- Email: [email protected]
b and microRNA therapeutics
Journal of Cardiology 67 (2016) 28–33
Contents lists available at ScienceDirect
Journal of Cardiology journal homepage: www.elsevier.com/locate/jjcc
[6_TD$IF]Review
Functions of microRNA-33a/b and microRNA therapeutics Koh Ono (MD, PhD, FJCC)*[4_TD$IF] Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 October 2015 Accepted 26 October 2015 Available online 29 November 2015
Atherosclerosis is a chronic inflammatory disease of the arterial wall. It is characterized by subendothelial accumulation of low-density lipoprotein cholesterol, and its subsequent modification at athero[12_TD$IF]-prone areas leads to further activation of the vascular wall and maintains vascular inflammation. An entirely new level of post-transcriptional gene regulation through microRNA (miR) expression has emerged recently as an important mechanism in the development and progression of numerous diseases, including atherosclerosis. Recently, miR-33a/b have been shown to act as posttranscriptional regulators of lipid metabolism, and their pharmacological inhibition diminished atherosclerosis by raising plasma high-density lipoprotein levels. This review summarizes the current understanding of the functions of miR-33a/b and the progress in miRNA therapeutics for treatment of various diseases, including atherosclerosis. ß 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
Keywords: ABCA1 HDL-C Plaque Antisense oligonucleotides
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . microRNA-33a/b regulates high-density lipoprotein cholesterol in vivo . microRNA-33 inhibition has a beneficial effect on atherosclerosis . . . . . Other functions of microRNA-33a/b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . microRNA modifications and chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of microRNA function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antisense oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . miRNA sponges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Restoration of microRNA levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonviral delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction MicroRNAs (miRNAs; miRs) are endogenous, small (approximately 20–22 nucleotides in length), non-protein-coding RNAs.
* [8_TD$IF]Correspondence to: Department of Cardiovascular Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 3190; fax: +81 75 751 3203. E-mail address: [email protected]
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
28 29 29 30 31 31 31 31 32 32 32 32 32 32 32
miRNAs bind to the 30 untranslated region (UTR) of specific mRNAs according to the complementarity of their sequences and inhibit translation or promote mRNA degradation. miRNAs were initially discovered in Caenorhabditis elegans by Victor Ambros’ group while studying the gene lin-14 [1], and they were later found to be evolutionarily conserved. More than 60% of human protein-coding genes have been shown to be regulated by miRNAs, and so far, approximately [13_TD$IF]2500 miRNAs have been identified in humans [2]. miRNAs are usually transcribed by RNA polymerase II (Pol II) in the nucleus as longer precursors with a hairpin structure, referred
http://dx.doi.org/10.1016/j.jjcc.2015.10.017 0914-5087/ß 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
[(Fig._1)TD$IG]
K. Ono / Journal of Cardiology 67 (2016) 28–33
to as [14_TD$IF]primary miRNAs (Pri-miRNAs), and these are subsequently processed by the Drosha (RNase III)/DGCR8 complex to form approximately 70-nucleotide pre-mature miRNAs (Pre-miRNAs). Pre-miRNAs are then translocated to the cytoplasm through exportin 5, where Dicer (an miRNA-specific nuclease) cleaves them, resulting in double-stranded miRNAs that form mature miRNAs of 18–25 nucleotides in length. Subsequently, mature miRNAs are incorporated into an RNA-inducing silencing complex (RISC) by associating with Argonaute (Ago) proteins. This complex with the miRNA targets the 30 -UTR of the mRNA, which is then responsible for the decrease in target gene expression, [15_TD$IF]via either degradation of the mRNA or inhibition of protein synthesis [3]. In addition to their existence in various tissues, recent studies have indicated that miRNAs also exist in serum, plasma, urine, and other body fluids in highly stable forms that are protected from endogenous RNase activity by exosomes and other proteins [4]. Circulating miRNA expression profiling has provided strong molecular markers for the detection of various diseases. Altered levels of circulating miRNAs have been observed in acute coronary syndrome [5], heart failure, essential hypertension, and stroke. Work over the past several years has demonstrated that miRNAs control the expression of most of the genes associated with high-density lipoprotein (HDL) metabolism, including the ATP transporters, ABCA1 and ABCG1, and the scavenger receptor SRB1 [3,6–8]. These findings suggested that miRNAs regulate HDL biogenesis, cellular cholesterol efflux, and HDL cholesterol (HDL-C) uptake in the liver, thus controlling all of the steps of reverse cholesterol transport (RCT). Elucidation of the function of miRNAs may provide novel strategies for the treatment of dyslipidemia. In particular, we have intensively investigated the functions of miR33a/b in vivo using genetically modified mice [9–12]. The present review summarizes the function of miR-33a/b and current strategies for modulating the function/levels of miRNAs. microRNA-33a/b regulates high-density lipoprotein cholesterol in vivo The miR-33 family consists of two intronic miRNAs, miR-33a and miR-33b, which are encoded in the introns of SREBF2 and SREBF1, respectively [13]. The sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate fatty acid and cholesterol synthesis. SREBF1 encodes SREBP-1a and -1c, which mainly regulate lipogenic genes, such as fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), and acyl-CoA carboxylase 1 (ACC1). SREBF2 encodes SREBP-2, which mainly regulates cholesterol-regulating genes, such as 3hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and lowdensity lipoprotein receptor (LDLR) [14]. In humans, miR-33a and miR-33b are encoded in the introns of SREBF2 and SREBF1, respectively [15], whereas in rodents there is a deletion in the miR33b encoding sequence and miR-33b cannot be expressed (Fig. 1). Furthermore, miR-33a and miR-33b share the same seed sequence and differ in only three nucleotides. The fact that miR-33a and miR33b are cotranscribed with their respective host genes suggests that they regulate lipid homeostasis with their host genes. Several groups have shown that miR-33a targets genes involved in cholesterol export, such as ATP-binding cassette transporter A1 (Abca1) and Abcg1 in vivo, using either antisense technology or by generating miR-33a-deficient (knockout) mice [9,13]. ABCA1 promotes the movement of excess free cholesterol out of the cell. In the liver, it is essential for efflux of cholesterol to lipid-poor apolipoprotein A-I (apoA-I) and forms nascent HDL. In peripheral cells, ABCA1 and ABCG1 efflux excess cholesterol to apoA-[16_TD$IF]I and HDL particles for delivery to the liver for excretion in the bile and feces. Therefore, ABCA1 is an essential molecule for HDL biogenesis and RCT. The ABCA1 mRNA and protein half-lives are short (1–2 h),
29
Rodents
Srebf2
Srebf1
miR-33a
miR-33a
Human SREBF1
miR-33b
miR-33b
SREBF2
miR-33a
miR-33a
Fig. 1. Mice have a single copy of microRNA (miR)-33, but humans have two copies (miR-33a and miR-33b).
suggesting that de novo transcription and translation are important for controlling its expression in response to stimuli. The 30 -UTRs of mouse and human ABCA1 and mouse ABCG1 contain binding sites for miR-33a/b, and transfection of miR-33a/b strongly repressed ABCA1 and ABCG1 mRNA and protein in a variety of cells. It was shown that mice treated with antisense oligonucleotides or antimiR-33a lentivirus exhibited increased ABCA1 expression in the liver and ABCA1 and ABCG1 expression in macrophages. Furthermore, inhibition of endogenous miR-33a in both these cell types promotes an increase in the expression of ABCA1 and ABCG1 protein and cholesterol efflux to apoAI and HDL-C, indicating the physiologically relevant role of this miRNA in regulating ABCA1 and ABCG1. Notably, in an anti-miR-33a-treated mouse model of atherosclerosis, plasma HDL levels increased by 35–50% without affecting other lipoproteins [13]. miR-33a knockout mice also showed a significant increase in the expression of ABCA1 and ABCG1 and a 25–40% increase in serum HDL-C [9] (Fig. 2A[17_TD$IF]). AntimiR-33a therapy has been shown to contribute to enhance the RCT pathway not only by increasing HDL-C through ABCA1 and ABCG1 upregulation but also by increasing bile secretion through upregulation of ABCB11 and ATP8B1, which are the other targets of miR-33a/b [16]. Using an in vivo assay to measure the efficiency of RCT, it was shown that the HDL-C generated by miR-33a inhibition was functional and increased the transport of cellular radiolabeled cholesterol to the plasma, liver, bile acid, and feces. Interestingly, in contrast to the results with miR-33a-deficient mice, HDL-C levels in miR-33b knock-in (KI) mice, which has miR33b in the same intron as in humans [12], were reduced by almost 35%, even in miR-33b KI [18_TD$IF]heteromice compared with the control mice (Fig. 2B). microRNA-33 inhibition has a beneficial effect on atherosclerosis The function of miR-33a inhibition in animal models of atherosclerosis has been examined by several groups. It has already been proven that antisense inhibition of miR-33a resulted in regression of atherosclerotic plaque in LDLR-deficient mice by promoting RCT [17]. This promotion of RCT was shown to be achieved in two ways: by directly increasing HDL-C biogenesis in the liver, and by increasing cellular cholesterol efflux from plaque macrophages. Moreover, miR-33a-deficient mice reduced the progression of atherosclerosis in apoE-deficient mice (Fig. 3) [10]. In our study, miR-33-deficient mice not only had higher and functional HDL-C but also had macrophages with higher cholesterol efflux capacity, resulting in the lower lipid accumulation in atherosclerotic areas [10]. Other possible beneficial properties of
[(Fig._2)TD$IG]
30
K. Ono / Journal of Cardiology 67 (2016) 28–33
Fig. 2. Lipid profiles of wild-type (WT), microRNA (miR)-33a-deficient mice (knockout, KO), and miR-33b knock-in (KI) mice ([1_TD$IF]cited and modified from Refs. [9,12]). (A) Representative high-performance liquid chromatography (HPLC) analysis of serum cholesterol from male wild-type and miR-33a-deficient mice. [2_TD$IF](B) Representative HPLC analysis of serum cholesterol from male wild-type and miR-33b KI+/+ mice.
anti-miR-33a/b therapy include an anti-inflammatory reaction [19_TD$IF]via upregulation of ABCA1. ABCA1 decreases cell-surface cholesterol levels, inhibits its partitioning into lipid rafts, and reduces the responsiveness of inflammatory signals from innate immune receptors. Furthermore, ABCA1 has been reported to act directly as an anti-inflammatory receptor independent of its lipid transport activities [18]. Although these preclinical studies of miR-33a inhibition in mice are encouraging, it has been shown that obesity and hepatic steatosis were observed in miR-33a-deficient mice at the age of 50 weeks or when fed a high-fat diet for 12 weeks [11]. We found that Srebf1 is a good target of miR-33a, and enhanced expression of SREBP-1 may have caused these phenotypic changes in miR-33adeficient mice [20_TD$IF][11]. Suppression of SREBP-1 by miR-33a indicated the existence of a more direct and fine regulatory mechanism between SREBPs. It was also shown that chronic inhibition of miR33a by antisense oligonucleotides increased the expression of genes involved in fatty acid synthesis such as ACC1 and FAS in the livers of mice [19]. It was also reported that anti-miR-33a therapy enhanced the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS.
[(Fig._3)TD$IG]
It is also of note that mice lack miR-33b, which is present in the Srebf1 gene of medium and large mammals, and that extrapolating the findings in rodents to humans is complicated. This difference between mice and humans may be particularly relevant under conditions in which the transcription of Srebf1 is upregulated, such as feeding after fasting and hyperinsulinemia. Because such a condition may lead to greater downregulation of cellular cholesterol efflux and plasma HDL-C levels, not complete but partial inhibition of miR-33a/b, which avoids the induction of further Srebf1, would be beneficial for the increase of HDL-C and prevention of atherosclerosis. Other functions of microRNA-33a/b miR-33a/b are known to have several other target genes including AMP-activated kinase (Ampka1) and Cpt1a, which are involved in the regulation of lipid and glucose metabolism [20]. However, no significant difference in these target genes has been observed in miR-33a-deficient and miR-33b-KI mice compared with their levels in control mice [11,12]. It has been suggested that miR-33a/b regulates stem cell selfrenewal [21_TD$IF]via downregulation of p53 [21]. p53 has two putative miR33a/b binding sites in the 30 -UTR, and miR-33s overexpression
Fig. 3. microRNA (miR)-33a-deficiency reduced atherosclerosis (cited and modified from Ref. [10]). (A) Representative microscopic images of cross-sections of the proximal aorta in miR-33+/+[3_TD$IF]Apoe/ and miR-33/Apoe/ male and female mice. Scale bars, 1 mm. (B) Quantification of the atherosclerotic plaque area in cross-sections of the proximal aorta in male and female mice. Values are the means S.E. ([5_TD$IF]n = 10–11 each, *p < 0.05).
K. Ono / Journal of Cardiology 67 (2016) 28–33
20 -fluoro (20 -F), and locked nucleic acid (LNA) modifications. In order to see the effect of miR-33a/b inhibition, 20 -F/MOE-modified phosphorothioate-backbone-modified antisense miR-33 was injected into African green monkeys [22]. It was reported that increased hepatic expression of ABCA1 and sustained increase in plasma HDL-C levels were observed over 12 weeks. Addition of LNAs to the ASOs increased the binding specificity. LNA-modified oligonucleotides contain one or more nucleotides in which an extra methylene bridge fixes the ribose moiety in the 30 [24_TD$IF]endoconformation [23]. This modification possesses high affinity and results in significant increases in melting temperature of up to several degrees per LNA residue and leads to the formation of a thermodynamically strong duplex with complementary RNA. In addition, this character of LNA oligonucleotides allows for the generation of ‘tiny LNAs’ that bind only to the seed region of target miRNAs. Tiny LNAs are 7–8 nucleotides long and can inhibit a family of miRNAs at the same time, an advantage over antagomiRs that can only target individual miRNAs. Rottiers et al. demonstrated that pharmacological inhibition of the miR-33 family by a subcutaneous delivered 8-mer LNA-modified antimiR into obese and insulin-resistant nonhuman primates, which resulted in derepression of miR-33 targets, such as ABCA1, and increases in circulating HDL-C [24]. Recently, another kind of miRNA inhibitor has been developed. These are referred to as ‘blockmirs,’ which target the miRNAbinding site in a specific target gene (Fig. 4B) [25]. They are designed to bind to the seed region with additional nucleotides to increase specificity. The unique feature of blockmirs is that they target specific mRNA while still allowing the miRNA to regulate other target genes.
repressed p53 expression and p53-mediated apoptosis. This study suggested that miR-33s may promote the repopulation capacity of hematopoietic stem cells. It is known that SREBP-1 and cellular cholesterol levels have also been shown to regulate cell-cycle progression. Therefore, miR-33a/b may cooperate with their host genes in regulating cell-cycle progression. microRNA modifications and chemistry The ability of miRNAs to regulate multiple genes in signal transduction pathways makes them attractive therapeutic targets. For this reason, miRNA therapeutics may be superior to other therapies that target a single protein or use small molecules. miRNA therapeutic approaches can be divided into two categories: miRNA inhibition (blockade) to reduce the expression of miRNAs upregulated in the setting of a pathological condition, and miRNA replacement to restore the expression of miRNAs repressed in the setting of a disease (Fig. 4). Inhibition of microRNA function Current strategies to inhibit miRNAs include the use of miRNA inhibitors and miRNA sponges. An ideal miRNA inhibitor needs to have several functions, including high affinity to the target genes, low toxicity, high specificity, resistance to exonucleases, and low cost for synthesis. Antisense oligonucleotides [2_TD$IF]miRNA inhibitors are chemically modified single-stranded antisense oligonucleotides[23_TD$IF] (ASOs) that are complimentary to the mature miRNA. To date, angtagomiRs (target-single miRNAs conjugated with cholesterol), antimiRs (target-single miRNAs), and tiny antimiRs (target miRNA families) have been synthesized (Fig. 4A). These ASOs can reduce the levels of pathogenically expressed miRNAs. A number of chemical modifications have been used to improve the pharmacodynamics and pharmacokinetics of ASOs. The modifications that are most commonly used are 20 -Omethoxyethyl (20 -MOE), 20 -O-methyl ribose-modified RNA (20 -OMe),
[25_TD$IF]miRNA sponges A recent clinical trial indicated that five weekly injections of an antimiR were sufficient to achieve inhibition for at least 6 weeks in humans. However, repeated injection may be necessary to allow for longer inhibition. To overcome these barriers, ‘miRNA sponges’ were developed to inhibit miRNA activity (Fig. 4C). miRNA sponges contain multiple miRNA[26_TD$IF]-binding sites, which act as competitive
[(Fig._4)TD$IG]
Normal miRNA funcon
31
A. AntagomiRs or anmiRs for miRNA inhibion
B. BlockmiRs for specific target inhibion cap
ORF
AAAAAAAAA(n)
Ribosome cap
ORF
AAAAAAAAA(n)
Ribosome
C. Sponges for miRNA inhibion cap
AAAAA(n)
ORF
miR-binding sites
D. miRNA mimics for miRNA replacement Fig. 4. microRNA (miRNA) manipulation strategies. Modification of miRNAs can be achieved by several strategies. Red lines indicate the methods to increase target gene expression and blue lines indicate approaches to reduce them. (A) AntagomiRs and antimiRs for miRNA inhibition. (B) BlockmiRs for specific target inhibition. (C) miRNA sponges for miRNA inhibition. (D) miRNA mimics for miRNA replacement.
32
K. Ono / Journal of Cardiology 67 (2016) 28–33
inhibitors for miRNA binding. This technology mainly utilizes viral gene transfer vectors to deliver the miRNA sponges into cells/ tissues. However, nonviral delivery systems can also be used, such as liposomes, polysaccharides, and artificial polymers. miRNA sponges have been used to regulate miRNAs specifically in skeletal muscle [26]. [27_TD$IF]Restoration of microRNA levels Although efforts to inhibit overexpressed miRNAs have been a major focus over the past years, several successful strategies have been employed to express depleted miRNAs in diseased cells/ tissues. miRNA levels are restored using oligonucleotide-based miRNA mimics, or by the use of viruses to drive expression of a given miRNA. miRNA mimics are double-stranded synthetic oligonucleotides that are processed into a single-stranded miRNA to regulate target genes in the cells (Fig. 4D). Montgomery et al. restored the functional miR-29 mimic to target fibrotic disease [27]. The miR-29 mimic was conjugated to cholesterol to facilitate cellular uptake and contained mismatches to prevent it from acting as an inhibitor. There are several reports that miRNA mimics were successfully used in clinical trials and the therapeutic potential of miRNA mimics has been discussed [28]. Nonviral delivery [28_TD$IF]Nonviral delivery of miRNAs is achieved by conjugation-based methods, liposomes, nanoparticles, or antibody-based methods. Lipid-conjugated dsRNAs, including palmitic acid, lauric acid, and cholesterol, in which these lipids modified the 50 -end of the sense strand[29_TD$IF], have been used to facilitate cellular uptake and to add a potent gene silencing effect for the delivery of siRNAs or miRNAs [29]. Liposome nanoparticles have been utilized for miRNA delivery applications. Nanoparticles can be targeted to specific cells and tissues by adding cell[30_TD$IF]-surface receptor, ligands, and antibodies [30]. [31_TD$IF]Viral delivery Retrovirus, lentivirus, adeno-associated virus (AAV), and adenovirus have been used for miRNA loss and gain of function research. An AAV vector that delivers miRNAs driven by tissuespecific promoters allows for specific regulation in the cell type of choice. However, there is a concern in using these viral vectors because of safety and efficacy issues. Pre-existing immunity against viral vectors may lead to rejection responses. [27_TD$IF]Conclusion miRNAs are able to directly regulate the expression of transcription factors and signaling molecules and play critical roles in numerous diseases. Given the role of miR-33a/b in repressing cholesterol efflux and atheroprotective effects, pharmacological targeting of miR-33a/b may be a promising strategy for the treatment of atherosclerosis. Recent reports have demonstrated the ability of exogenously administered miRNA inhibitors or miRNA mimics to modulate these pathological processes, thereby ameliorating disease, which opens the door for novel therapeutic approaches in the future. Funding This work was supported in part by grants from the Global COE program ‘‘Center for Frontier Medicine’’, the [32_TD$IF]Japan Society for the Promotion of Science[3_TD$IF], and the Ministry of Education, Culture, Sports, Science, and Technology of Japan[34_TD$IF].
Conflict of interest The author has no conflict of interest to disclose. References [1] Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75:843–54. [2] Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009;19:92–105. [3] Rottiers V, Naar AM. MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol 2012;13:239–50. [4] Chim SS, Shing TK, Hung EC, Leung TY, Lau TK, Chiu RW, Lo YM. Detection and characterization of placental microRNAs in maternal plasma. Clin Chem 2008;54:482–90. [5] Kuwabara Y, Ono K, Horie T, Nishi H, Nagao K, Kinoshita M, Watanabe S, Baba O, Kojima Y, Shizuta S, Imai M, Tamura T, Kita T, Kimura T. Increased microRNA-1 and microRNA-133a levels in serum of patients with cardiovascular disease indicate myocardial damage. Circ Cardiovasc Genet 2011;4:446–54. [6] Moore KJ, Rayner KJ, Suarez Y, Fernandez-Hernando C. microRNAs and cholesterol metabolism. Trends Endocrinol Metab 2010;21:699–706. [7] Norata GD, Sala F, Catapano AL, Fernandez-Hernando C. MicroRNAs and lipoproteins: a connection beyond atherosclerosis? Atherosclerosis 2013; 227:209–15. [8] Wang L, Jia XJ, Jiang HJ, Du Y, Yang F, Si SY, Hong B. MicroRNAs 185, 96, and 223 repress selective high-density lipoprotein cholesterol uptake through posttranscriptional inhibition. Mol Cell Biol 2013;33:1956–64. [9] Horie T, Ono K, Horiguchi M, Nishi H, Nakamura T, Nagao K, Kinoshita M, Kuwabara Y, Marusawa H, Iwanaga Y, Hasegawa K, Yokode M, Kimura T, Kita T. MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo. Proc Natl Acad Sci U S A 2010; 107:17321–26. [10] Horie T, Baba O, Kuwabara Y, Chujo Y, Watanabe S, Kinoshita M, Horiguchi M, Nakamura T, Chonabayashi K, Hishizawa M, Hasegawa K, Kume N, Yokode M, Kita T, Kimura T, et al. MicroRNA-33 deficiency reduces the progression of atherosclerotic plaque in ApoE(/) mice. J Am Heart Assoc 2012;1:e003376. [11] Horie T, Nishino T, Baba O, Kuwabara Y, Nakao T, Nishiga M, Usami S, Izuhara M, Sowa N, Yahagi N, Shimano H, Matsumura S, Inoue K, Marusawa H, Nakamura T, et al. MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice. Nat Commun 2013;4:2883. [12] Horie T, Nishino T, Baba O, Kuwabara Y, Nakao T, Nishiga M, Usami S, Izuhara M, Nakazeki F, Ide Y, Koyama S, Sowa N, Yahagi N, Shimano H, Nakamura T, et al. MicroRNA-33b knock-in mice for an intron of sterol regulatory elementbinding factor 1 (Srebf1) exhibit reduced HDL-C in vivo. Sci Rep 2014;4:5312. [13] Rayner KJ, Suarez Y, Davalos A, Parathath S, Fitzgerald ML, Tamehiro N, Fisher EA, Moore KJ, Fernandez-Hernando C. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 2010;328:1570–3. [14] Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997; 89:331–40. [15] Najafi-Shoushtari SH, Kristo F, Li Y, Shioda T, Cohen DE, Gerszten RE, Naar AM. MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis. Science 2010;328:1566–9. [16] Allen RM, Marquart TJ, Albert CJ, Suchy FJ, Wang DQ, Ananthanarayanan M, Ford DA, Baldan A. miR-33 controls the expression of biliary transporters, and mediates statin- and diet-induced hepatotoxicity. EMBO Mol Med 2012;4:882–95. [17] Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, van Gils JM, Rayner AJ, Chang AN, Suarez Y, Fernandez-Hernando C, Fisher EA, Moore KJ. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest 2011;121:2921–31. [18] Tang C, Liu Y, Kessler PS, Vaughan AM, Oram JF. The macrophage cholesterol exporter ABCA1 functions as an anti-inflammatory receptor. J Biol Chem 2009;284:32336–43. [19] Goedeke L, Salerno A, Ramirez CM, Guo L, Allen RM, Yin X, Langley SR, Esau C, Wanschel A, Fisher EA, Suarez Y, Baldan A, Mayr M, Fernandez-Hernando C. Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice. EMBO Mol Med 2014;6: 1133–41. [20] Davalos A, Goedeke L, Smibert P, Ramirez CM, Warrier NP, Andreo U, CireraSalinas D, Rayner K, Suresh U, Pastor-Pareja JC, Esplugues E, Fisher EA, Penalva LO, Moore KJ, Suarez Y, et al. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc Natl Acad Sci U S A 2011;108: 9232–7. [21] Herrera-Merchan A, Cerrato C, Luengo G, Dominguez O, Piris MA, Serrano M, Gonzalez S. miR-33-mediated downregulation of p53 controls hematopoietic stem cell self-renewal. Cell Cycle 2010;9:3277–85. [22] Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, van Gils JM, Ray TD, Sheedy FJ, Goedeke L, Liu X, Khatsenko OG, Kaimal V, Lees CJ, FernandezHernando C, Fisher EA, et al. Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature 2011;478:404–7. [23] Grunweller A, Hartmann RK. Locked nucleic acid oligonucleotides: the next generation of antisense agents? BioDrugs 2007;21:235–43. [24] Rottiers V, Obad S, Petri A, McGarrah R, Lindholm MW, Black JC, Sinha S, Goody RJ, Lawrence MS, Delemos AS, Hansen HF, Whittaker S, Henry S, Brookes R,
K. Ono / Journal of Cardiology 67 (2016) 28–33 Najafi-Shoushtari SH, et al. Pharmacological inhibition of a microRNA family in nonhuman primates by a seed-targeting 8-mer antimiR. Sci Transl Med 2013;5:212ra162. [25] Young JA, Ting KK, Li J, Moller T, Dunn L, Lu Y, Moses J, Prado-Lourenco L, Khachigian LM, Ng M, Gregory PA, Goodall GJ, Tsykin A, Lichtenstein I, Hahn CN, et al. Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27. Blood 2013;122:2911–9. [26] Winbanks CE, Beyer C, Hagg A, Qian H, Sepulveda PV, Gregorevic P. miR-206 represses hypertrophy of myogenic cells but not muscle fibers via inhibition of HDAC4. PLOS ONE 2013;8:e73589.
33
[27] Montgomery RL, Yu G, Latimer PA, Stack C, Robinson K, Dalby CM, Kaminski N, van Rooij E. MicroRNA mimicry blocks pulmonary fibrosis. EMBO Mol Med 2014;6:1347–56. [28] Bader AG. miR-34 – a microRNA replacement therapy is headed to the clinic. Front Genet 2012;3:120. [29] Kubo T, Yanagihara K, Takei Y, Mihara K, Sato Y, Seyama T. Lipid-conjugated 27-nucleotide double-stranded RNAs with dicer-substrate potency enhance RNAi-mediated gene silencing. Mol Pharm 2012;9:1374–83. [30] Chen Y, Zhu X, Zhang X, Liu B, Huang L. Nanoparticles modified with tumortargeting scFv deliver siRNA and miRNA for cancer therapy. Mol Ther 2010; 18:1650–6.
Contents lists available at ScienceDirect
Journal of Cardiology journal homepage: www.elsevier.com/locate/jjcc
[6_TD$IF]Review
Functions of microRNA-33a/b and microRNA therapeutics Koh Ono (MD, PhD, FJCC)*[4_TD$IF] Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 October 2015 Accepted 26 October 2015 Available online 29 November 2015
Atherosclerosis is a chronic inflammatory disease of the arterial wall. It is characterized by subendothelial accumulation of low-density lipoprotein cholesterol, and its subsequent modification at athero[12_TD$IF]-prone areas leads to further activation of the vascular wall and maintains vascular inflammation. An entirely new level of post-transcriptional gene regulation through microRNA (miR) expression has emerged recently as an important mechanism in the development and progression of numerous diseases, including atherosclerosis. Recently, miR-33a/b have been shown to act as posttranscriptional regulators of lipid metabolism, and their pharmacological inhibition diminished atherosclerosis by raising plasma high-density lipoprotein levels. This review summarizes the current understanding of the functions of miR-33a/b and the progress in miRNA therapeutics for treatment of various diseases, including atherosclerosis. ß 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
Keywords: ABCA1 HDL-C Plaque Antisense oligonucleotides
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . microRNA-33a/b regulates high-density lipoprotein cholesterol in vivo . microRNA-33 inhibition has a beneficial effect on atherosclerosis . . . . . Other functions of microRNA-33a/b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . microRNA modifications and chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibition of microRNA function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Antisense oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . miRNA sponges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Restoration of microRNA levels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nonviral delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Viral delivery. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction MicroRNAs (miRNAs; miRs) are endogenous, small (approximately 20–22 nucleotides in length), non-protein-coding RNAs.
* [8_TD$IF]Correspondence to: Department of Cardiovascular Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 3190; fax: +81 75 751 3203. E-mail address: [email protected]
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
. . . . . . . . . . . . . . .
28 29 29 30 31 31 31 31 32 32 32 32 32 32 32
miRNAs bind to the 30 untranslated region (UTR) of specific mRNAs according to the complementarity of their sequences and inhibit translation or promote mRNA degradation. miRNAs were initially discovered in Caenorhabditis elegans by Victor Ambros’ group while studying the gene lin-14 [1], and they were later found to be evolutionarily conserved. More than 60% of human protein-coding genes have been shown to be regulated by miRNAs, and so far, approximately [13_TD$IF]2500 miRNAs have been identified in humans [2]. miRNAs are usually transcribed by RNA polymerase II (Pol II) in the nucleus as longer precursors with a hairpin structure, referred
http://dx.doi.org/10.1016/j.jjcc.2015.10.017 0914-5087/ß 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.
[(Fig._1)TD$IG]
K. Ono / Journal of Cardiology 67 (2016) 28–33
to as [14_TD$IF]primary miRNAs (Pri-miRNAs), and these are subsequently processed by the Drosha (RNase III)/DGCR8 complex to form approximately 70-nucleotide pre-mature miRNAs (Pre-miRNAs). Pre-miRNAs are then translocated to the cytoplasm through exportin 5, where Dicer (an miRNA-specific nuclease) cleaves them, resulting in double-stranded miRNAs that form mature miRNAs of 18–25 nucleotides in length. Subsequently, mature miRNAs are incorporated into an RNA-inducing silencing complex (RISC) by associating with Argonaute (Ago) proteins. This complex with the miRNA targets the 30 -UTR of the mRNA, which is then responsible for the decrease in target gene expression, [15_TD$IF]via either degradation of the mRNA or inhibition of protein synthesis [3]. In addition to their existence in various tissues, recent studies have indicated that miRNAs also exist in serum, plasma, urine, and other body fluids in highly stable forms that are protected from endogenous RNase activity by exosomes and other proteins [4]. Circulating miRNA expression profiling has provided strong molecular markers for the detection of various diseases. Altered levels of circulating miRNAs have been observed in acute coronary syndrome [5], heart failure, essential hypertension, and stroke. Work over the past several years has demonstrated that miRNAs control the expression of most of the genes associated with high-density lipoprotein (HDL) metabolism, including the ATP transporters, ABCA1 and ABCG1, and the scavenger receptor SRB1 [3,6–8]. These findings suggested that miRNAs regulate HDL biogenesis, cellular cholesterol efflux, and HDL cholesterol (HDL-C) uptake in the liver, thus controlling all of the steps of reverse cholesterol transport (RCT). Elucidation of the function of miRNAs may provide novel strategies for the treatment of dyslipidemia. In particular, we have intensively investigated the functions of miR33a/b in vivo using genetically modified mice [9–12]. The present review summarizes the function of miR-33a/b and current strategies for modulating the function/levels of miRNAs. microRNA-33a/b regulates high-density lipoprotein cholesterol in vivo The miR-33 family consists of two intronic miRNAs, miR-33a and miR-33b, which are encoded in the introns of SREBF2 and SREBF1, respectively [13]. The sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate fatty acid and cholesterol synthesis. SREBF1 encodes SREBP-1a and -1c, which mainly regulate lipogenic genes, such as fatty acid synthase (FAS), stearoyl-CoA desaturase (SCD), and acyl-CoA carboxylase 1 (ACC1). SREBF2 encodes SREBP-2, which mainly regulates cholesterol-regulating genes, such as 3hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and lowdensity lipoprotein receptor (LDLR) [14]. In humans, miR-33a and miR-33b are encoded in the introns of SREBF2 and SREBF1, respectively [15], whereas in rodents there is a deletion in the miR33b encoding sequence and miR-33b cannot be expressed (Fig. 1). Furthermore, miR-33a and miR-33b share the same seed sequence and differ in only three nucleotides. The fact that miR-33a and miR33b are cotranscribed with their respective host genes suggests that they regulate lipid homeostasis with their host genes. Several groups have shown that miR-33a targets genes involved in cholesterol export, such as ATP-binding cassette transporter A1 (Abca1) and Abcg1 in vivo, using either antisense technology or by generating miR-33a-deficient (knockout) mice [9,13]. ABCA1 promotes the movement of excess free cholesterol out of the cell. In the liver, it is essential for efflux of cholesterol to lipid-poor apolipoprotein A-I (apoA-I) and forms nascent HDL. In peripheral cells, ABCA1 and ABCG1 efflux excess cholesterol to apoA-[16_TD$IF]I and HDL particles for delivery to the liver for excretion in the bile and feces. Therefore, ABCA1 is an essential molecule for HDL biogenesis and RCT. The ABCA1 mRNA and protein half-lives are short (1–2 h),
29
Rodents
Srebf2
Srebf1
miR-33a
miR-33a
Human SREBF1
miR-33b
miR-33b
SREBF2
miR-33a
miR-33a
Fig. 1. Mice have a single copy of microRNA (miR)-33, but humans have two copies (miR-33a and miR-33b).
suggesting that de novo transcription and translation are important for controlling its expression in response to stimuli. The 30 -UTRs of mouse and human ABCA1 and mouse ABCG1 contain binding sites for miR-33a/b, and transfection of miR-33a/b strongly repressed ABCA1 and ABCG1 mRNA and protein in a variety of cells. It was shown that mice treated with antisense oligonucleotides or antimiR-33a lentivirus exhibited increased ABCA1 expression in the liver and ABCA1 and ABCG1 expression in macrophages. Furthermore, inhibition of endogenous miR-33a in both these cell types promotes an increase in the expression of ABCA1 and ABCG1 protein and cholesterol efflux to apoAI and HDL-C, indicating the physiologically relevant role of this miRNA in regulating ABCA1 and ABCG1. Notably, in an anti-miR-33a-treated mouse model of atherosclerosis, plasma HDL levels increased by 35–50% without affecting other lipoproteins [13]. miR-33a knockout mice also showed a significant increase in the expression of ABCA1 and ABCG1 and a 25–40% increase in serum HDL-C [9] (Fig. 2A[17_TD$IF]). AntimiR-33a therapy has been shown to contribute to enhance the RCT pathway not only by increasing HDL-C through ABCA1 and ABCG1 upregulation but also by increasing bile secretion through upregulation of ABCB11 and ATP8B1, which are the other targets of miR-33a/b [16]. Using an in vivo assay to measure the efficiency of RCT, it was shown that the HDL-C generated by miR-33a inhibition was functional and increased the transport of cellular radiolabeled cholesterol to the plasma, liver, bile acid, and feces. Interestingly, in contrast to the results with miR-33a-deficient mice, HDL-C levels in miR-33b knock-in (KI) mice, which has miR33b in the same intron as in humans [12], were reduced by almost 35%, even in miR-33b KI [18_TD$IF]heteromice compared with the control mice (Fig. 2B). microRNA-33 inhibition has a beneficial effect on atherosclerosis The function of miR-33a inhibition in animal models of atherosclerosis has been examined by several groups. It has already been proven that antisense inhibition of miR-33a resulted in regression of atherosclerotic plaque in LDLR-deficient mice by promoting RCT [17]. This promotion of RCT was shown to be achieved in two ways: by directly increasing HDL-C biogenesis in the liver, and by increasing cellular cholesterol efflux from plaque macrophages. Moreover, miR-33a-deficient mice reduced the progression of atherosclerosis in apoE-deficient mice (Fig. 3) [10]. In our study, miR-33-deficient mice not only had higher and functional HDL-C but also had macrophages with higher cholesterol efflux capacity, resulting in the lower lipid accumulation in atherosclerotic areas [10]. Other possible beneficial properties of
[(Fig._2)TD$IG]
30
K. Ono / Journal of Cardiology 67 (2016) 28–33
Fig. 2. Lipid profiles of wild-type (WT), microRNA (miR)-33a-deficient mice (knockout, KO), and miR-33b knock-in (KI) mice ([1_TD$IF]cited and modified from Refs. [9,12]). (A) Representative high-performance liquid chromatography (HPLC) analysis of serum cholesterol from male wild-type and miR-33a-deficient mice. [2_TD$IF](B) Representative HPLC analysis of serum cholesterol from male wild-type and miR-33b KI+/+ mice.
anti-miR-33a/b therapy include an anti-inflammatory reaction [19_TD$IF]via upregulation of ABCA1. ABCA1 decreases cell-surface cholesterol levels, inhibits its partitioning into lipid rafts, and reduces the responsiveness of inflammatory signals from innate immune receptors. Furthermore, ABCA1 has been reported to act directly as an anti-inflammatory receptor independent of its lipid transport activities [18]. Although these preclinical studies of miR-33a inhibition in mice are encouraging, it has been shown that obesity and hepatic steatosis were observed in miR-33a-deficient mice at the age of 50 weeks or when fed a high-fat diet for 12 weeks [11]. We found that Srebf1 is a good target of miR-33a, and enhanced expression of SREBP-1 may have caused these phenotypic changes in miR-33adeficient mice [20_TD$IF][11]. Suppression of SREBP-1 by miR-33a indicated the existence of a more direct and fine regulatory mechanism between SREBPs. It was also shown that chronic inhibition of miR33a by antisense oligonucleotides increased the expression of genes involved in fatty acid synthesis such as ACC1 and FAS in the livers of mice [19]. It was also reported that anti-miR-33a therapy enhanced the expression of nuclear transcription Y subunit gamma (NFYC), a transcriptional regulator required for DNA binding and full transcriptional activation of SREBP-responsive genes, including ACC and FAS.
[(Fig._3)TD$IG]
It is also of note that mice lack miR-33b, which is present in the Srebf1 gene of medium and large mammals, and that extrapolating the findings in rodents to humans is complicated. This difference between mice and humans may be particularly relevant under conditions in which the transcription of Srebf1 is upregulated, such as feeding after fasting and hyperinsulinemia. Because such a condition may lead to greater downregulation of cellular cholesterol efflux and plasma HDL-C levels, not complete but partial inhibition of miR-33a/b, which avoids the induction of further Srebf1, would be beneficial for the increase of HDL-C and prevention of atherosclerosis. Other functions of microRNA-33a/b miR-33a/b are known to have several other target genes including AMP-activated kinase (Ampka1) and Cpt1a, which are involved in the regulation of lipid and glucose metabolism [20]. However, no significant difference in these target genes has been observed in miR-33a-deficient and miR-33b-KI mice compared with their levels in control mice [11,12]. It has been suggested that miR-33a/b regulates stem cell selfrenewal [21_TD$IF]via downregulation of p53 [21]. p53 has two putative miR33a/b binding sites in the 30 -UTR, and miR-33s overexpression
Fig. 3. microRNA (miR)-33a-deficiency reduced atherosclerosis (cited and modified from Ref. [10]). (A) Representative microscopic images of cross-sections of the proximal aorta in miR-33+/+[3_TD$IF]Apoe/ and miR-33/Apoe/ male and female mice. Scale bars, 1 mm. (B) Quantification of the atherosclerotic plaque area in cross-sections of the proximal aorta in male and female mice. Values are the means S.E. ([5_TD$IF]n = 10–11 each, *p < 0.05).
K. Ono / Journal of Cardiology 67 (2016) 28–33
20 -fluoro (20 -F), and locked nucleic acid (LNA) modifications. In order to see the effect of miR-33a/b inhibition, 20 -F/MOE-modified phosphorothioate-backbone-modified antisense miR-33 was injected into African green monkeys [22]. It was reported that increased hepatic expression of ABCA1 and sustained increase in plasma HDL-C levels were observed over 12 weeks. Addition of LNAs to the ASOs increased the binding specificity. LNA-modified oligonucleotides contain one or more nucleotides in which an extra methylene bridge fixes the ribose moiety in the 30 [24_TD$IF]endoconformation [23]. This modification possesses high affinity and results in significant increases in melting temperature of up to several degrees per LNA residue and leads to the formation of a thermodynamically strong duplex with complementary RNA. In addition, this character of LNA oligonucleotides allows for the generation of ‘tiny LNAs’ that bind only to the seed region of target miRNAs. Tiny LNAs are 7–8 nucleotides long and can inhibit a family of miRNAs at the same time, an advantage over antagomiRs that can only target individual miRNAs. Rottiers et al. demonstrated that pharmacological inhibition of the miR-33 family by a subcutaneous delivered 8-mer LNA-modified antimiR into obese and insulin-resistant nonhuman primates, which resulted in derepression of miR-33 targets, such as ABCA1, and increases in circulating HDL-C [24]. Recently, another kind of miRNA inhibitor has been developed. These are referred to as ‘blockmirs,’ which target the miRNAbinding site in a specific target gene (Fig. 4B) [25]. They are designed to bind to the seed region with additional nucleotides to increase specificity. The unique feature of blockmirs is that they target specific mRNA while still allowing the miRNA to regulate other target genes.
repressed p53 expression and p53-mediated apoptosis. This study suggested that miR-33s may promote the repopulation capacity of hematopoietic stem cells. It is known that SREBP-1 and cellular cholesterol levels have also been shown to regulate cell-cycle progression. Therefore, miR-33a/b may cooperate with their host genes in regulating cell-cycle progression. microRNA modifications and chemistry The ability of miRNAs to regulate multiple genes in signal transduction pathways makes them attractive therapeutic targets. For this reason, miRNA therapeutics may be superior to other therapies that target a single protein or use small molecules. miRNA therapeutic approaches can be divided into two categories: miRNA inhibition (blockade) to reduce the expression of miRNAs upregulated in the setting of a pathological condition, and miRNA replacement to restore the expression of miRNAs repressed in the setting of a disease (Fig. 4). Inhibition of microRNA function Current strategies to inhibit miRNAs include the use of miRNA inhibitors and miRNA sponges. An ideal miRNA inhibitor needs to have several functions, including high affinity to the target genes, low toxicity, high specificity, resistance to exonucleases, and low cost for synthesis. Antisense oligonucleotides [2_TD$IF]miRNA inhibitors are chemically modified single-stranded antisense oligonucleotides[23_TD$IF] (ASOs) that are complimentary to the mature miRNA. To date, angtagomiRs (target-single miRNAs conjugated with cholesterol), antimiRs (target-single miRNAs), and tiny antimiRs (target miRNA families) have been synthesized (Fig. 4A). These ASOs can reduce the levels of pathogenically expressed miRNAs. A number of chemical modifications have been used to improve the pharmacodynamics and pharmacokinetics of ASOs. The modifications that are most commonly used are 20 -Omethoxyethyl (20 -MOE), 20 -O-methyl ribose-modified RNA (20 -OMe),
[25_TD$IF]miRNA sponges A recent clinical trial indicated that five weekly injections of an antimiR were sufficient to achieve inhibition for at least 6 weeks in humans. However, repeated injection may be necessary to allow for longer inhibition. To overcome these barriers, ‘miRNA sponges’ were developed to inhibit miRNA activity (Fig. 4C). miRNA sponges contain multiple miRNA[26_TD$IF]-binding sites, which act as competitive
[(Fig._4)TD$IG]
Normal miRNA funcon
31
A. AntagomiRs or anmiRs for miRNA inhibion
B. BlockmiRs for specific target inhibion cap
ORF
AAAAAAAAA(n)
Ribosome cap
ORF
AAAAAAAAA(n)
Ribosome
C. Sponges for miRNA inhibion cap
AAAAA(n)
ORF
miR-binding sites
D. miRNA mimics for miRNA replacement Fig. 4. microRNA (miRNA) manipulation strategies. Modification of miRNAs can be achieved by several strategies. Red lines indicate the methods to increase target gene expression and blue lines indicate approaches to reduce them. (A) AntagomiRs and antimiRs for miRNA inhibition. (B) BlockmiRs for specific target inhibition. (C) miRNA sponges for miRNA inhibition. (D) miRNA mimics for miRNA replacement.
32
K. Ono / Journal of Cardiology 67 (2016) 28–33
inhibitors for miRNA binding. This technology mainly utilizes viral gene transfer vectors to deliver the miRNA sponges into cells/ tissues. However, nonviral delivery systems can also be used, such as liposomes, polysaccharides, and artificial polymers. miRNA sponges have been used to regulate miRNAs specifically in skeletal muscle [26]. [27_TD$IF]Restoration of microRNA levels Although efforts to inhibit overexpressed miRNAs have been a major focus over the past years, several successful strategies have been employed to express depleted miRNAs in diseased cells/ tissues. miRNA levels are restored using oligonucleotide-based miRNA mimics, or by the use of viruses to drive expression of a given miRNA. miRNA mimics are double-stranded synthetic oligonucleotides that are processed into a single-stranded miRNA to regulate target genes in the cells (Fig. 4D). Montgomery et al. restored the functional miR-29 mimic to target fibrotic disease [27]. The miR-29 mimic was conjugated to cholesterol to facilitate cellular uptake and contained mismatches to prevent it from acting as an inhibitor. There are several reports that miRNA mimics were successfully used in clinical trials and the therapeutic potential of miRNA mimics has been discussed [28]. Nonviral delivery [28_TD$IF]Nonviral delivery of miRNAs is achieved by conjugation-based methods, liposomes, nanoparticles, or antibody-based methods. Lipid-conjugated dsRNAs, including palmitic acid, lauric acid, and cholesterol, in which these lipids modified the 50 -end of the sense strand[29_TD$IF], have been used to facilitate cellular uptake and to add a potent gene silencing effect for the delivery of siRNAs or miRNAs [29]. Liposome nanoparticles have been utilized for miRNA delivery applications. Nanoparticles can be targeted to specific cells and tissues by adding cell[30_TD$IF]-surface receptor, ligands, and antibodies [30]. [31_TD$IF]Viral delivery Retrovirus, lentivirus, adeno-associated virus (AAV), and adenovirus have been used for miRNA loss and gain of function research. An AAV vector that delivers miRNAs driven by tissuespecific promoters allows for specific regulation in the cell type of choice. However, there is a concern in using these viral vectors because of safety and efficacy issues. Pre-existing immunity against viral vectors may lead to rejection responses. [27_TD$IF]Conclusion miRNAs are able to directly regulate the expression of transcription factors and signaling molecules and play critical roles in numerous diseases. Given the role of miR-33a/b in repressing cholesterol efflux and atheroprotective effects, pharmacological targeting of miR-33a/b may be a promising strategy for the treatment of atherosclerosis. Recent reports have demonstrated the ability of exogenously administered miRNA inhibitors or miRNA mimics to modulate these pathological processes, thereby ameliorating disease, which opens the door for novel therapeutic approaches in the future. Funding This work was supported in part by grants from the Global COE program ‘‘Center for Frontier Medicine’’, the [32_TD$IF]Japan Society for the Promotion of Science[3_TD$IF], and the Ministry of Education, Culture, Sports, Science, and Technology of Japan[34_TD$IF].
Conflict of interest The author has no conflict of interest to disclose. References [1] Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75:843–54. [2] Friedman RC, Farh KK, Burge CB, Bartel DP. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res 2009;19:92–105. [3] Rottiers V, Naar AM. MicroRNAs in metabolism and metabolic disorders. Nat Rev Mol Cell Biol 2012;13:239–50. [4] Chim SS, Shing TK, Hung EC, Leung TY, Lau TK, Chiu RW, Lo YM. Detection and characterization of placental microRNAs in maternal plasma. Clin Chem 2008;54:482–90. [5] Kuwabara Y, Ono K, Horie T, Nishi H, Nagao K, Kinoshita M, Watanabe S, Baba O, Kojima Y, Shizuta S, Imai M, Tamura T, Kita T, Kimura T. Increased microRNA-1 and microRNA-133a levels in serum of patients with cardiovascular disease indicate myocardial damage. Circ Cardiovasc Genet 2011;4:446–54. [6] Moore KJ, Rayner KJ, Suarez Y, Fernandez-Hernando C. microRNAs and cholesterol metabolism. Trends Endocrinol Metab 2010;21:699–706. [7] Norata GD, Sala F, Catapano AL, Fernandez-Hernando C. MicroRNAs and lipoproteins: a connection beyond atherosclerosis? Atherosclerosis 2013; 227:209–15. [8] Wang L, Jia XJ, Jiang HJ, Du Y, Yang F, Si SY, Hong B. MicroRNAs 185, 96, and 223 repress selective high-density lipoprotein cholesterol uptake through posttranscriptional inhibition. Mol Cell Biol 2013;33:1956–64. [9] Horie T, Ono K, Horiguchi M, Nishi H, Nakamura T, Nagao K, Kinoshita M, Kuwabara Y, Marusawa H, Iwanaga Y, Hasegawa K, Yokode M, Kimura T, Kita T. MicroRNA-33 encoded by an intron of sterol regulatory element-binding protein 2 (Srebp2) regulates HDL in vivo. Proc Natl Acad Sci U S A 2010; 107:17321–26. [10] Horie T, Baba O, Kuwabara Y, Chujo Y, Watanabe S, Kinoshita M, Horiguchi M, Nakamura T, Chonabayashi K, Hishizawa M, Hasegawa K, Kume N, Yokode M, Kita T, Kimura T, et al. MicroRNA-33 deficiency reduces the progression of atherosclerotic plaque in ApoE(/) mice. J Am Heart Assoc 2012;1:e003376. [11] Horie T, Nishino T, Baba O, Kuwabara Y, Nakao T, Nishiga M, Usami S, Izuhara M, Sowa N, Yahagi N, Shimano H, Matsumura S, Inoue K, Marusawa H, Nakamura T, et al. MicroRNA-33 regulates sterol regulatory element-binding protein 1 expression in mice. Nat Commun 2013;4:2883. [12] Horie T, Nishino T, Baba O, Kuwabara Y, Nakao T, Nishiga M, Usami S, Izuhara M, Nakazeki F, Ide Y, Koyama S, Sowa N, Yahagi N, Shimano H, Nakamura T, et al. MicroRNA-33b knock-in mice for an intron of sterol regulatory elementbinding factor 1 (Srebf1) exhibit reduced HDL-C in vivo. Sci Rep 2014;4:5312. [13] Rayner KJ, Suarez Y, Davalos A, Parathath S, Fitzgerald ML, Tamehiro N, Fisher EA, Moore KJ, Fernandez-Hernando C. MiR-33 contributes to the regulation of cholesterol homeostasis. Science 2010;328:1570–3. [14] Brown MS, Goldstein JL. The SREBP pathway: regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor. Cell 1997; 89:331–40. [15] Najafi-Shoushtari SH, Kristo F, Li Y, Shioda T, Cohen DE, Gerszten RE, Naar AM. MicroRNA-33 and the SREBP host genes cooperate to control cholesterol homeostasis. Science 2010;328:1566–9. [16] Allen RM, Marquart TJ, Albert CJ, Suchy FJ, Wang DQ, Ananthanarayanan M, Ford DA, Baldan A. miR-33 controls the expression of biliary transporters, and mediates statin- and diet-induced hepatotoxicity. EMBO Mol Med 2012;4:882–95. [17] Rayner KJ, Sheedy FJ, Esau CC, Hussain FN, Temel RE, Parathath S, van Gils JM, Rayner AJ, Chang AN, Suarez Y, Fernandez-Hernando C, Fisher EA, Moore KJ. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis. J Clin Invest 2011;121:2921–31. [18] Tang C, Liu Y, Kessler PS, Vaughan AM, Oram JF. The macrophage cholesterol exporter ABCA1 functions as an anti-inflammatory receptor. J Biol Chem 2009;284:32336–43. [19] Goedeke L, Salerno A, Ramirez CM, Guo L, Allen RM, Yin X, Langley SR, Esau C, Wanschel A, Fisher EA, Suarez Y, Baldan A, Mayr M, Fernandez-Hernando C. Long-term therapeutic silencing of miR-33 increases circulating triglyceride levels and hepatic lipid accumulation in mice. EMBO Mol Med 2014;6: 1133–41. [20] Davalos A, Goedeke L, Smibert P, Ramirez CM, Warrier NP, Andreo U, CireraSalinas D, Rayner K, Suresh U, Pastor-Pareja JC, Esplugues E, Fisher EA, Penalva LO, Moore KJ, Suarez Y, et al. miR-33a/b contribute to the regulation of fatty acid metabolism and insulin signaling. Proc Natl Acad Sci U S A 2011;108: 9232–7. [21] Herrera-Merchan A, Cerrato C, Luengo G, Dominguez O, Piris MA, Serrano M, Gonzalez S. miR-33-mediated downregulation of p53 controls hematopoietic stem cell self-renewal. Cell Cycle 2010;9:3277–85. [22] Rayner KJ, Esau CC, Hussain FN, McDaniel AL, Marshall SM, van Gils JM, Ray TD, Sheedy FJ, Goedeke L, Liu X, Khatsenko OG, Kaimal V, Lees CJ, FernandezHernando C, Fisher EA, et al. Inhibition of miR-33a/b in non-human primates raises plasma HDL and lowers VLDL triglycerides. Nature 2011;478:404–7. [23] Grunweller A, Hartmann RK. Locked nucleic acid oligonucleotides: the next generation of antisense agents? BioDrugs 2007;21:235–43. [24] Rottiers V, Obad S, Petri A, McGarrah R, Lindholm MW, Black JC, Sinha S, Goody RJ, Lawrence MS, Delemos AS, Hansen HF, Whittaker S, Henry S, Brookes R,
K. Ono / Journal of Cardiology 67 (2016) 28–33 Najafi-Shoushtari SH, et al. Pharmacological inhibition of a microRNA family in nonhuman primates by a seed-targeting 8-mer antimiR. Sci Transl Med 2013;5:212ra162. [25] Young JA, Ting KK, Li J, Moller T, Dunn L, Lu Y, Moses J, Prado-Lourenco L, Khachigian LM, Ng M, Gregory PA, Goodall GJ, Tsykin A, Lichtenstein I, Hahn CN, et al. Regulation of vascular leak and recovery from ischemic injury by general and VE-cadherin-restricted miRNA antagonists of miR-27. Blood 2013;122:2911–9. [26] Winbanks CE, Beyer C, Hagg A, Qian H, Sepulveda PV, Gregorevic P. miR-206 represses hypertrophy of myogenic cells but not muscle fibers via inhibition of HDAC4. PLOS ONE 2013;8:e73589.
33
[27] Montgomery RL, Yu G, Latimer PA, Stack C, Robinson K, Dalby CM, Kaminski N, van Rooij E. MicroRNA mimicry blocks pulmonary fibrosis. EMBO Mol Med 2014;6:1347–56. [28] Bader AG. miR-34 – a microRNA replacement therapy is headed to the clinic. Front Genet 2012;3:120. [29] Kubo T, Yanagihara K, Takei Y, Mihara K, Sato Y, Seyama T. Lipid-conjugated 27-nucleotide double-stranded RNAs with dicer-substrate potency enhance RNAi-mediated gene silencing. Mol Pharm 2012;9:1374–83. [30] Chen Y, Zhu X, Zhang X, Liu B, Huang L. Nanoparticles modified with tumortargeting scFv deliver siRNA and miRNA for cancer therapy. Mol Ther 2010; 18:1650–6.