment of dyslipidemias, including miR-30c-5p and well as locked nucleic acid (LNA) modified nucleotides miR-148a-3p. The overexpression of miR-30c-5p has been or by modifying linkages between nucleotides [191, 192]. showed to reduce plaque formation in an atherosclerotic Also, adding a cholesterol molecule to the original antago‑ mouse model and, alternatively, its inhibition has induced miR structure has already been used to increase its cel‑ hyperlipidemia and increased atherosclerotic plaque for‑ lular uptake (more specifically in the cardiac tissue) [191]. mation [80]. The action of miR-30c-5p was mediated by the Another strategy to downregulate a miRNA function is the inhibition of MTP, which regulates the secretion of VLDL use of miRNA sponges, which harbor multiple comple‑ by the liver, even though miR-30c-5p can also mediate its mentary sites to the targeted miRNA [193]. action in a MTP-independent way. miR-148a-3p is also an Furthermore, the levels of miRNAs downregulated in interesting target for a miRNA-based treatment, because disease may be normalized using miRNA mimics, which this miRNA has been found to downregulate two genes are synthetic double-stranded oligonucleotides that important in lipoprotein metabolism: LDLR and ABCA1 mimic the miRNA function they replace [188, 189]. Similar [115]. The expression of miR-148a-3p was increased in the to antagomiRs, miRNA mimics are chemically modified to liver of high-fat diet mice and rhesus monkeys [105], and improve their stability and cellular uptake [188]. However, its inhibition in huh7 cells and mice models was shown only the “passenger strand” is usually modified, while the to significantly increase hepatic LDLR and ABCA1 expres‑ “guide strand” remains identical to the mature miRNA sion, concomitant with increased HDL-C and decreased of interest [189]. Alternatively, the expression of a down‑ LDL-C levels in circulation [105, 115]. Moreover, miR- regulated target miRNA may be stably normalized by the 148a-3p was also able to repress ABCA1 and the cholesterol use of adenovirus-associated vectors and tissue-specific efflux capacity in mice macrophages [105]. Also, a large ­promoters [194]. genome-wide association study (GWAS) meta-analysis Various methods of miRNA-based treatment deliv‑ (> 188,000 individuals) identified two polymorphisms ery have been described [188]. It is clear that the specific within the MIR148a promoter region that were associ‑ delivery of the molecule to a particular site of action is ated with plasma levels of total cholesterol, LDL-C and 680 Desgagné et al.: microRNAs in lipoprotein and lipid metabolism triglycerides, suggesting that this miRNA is involved in an exciting research avenue to explore in regard of lipo‑ lipid homeostasis in humans [105]. These miRNAs have protein functionality and potential miRNA-based clinical been linked to the lipid metabolism and atherosclerosis applications for the prevention and treatment of cardio‑ and are thus very promising novel therapeutic targets. metabolic diseases. The demonstration that HDLs could transport a spe‑ cific and metabolically sensitive set of functional miRNAs Acknowledgments: We express our gratitude to Céline and deliver them to recipient cells led us to an endocrine- Bélanger (CIUSSS du Saguenay–Lac-St-Jean, Hôpital de like signaling pathway which still needs to be validated in Chicoutimi) for her thoughtful revision of the manuscript. vivo [6]. This discovery could improve our understanding Author contributions: All the authors have accepted of the functionality of HDLs in cardioprotection. It could responsibility for the entire content of this submitted support the interesting possibility of using HDL particles manuscript and approved submission. to deliver specific miRNA therapies. Infusion of synthetic Research funding: VD was recipient of a doctoral research HDL particles is presently in phase II trial [184]. These award from the Fonds de recherche du Québec en santé particles are composed of ApoA1 and phospholipids, (FRQS). LB is junior research scholar from the FRQS and mimicking nascent HDLs. Loading these synthetic HDLs member of the FRQS-funded Centre de recherche du with functional miRNAs is an exciting avenue to improve CHUS (affiliated to the Centre hospitalier universitaire de HDL-directed therapy, by which HDLs could stabilize and Sherbrooke). deliver functional antagonist or mimetic miRNAs. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and Conclusions interpretation of data; in the writing of the report; or in the decision to submit the report for publication. The involvement of miRNAs as central regulators of the lipoprotein metabolism and cholesterol homeostasis is now well established. The role of lipoprotein-carried References miRNAs as functional regulators or useful biomarkers is emerging. HDLs have been shown to transport and 1. 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