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Received Date : 30-Jan-2014
Revision Requested: 27-Feb-2014 Revised Date : 30-Apr-2014 Accepted Date : 12-May-2014 Article type
: Review Article
MicroRNAs: potential therapeutic targets in diabetic complications of the cardiovascular and renal systems
Miriam Frankenthal Figueira1,2,*, Gustavo Monnerat-Cahli1,*, Emiliano Medei1, Adriana Bastos Carvalho1, Marcelo Marcos Morales1, Marcelo Einicker Lamas1, Rodrigo Nunes da Fonseca2, Jackson Souza-Menezes1,2
1
Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal
do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil 2
Laboratório Integrado de Ciências Morfofuncionais, Núcleo em Ecologia e Desenvolvimento
Sócio-Ambiental de Macaé, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Macaé, RJ, 27965-045, Brazil
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/apha.12316 This article is protected by copyright. All rights reserved.
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*Both authors contributed equally to this work. Correspondence: Jackson de Souza Menezes, Laboratório Integrado de Ciências Morfofuncionais, Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Avenida São José do Barreto, 764, Bairro Barreto, Macaé, RJ, 27965-045, Brazil. E-mail: [email protected] or [email protected]
Running head: MicroRNAs in diabetic cardiomyopathy and nephropathy
Abstract Diabetes mellitus is a serious health problem that can lead to several pathologic complications in numerous organs and tissues. The most important and most prevalent organs affected by this disease are the heart and the kidneys and these complications are the major causes of death in patients with diabetes. MicroRNAs (miRNAs), short non-coding RNAs, have been found to be functionally important in the regulation of several pathologic processes and they are emerging as an important therapeutic tool to avoid the complications of diabetes mellitus. This review summarizes the knowledge on the effects of miRNAs in diabetes. The use of miRNAs in diabetes from a clinical perspective is also discussed, focusing on their potential role to repair cardiovascular and renal complications.
Keywords: diabetes, nephropathy, cardiomyopathy, miRNAs, hyperglycaemia.
Introduction Diabetes mellitus is a metabolic disease; the main symptom is a high concentration of plasma glucose. The number of individuals developing this condition is increasing at an alarming rate
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worldwide. It is a serious health problem that can lead to several pathologic complications in many organs and tissues. In 2010, it was estimated that by 2030 there will be a 69% increase in the number of adults with diabetes in developing countries and a 20% increase in developed countries, which represents 439 million adults worldwide with diabetes (Shaw et al. 2010). During the development of the disease, the cardiovascular system is often damaged; at least 65% of patients with diabetes mellitus die of some form of heart disease or stroke (Schramm et al. 2008). The renal system is also severely impaired by diabetes. About 30–40% of patients with diabetes develop chronic renal disease, which is associated with increased mortality (Fig. 1) (Gross et al. 2002).
The pathophysiologic mechanisms of diabetes are not completely understood. In this context, several studies have attempted to understand the molecular basis of these events in order to create more effective treatments with fewer side effects (Li et al. 2009, Couzin 2008, Xiao et al. 2007). Recent studies have shown that microRNAs (miRNAs) could play an important role in different diseases, opening a new perspective for basic science and treatments. MiRNAs are small 19–23 nucleotide RNA molecules that act as regulators of protein expression in eukaryotic cells by inducing the translational arrest and degradation of messenger RNAs (Shantikumar et al. 2011, Cai et al. 2010, Feng et al. 2010, Lu et al. 2010, Zampetaki et al. 2010). Recently, this group of non-coding RNAs (ncRNAs) has received considerable attention in biomedical research as a possible alternative for treatment of disease. The basic features of miRNA biogenesis unveiled in recent years are described in the following.
In recent years, several bioinformatics tools have been developed to predict miRNA targets in human and mouse genomes (reviewed by Peterson et al. 2014). miRNA target prediction tools take into account several features of miRNA and mRNA target sequence including the seed match, the first 2–8 nucleotides starting at the 5′ end and counting towards the 3′ end, conservation across species, which is presumably higher at the seed region, and
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Gibbs free energy, where the binding of miRNA to a candidate target mRNA is predicted to be stable. These features have been used successfully to predict functional interactions (reviewed by Peterson et al. 2014). The putative miRNA regulators involved in kidney disease can be investigated using one of the following tools: DIANA-microT-CDS (Maragkakis et al. 2009), miRanda-mirSVR (Betel et al. 2010), and TargetScan (Lewis et al. 2005).
MiRNAs can be divided into two broad categories, canonical and non-canonical miRNAs, The canonical miRNA pathway involves step-wise processing of long genome-encoded primary miRNA (pri-miRNA) transcripts into short hairpin-like double-stranded miRNA duplexes of 19–21nt with 2-nt 3 overhangs. These double-stranded miRNAs are also referred to as short-interfering RNAs (siRNAs), which act as substrates for the gene silencing machinery (Chong et al. 2010, Westholm & Lai 2011). However, deviations from this paradigm have been observed: subclasses of miRNAs, non-canonical miRNA, undergo alternative biogenesis pathways that do not involve Drosha, thereby providing an additional level of complexity to miRNA-dependent regulation of gene expression (Fig. 2).
At the nucleus, canonical miRNAs biogenesis is characterized by a cleavage process catalysed by Drosha, an RNase III, and its essential cofactor known as DGCR8 (DiGeorge syndrome critical region) (Lee et al. 2004, Han et al. 2004, Gregory et al. 2004). After the cleavage process, an intermediate stem loop is released, known as the miRNA precursor or pre-miRNA. Pre-miRNA hairpins are then recognized by Exportin-5 for nuclear export. At the cytoplasm, the pre-miRNA is cleaved by Dicer, an RNase III, into the mature form of 20–22 nucleotides. The miRNA duplex is then unrolled and the functional strand is incorporated into the Argonaute (AGO) protein family and then loaded into the RNA-induced silencing complex (RISC). The RISC is guided by AGO and the mature miRNA to an almost complementary sequence, usually in the untranslated region (UTR) of a target messenger RNA (mRNA).
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Translational repression of imperfectly matched targets or target cleavage of perfectly matched targets takes place. Thus, miRNAs are generally negative regulators of gene expression (Lee et al. 2003, Yi et al. 2003, Farazi et al. 2013).
However, miRNAs have also been shown to be involved in the targeting of the promoter region, which leads to transcriptional gene silencing. In addition, miRNAs also have the ability to positively control global protein synthesis through ribosome biogenesis and ribosomal protein mRNA translation stimulation. Thus, miRNAs are now known to promote as well as inhibit gene expression (Du & Zamore 2005, Martinez & Tuschl 2004, Ørom et al. 2008, Schamberger et al. 2012).
An increasing number of non-canonical miRNA pathways have been described in recent years (Chong et al. 2010, Westholm & Lai 2011, Sand 2014). The first and most important alternative miRNA maturation pathway described is the mirtron pathway. Mirtrons are directly spliced out from host genes as pre-miRNAs with hairpin potential and do not depend on pri-miRNA Drosha/DGCR8 processing. Mirtron biogenesis is instead catalysed by the spliceosome. The product of intron splicing is not linear but mimics a transient lariat shape wherein the 3′ branch point is ligated to the 5′ end of the intron. After the lariat-shaped intron is processed by the lariat debranching enzyme (Ldbr), it refolds into a pre-miRNA shape, which is recognized and transferred to the cytoplasm by Exp-5, similar to the canonical pathway. Following regular Dicer cleavage, the mature miRNA can be loaded on the RISC and exerts its effect (Chong et al. 2010, Westholm & Lai 2011, Farazi et al. 2013, Sand 2014). Canonical and non-canonical miRNA biogenesis is summarized in Fig. 2.
The role of miRNAs has been demonstrated in many biological processes, including proliferation, differentiation, apoptosis, and development (Bartel 2004, Bentwich et al. 2005). Each miRNA has the capacity to regulate multiple genes, therefore deregulation of miRNAs can certainly lead to changes in several physiologic processes. This review summarizes the
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knowledge on the effects of miRNAs in diabetes. The use of miRNAs in diabetes from a clinical perspective is also discussed, focusing on their potential role in cardiovascular and renal complications.
Diabetic cardiomyopathy and miRNAs Cardiomyopathy caused by diabetes was first described in 1972 (Rubler et al. 1972). The pathology of the cardiac system induced by diabetes includes interstitial fibrosis, imbalance of cardiac ionic currents, apoptosis of cardiomyocytes, abnormal energy use, and cardiac neuropathy. Moreover, diabetes can cause severe vascular defects, leading to microand macroangiopathy (Aragno et al. 2008, McVeigh et al. 1992, Casis et al. 2000, TorresJacome et al. 2013). However, the mechanisms of this pathology are not entirely understood. Recent studies have shown an association between miRNAs and diabetic cardiomyopathy.
One of the most crucial clinical manifestations of diabetic cardiomyopathy is the high incidence of cardiac arrhythmias, including ventricular fibrillation and cardiac sudden death. Electrocardiograms of patients with diabetes show changes affecting the process of ventricular repolarization, such as QT prolongation (Kahn et al. 1987). The autonomic neuropathy caused by diabetes is also associated as a mechanism underlying the high incidence of arrhythmias (Ewing et al. 1991). An overview of miRNA expression and targets during the development of diabetic cardiomyopathy is presented in Fig. 3.
Xiao et al. (2007) showed remarkable upregulation of miR-133 expression in the heart in a rabbit model of diabetes. This study reported that this upregulation of miR-133 expression could decrease a rectifier K+ current by reducing the expression of this ion channel. Since potassium ionic currents are essential to physiologic repolarization, this change contributes to long QT syndromes, which are directly associated with cardiac arrhythmias (Xiao et al. 2007).
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During the progression of diabetes, a high percentage of apoptotic cardiomyocytes is observed, intensifying cardiac remodelling and leading to heart failure (HF) (Fiordaliso et al. 2000). A group of chaperones known as heat shock proteins (Hsp) can prevent several dysfunctional events that can lead to cell apoptosis. The increase in Hsp expression can be induced by different stress events such as depletion of oxygen or ATP, oxidative stress, or hypothermia. In the heart, Hsp60 is an important component of the defence mechanism against myocardial injury but its expression is reduced in the diabetic myocardium. Shan et al. (2010) demonstrated that the high expression of miR-1 and miR-206 in cardiomyocytes stimulated with high glucose concentrations was high. Moreover, this study proved that miR-1 and miR-206 could downregulate Hsp60 expression by directly targeting the 3′ UTR of Hsp60 mRNA, contributing to high glucose-mediated apoptosis in cardiomyocytes.
The risk of developing cardiovascular disease in diabetes is also related to myocardial insulin resistance, which promotes downregulation of the glucose transporter 4 (GLUT4) protein at the plasma membrane level. In 2009, Lu et al. (2010) showed that miR-223 plays an important role in the regulation of GLUT4 expression and in the glucose metabolism of cardiomyocytes. This study shows that miR-223 is consistently upregulated in the insulinresistant heart. They also evaluated the molecular mechanism of miR-223 and found that this miRNA could increase nuclear factor IA expression, but had no effect on myocyte enhancer factor 2c or insulin-like growth factor 1 receptor. They also examined insulin signalling and glucose transport proteins and found that phosphoinositide 3-kinase signalling and AMP kinase activity were not affected by miR-223 overexpression; however, GLUT4 protein expression was increased by miR-223 overexpression. The data obtained in this study show that miR-223 can upregulate target genes such as GLUT4 in adult cardiomyocytes, suggesting an important target for treatment in patients with type 2 diabetes. In diabetic human and mouse hearts,
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GLUT4 is markedly downregulated at the plasma membrane and the investigators suggested that the increased miR-223 levels observed in the diabetic heart are an adaptive/homeostatic response to restore GLUT4 expression and normal glucose uptake. This model is in agreement with the idea that miRNAs act as stress response genes and are important for maintaining the robustness of physiologic processes in the face of a pathophysiologic condition (Lu et al. 2010).
Ischaemic HF is one of the major causes of death in patients with diabetes. In a recent work (Greco et al. 2012), the expression profile of miRNAs was measured in the left ventricle of patients with HF; one group with type 2 diabetes (D-HF) and the other without diabetes (ND-HF). This study demonstrated that 17 miRNAs were modulated in both groups of patients compared with a control group (non-diabetic and no HF). This study revealed that the expression of 6 miRNAs was different in D-HF and ND-HF patients: miR-34b, miR-34c, miR199b, miR-210, miR-650, and miR-223. These observations are important to highlight the specific miRNAs involved in the diabetes disease mechanism during ischaemic cardiomyopathy. The authors also showed that miR-216a is the miRNA with the highest expression in both HF patient classes and that TGF-β may trigger miR-216a expression. For the first time, miR-216a overexpression has been found to be related to human HF. MiR-216a acts in many ways; one of them is to inhibit CAV2, a member of the caveolin family of scaffolding proteins, which regulates lipid metabolism and signal transduction pathways. The absence of caveolin expression leads to deleterious cardiac activity and progressive hypertrophy in cardiac myocytes (Greco et al. 2012).
Another complication of diabetes is mitochondrial dysfunction, which can lead to an increased oxidative stress state and a decrease in ATP production. Proteomic alterations in mitochondria resulting from diabetes have been reported, however, the mechanisms underlying the changes in the proteomic signatures are unknown. In this context, it was demonstrated that miR-141 upregulation can modulate Slc25a3 gene expression (the protein
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encoded by this gene catalyses the transport of phosphate into the mitochondrial matrix, either by proton cotransport or in exchange for hydroxyl ions) in type 1 diabetic hearts of mice, which could explain, at least in part, why diabetic cardiomyocytes have a low rate of ATP production and a decrease in ATP synthase activity (Baseler et al. 2012).
Diabetic nephropathy and miRNAs Diabetic nephropathy (DN) is an incurable kidney disease and one of the most important and disabling complications of diabetes. There are some therapies available for DN, but they are not effective enough to prevent many patients with diabetes reaching end-stage renal disease (Kato et al. 2009).
When DN is diagnosed by classic methods, such as the detection of microalbuminuria (urinary excretion of albumin between 20 and 200 μg min−1 in humans) or a reduction in the glomerular filtration rate (GFR), little can be done to prevent the progressive course towards renal failure. Research on markers and new therapeutic options for diabetic kidney disease is ongoing (Hong & Chia 1998, Swärd & Rippe 2012). Many studies have addressed miRNAs as a promising therapeutic target and potential diagnostic tool for this condition (Elmén et al. 2008, Zarjou et al. 2011, Long et al. 2010, Krupa et al. 2010). DN is classically characterized by albuminuria, thickening of the glomerular and tubular basal membrane, expansion of the mesangial extracellular matrix, microvascular damage, and tubulointerstitial fibrosis (Bohlender et al. 2005). The mechanisms by which these disease processes occur are not yet fully understood. Here, we summarize some features of this pathologic process in DN and the involvement of miRNA, emphasizing two main and correlated mechanisms underlying this disease: fibrogenesis and expansion of the mesangial extracellular matrix.
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Fibrogenesis and miRNA Tubulointerstitial fibrosis and fibrogenesis are the major pathologic problems in DN (Swärd & Rippe 2012). One of the most important characteristics of fibrogenesis is the production of collagen IV, collagen 1-α1, collagen 1-α2, and fibronectin. In this context, Long et al. (2010) showed a correlation between miR-93 and the fibrogenic process. They showed in vitro and in vivo that vascular endothelial growth factor (VEGF), which is a key factor involved in fibrogenesis and other microvascular complications of diabetes, is regulated by miR-93. Using diabetic mice, under hyperglycaemic conditions, the group demonstrated that miR-93 is downregulated in the glomeruli and this leads to upregulation of VEGF-A expression. These data highlight miR-193 and the VEGF signalling pathway as important targets in preventing the progression of DN.
Another miRNA associated with fibrogenesis is miR-192. In 2006, Kato et al. (2007) demonstrated that miR-192 is significantly increased in glomeruli isolated from diabetic db/db mice (an animal model of obesity, diabetes and dyslipidemia; the activity of the leptin receptor is deficient in these mice because they are homozygous for a point mutation in the leptin receptor gene) as well in mice with diabetes induced by streptozotocin (an established model of type 1 diabetes). High levels of collagen 1-α1, collagen-2, and TGF-β expression were also observed in both animal models. In mouse mesangial cells, the authors showed that high expression of miR-192 downregulated the expression of E-box repressors (ZEB1 and ZEB2). Conversely, in 2010, Krupa et al. (2010) showed that patients with DN have lower expression of miR-192 in the kidney, which is directly related to fibroneogenesis and a low GFR. The in vitro data showed that the lower expression of miR-192 occurs by the action of increased transforming growth factor β (TGF-β), which is a key cytokine involved in tubulointerstitial
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fibrosis. In addition, in accordance with Kato et al. (2006), they found that overexpression of miR-192 decreased the expression of the E-Box repressors ZEB1 and ZEB2. Despite these two seemingly contradictory reports, it can be assumed that both extremes, low and high levels of miR-192, may be associated with the progression of the complications associated with DN. The contradictory reports could be also due to the different models and methods used by the authors.
Dey et al. (2011) showed that a high glucose concentration enhances the expression of miR-21 in cultured proximal tubule cells and glomerular mesangial cells. In addition, the increased expression of miR-21 by a high glucose concentration generates an increase in TORC1 (target of rapamycin complex-1) activity, leading to fibronectin expression and renal cell hypertrophy. In addition, phosphatase and tensin homologue deleted in chromosome 10 (PTEN) is associated with this signalling pathway and is downregulated by miR-21. Thus, their findings identify a previously unknown action of miR-21, the reciprocal control of Akt/TORC1 and PTEN activity, that mediates critical pathologic processes in DN. Recently, Zhong et al. (2013) also showed a strong association between miR-21 and fibrogenesis. miR-21 was shown to be increased in the kidneys of mice with type 2 diabetes. In addition, the knockdown of miR-21 in diabetic mice restored Smad7 levels and repressed activation of TGF-β and NF-κB signalling pathways. The miR-21 knockdown also revealed a significant improvement in inflammation, microalbuminuria and renal fibrosis.
Wang et al. (2008) studied fibronectin production by exposing mouse and human mesangial cells to high glucose levels. Under these conditions, and in mouse models of DN, miR-377 displayed higher expression compared with controls. In addition, higher expression of miR-377 decreased the expression of superoxide dismutase and p21-activated kinase (PAK1), which increased fibronectin protein production. Thus, miR-377 upregulation in the diabetic kidney has a critical role in enhancing fibronectin protein production.
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Recent studies have shown that other miRNAs, such as miR-29, miR-324-3p, and miR200, are involved in renal fibrosis production (Macconi et al. 2012; Wang et al. 2012; Xiong et al. 2012). They have different targets and pathways that ultimately lead to the common point of fibrosis regulation. MiR-29 is negatively regulated by TGF-β1 and targets collagen gene leading to renal fibrosis (Wang et al. 2012). MiR-324-3p is involved in the modulation of a serine peptidase, prolyl endopeptidase (Prep), important in the synthesis of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) and the metabolism of angiotensin. In cultured tubular cells, overexpression of miR-324-3p is linked to a very low expression of Prep and increased deposition of collagen (Macconi et al. 2012). The miR-200 family was involved in inhibition of renal tubule dedifferentiation and renal fibrosis progression, which occur through the downregulation of E-cadherin transcriptional repressors, such as zinc finger and E-boxbinding proteins (ZEB1 and ZEB2) (Xiong et al. 2012).
Expansion of the mesangial extracellular matrix and miRNA Expansion of the mesangial extracellular matrix is also an important pathologic process typical of DN. Zhanga et al. (2009) examined miRNA expression involved in expansion of the mesangial extracellular matrix during the initial phase of DN. They showed that miR-21 expression is downregulated in the early stages of DN in mouse mesangial cells and in type 2 diabetic mice. In addition, overexpression of miR-21 was reported to downregulate PTEN in vivo and in vitro, prevent proliferation of mesangial cells and decrease the 24-hour urine albumin excretion in vivo.
Another miRNA involved in the expansion of the mesangial extracellular matrix is miR-29c. Long et al. (2011) examined the expression of miR-29c in microvascular endothelial cells from kidney glomeruli and podocytes treated with a high concentration of glucose in vitro and in
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db/db mice. Functionally, they found that overexpression of miR-29c downregulates Sprouty homologue 1 (Spry1), promotes Rho kinase activation, enhances extracellular matrix protein accumulation and induces cell apoptosis. In addition, knockdown of miR-29c by a specific antisense oligonucleotide significantly decreased kidney mesangial matrix accumulation and albuminuria in the db/db mice model.
A more recent study by Zhang et al. (2012) focused on miR-451. Using computational approaches, they identified some miR-451 targets. One of these targets is Ywhaz (tyrosine3monooxy-genase/tryptophan 5-monooxygenase activation protein, zeta), which is downregulated and is necessary for the miR-451-mediated negative regulation of MAP kinase kinase 3 (MKK3) and p38 mitogen-activated protein kinase (p38 MAPK). They also showed that miR-451 overexpression inhibits expansion of glomerular mesangial cells. These findings suggest that miR-451 has an important role in mediating the complex pathways involved in the early stages of DN.
Alvarez et al. (2013) showed a critical role of miR-1207-3p in the expansion of the mesangial extracellular matrix. The authors showed that miR-1207-3p is upregulated when exposed to high glucose levels using three different cell lines: podocytes, normal human renal proximal tubule epithelial cells (RPTEC), and normal human mesangial cells. MiR-1207-5p is a PVT1derived miRNA that was shown to be independent of its host gene, PCT1. In addition, miR1207-3p was shown to downregulate several targets such as G6PD, PMEPA1, PDPK1, and SMAD7 mRNA.
Thus, it is possible to suggest that some miRNAs are more correlated with fibrogenesis and others with the expansion of the mesangial extracellular matrix. Moreover, there is some
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overlap of miRNAs involved with both pathologic processes of DN. An overview of miRNA expressions and targets during DN is presented in Fig. 4
Clinical perspectives Many studies show extensive characterization of miRNA expression in malignant diseases such as cancer. MiRNA profiling is promising as a clinical tool for therapy development and diagnostic and prognostic evaluation (Bartel 2004). However, in non-malignant diseases such as diabetes, therapeutic targets are not well explored.
As mentioned previously, miRNA might be used as a clinical tool in different ways, one of which is its diagnostic value. Zampetaki et al. (2010) highlighted the value of miRNA as a potential biomarker. For the first time, the authors found 13 plasma miRNAs deregulated in patients with type 2 diabetes, including endothelial miR-126, using microarray screening and miRNA network inference. MiR-126 is known to maintain vascular integrity. The data demonstrated that in patients with diabetes, the loss of MiR-126 is correlated with subclinical and manifest peripheral artery disease. These results enhance the possibility of novel biomarkers for vascular complications associated with diabetes at an early stage. (Zampetaki et al. 2010).
Another clinical application of miRNA is the specific inhibition of deleterious miRNA. Different tools are used to block specific miRNAs such as the use of RNA interference (RNAi), a mechanism that controls gene expression using small double-stranded RNA (dsRNA) molecules (Aagaard and Rossi 2007). The use of vector-based miRNA inhibitors such as microRNA sponges and microRNA Decoy is a powerful tool; both can inhibit specifically an entire miRNA seed family (Bak et al. 2013). The use of locked nucleic acids (LNA) or LNA-antimiR is being
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widely studied. LNA-antimiR is an antisense chemically modified oligonucleotide capable of inhibiting binding of mature miRNA to its target sequence (Stenvang et al. 2012).
The new miRNA technologies can be exemplified, in the cardiovascular field, by the work of Caporali et al. (2011), in which adenovirus-mediated transfer of a miR-503 decoy (Ad.decoymiR-503) was used to inhibit miR-503 function in vivo. The authors demonstrated that the inhibition of miR-503 promotes benefits for ischaemic injury in a diabetic animal model. They concluded that miR-503 plays an important role in ischaemic disease, which is a major cause of morbidity and mortality in patients with diabetes. Although many mechanisms and pathways still have to be studied and better understood, their data demonstrate a possible and successful pathway for the clinical treatment of ischaemic disease, mainly for patients with diabetes.
Some important strategies have been studied for DN, such as the use of LNA or LNA-antimiR. LNA-antimiR is an antisense chemically modified oligonucleotide capable of inhibiting the binding of mature miRNA to its target sequence. Elmén et al. (2008) showed that LNA-antimiR effectively antagonizes liver-expressed miR-122 in non-human primates. Using three doses of LNA-antimiR (10 mg kg−1), they efficiently reached silencing of miR-122, which led to a reversible and long-lasting decrease in total plasma cholesterol. There was no evidence of histopathologic changes or LNA-associated toxicities in the animals used in the study. This study had the advantage of using systemically administered LNA-antimiR in primates to better understand the function of miRNA, which could lead to new strategies for treatment, establishing LNA-antimiR as a promising agent for treating disease-associated miRNAs.
Zarjou et al. (2011) also used LNA-antimiR technology. They found that miR-21 blockage, using LNA-antimiR-21, attenuated renal fibrosis induced by unilateral ureteral obstruction (UUO). This attenuation is probably due to decreased expression of profibrotic proteins and decreased infiltration of inflammatory macrophages in UUO kidneys. Their data demonstrate that
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targeting specific miRNAs could be an important new therapeutic approach to treat renal fibrosis.
In conclusion, miRNA therapy may help to prevent or stimulate the development of pathologic processes in both diabetic complications, cardiomyopathy and nephropathy. Thus, specific inhibition of one miRNA, using different tools, can contribute greatly to prevent worsening of the disease.
There is mounting evidence of an important role for miRNA in the development of human diseases, despite many fundamental questions remaining unanswered regarding its biology. Thus, the ability to restore and inhibit miRNA functions could be a useful approach in the treatment of many diseases such as diabetes and its complications in the heart and kidneys, but new strategies and studies in this area are still necessary.
Conflict of interest The authors have no conflicts of interest to declare.
Financial support FAPERJ, CNPq, CAPES, INCTEM
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Rubler, S., Dlugash, J., Yuceoglu, Y.Z., Kumral, T., Branwood, A.W. & Grishman, A. 1972. New type of cardiomyopathy associated with diabetic glomerulosclerosis. Am J Cardiol 30, 595–602. Sand, M. 2014. The pathway of miRNA maturation. Methods Mol Biol 1095, 3–10. Schamberger, A., Sarkadi, B. & Orban, T.I. 2012. Human mirtrons can express functional microRNAs simultaneously from both arms in a flanking exon-independent manner. RNA Biol 9, 1177–1185. Schramm, T.K., Gislason, G.H., Køber, L., Rasmussen, S., Rasmussen, J.N., Abildstrøm, S.Z., Hansen, M.L., Folke, F., Buch, P., Madsen, M., Vaag, A. & Torp-Pedersen, C. 2008. Diabetes patients requiring glucose-lowering therapy and nondiabetics with a prior myocardial infarction carry the same cardiovascular risk: a population study of 3.3 million people. Circulation 117, 1945–1954. Shan, Z.X., Lin, Q.X., Deng, C.Y., Zhu, J.N., Mai, L.P., Liu, J.L., Fu, Y.H., Liu, X.Y., Li, Y.X., Zhang, Y.Y., Lin, S.G. & Yu, X.Y. 2010. miR-1/miR-206 regulate Hsp60 expression contributing to glucose-mediated apoptosis in cardiomyocytes. FEBS Lett 584, 3592–3600. Shantikumar, S., Caporali, A. & Emanueli, C. 2011. Role of microRNAs in diabetes and its cardiovascular complications. Cardiovasc Res, 93, 583–593. Shaw, J.E., Sicree, R.A. & Zimmet, P.Z. 2010. Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res Clin Pract 87, 4–14. Stenvang, J., Petri, A., Lindow, M., Obad, S. & Kauppinen, S. 2012. Inhibition of microRNA function by antimiR oligonucleotides. Silence 3, 1. Swärd, P. & Rippe, B. 2012. Acute and sustained actions of hyperglycaemia on endothelial and glomerular barrier permeability. Acta Physiol (Oxf) 204, 294–307.
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Torres-Jacome, J., Gallego, M., Rodríguez-Robledo, J.M., Sanchez-Chapula, J.A. & Casis, O. 2013. Improvement of the metabolic status recovers cardiac potassium channel synthesis in experimental diabetes. Acta Physiol (Oxf) 207, 447–459. Wang, B., Komers, R., Carew, R., Winbanks, C.E., Xu, B., Herman-Edelstein, M., Koh, P., Thomas, M., Jandeleit-Dahm, K., Gregorevic, P., Cooper, M.E. & Kantharidis, P. 2012. Suppression of microRNA-29 Expression by TGF-β1 promotes collagen expression and renal fibrosis. J Am Soc Nephrol 23, 252–265. Wang, Q., Wang, Y., Minto, A.W., Wang, J., Shi, Q., Li, X. & Quigg, R.J. 2008. MicroRNA-377 is up-regulated and can lead to increased fibronectin production in diabetic nephropathy. FASEB J 22, 4126–4135. Westholm, J.O. & Lai, E.C. 2011. Mirtrons: microRNA biogenesis via splicing. Biochimie 93, 1897–1904. Xiao, J., Luo, X., Lin, H., Zhang, Y., Lu, Y., Wang, N., Yang, B. & Wang, Z. 2007. MicroRNA miR133 represses HERG K+ channel expression contributing to QT prolongation in diabetic hearts. J Biol Chem 282, 12363–12367. Xiong, M., Jiang, L., Zhou, Y., Qiu, W., Fang, L., Tan, R., Wen, P. & Yang, J. 2012. The miR-200 family regulates TGF-β1-induced renal tubular epithelial to mesenchymal transition through Smad pathway by targeting ZEB1 and ZEB2 expression. Am J Physiol Renal Physiol 302, F369–379. Yi, R., Qin, Y., Macara, I.G. & Cullen, B.R. 2003. Exportin-5 mediates the nuclear export of premicroRNAs and short hairpin RNAs. Genes Dev 17, 3011–3016. Zampetaki, A., Kiechl, S., Drozdov, I., Willeit, P., Mayr, U., Prokopi, M., Mayr, A., Weger, S., Oberhollenzer, F., Bonora, E., Shah, A., Willeit, J. & Mayr, M. 2010. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circ Res 107, 810–817.
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Zarjou, A., Yang, S., Abraham, E., Agarwal, A. & Liu, G. 2011. Identification of a microRNA signature in renal fibrosis: role of miR-21. Am J Physiol Renal Physiol 301, F793–801. Zhang, Z., Luo, X., Ding, S., Chen, J., Chen, T., Chen, X., Zha, H., Yao, L., He, X. & Peng, H. 2012. MicroRNA-451 regulates p38 MAPK signaling by targeting of Ywhaz and suppresses the mesangial hypertrophy in early diabetic nephropathy. FEBS Lett 586, 20–26. Zhang, Z., Peng, H., Chen, J., Chen, X., Han, F., Xu, X., He, X. & Yan, N. 2009. MicroRNA-21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice. FEBS Lett 583, 2009–2014. Zhong, X., Chung, A.C., Chen, H.Y., Dong, Y., Meng, X.M., Li, R., Yang, W., Hou, F.F. & Lan, H.Y. 2013. miR-21 is a key therapeutic target for renal injury in a mouse model of type 2 diabetes. Diabetologia 56, 663–674.
Figure 1 Diabetes mellitus and its major complications. There are two forms of diabetes mellitus: type 1 and type 2. Hyperglycaemia is the main feature in both types. Hyperglycaemia is also the main factor responsible for diabetic complications such as cardiomyopathy and nephropathy, which increase the risk of morbidity in these patients. Figure 2 Steps of canonical and non-canonical miRNA biogenesis in the nucleus and cytoplasm. 1. miRNA biogenesis consists of cleavage of the primary miRNA, and then an intermediate stem loop is released, known as the miRNA precursor or pre-miRNA. 2. During canonical miRNA biogenesis, this process is catalysed by Drosha, an RNase III, and its essential cofactor known as DGCR8. Mirtron biogenesis is catalysed by the spliceosome. 3. The catalysts are responsible for cleaving the RNA duplex with the premiRNAs, which are carried from the nucleus to the cytosol by the export complex Exportin-5 and Ran-GTP. 4. When reaching the cytoplasm, they are cleaved by Dicer,
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another RNase III, into their mature form of 20–22 nucleotides. 5. Mature miRNAs (indicated in yellow) are then incorporated into the Argonaute (AGO) protein family and then loaded into the RNA-induced silencing complex (RISC). 6. RISC is guided by AGO and the mature miRNA to an almost complementary sequence, usually in the untranslated region (UTR) of a target messenger RNA (mRNA). 7. After ligation, RISC causes post-transcriptional silencing of genes by cleaving the target mRNA or blocking its translation. miRNAs may also positively control global protein synthesis through ribosome biogenesis and ribosomal protein mRNA translation stimulation.
Figure 3 Main miRNAs in diabetic heart complications. About 65% of patients with diabetes mellitus develop diabetic cardiomyopathy. Specific miRNAs are correlated with pathologic processes in diabetic cardiomyopathy through the modulation of target genes or proteins. MiRNA expression (yellow boxes), the miRNA target (blue boxes) and the pathologic processes associated with diabetic cardiomyopathy (gray boxes) are represented. ↑ means high expression and ↓ means low expression. 1. High expression of miR-133 reduces the expression of K+ channels and is correlated with arrhythmia. 2. High expression of miR-1 and miR-206 downregulates Hsp60 and is correlated with apoptotic cardiomyocytes. 3. High expression of miR-223 increases GLUT4 expression and is correlated with myocardial insulin resistance. 4. High expression of miR-216a inhibits CAV2 expression and is correlated with ischaemic heart failure. 5. High expression of miR-141 regulates Slc25a3 gene expression and is correlated with mitochondrial dysfunction.
Figure 4 Main miRNAs in diabetic kidney complications. About 40% of patients with diabetes mellitus develop diabetic nephropathy. Specific miRNAs are correlated with pathologic processes in diabetic nephropathy through the modulation of target genes or proteins. MiRNA expression (yellow boxes), the miRNA target (blue boxes) and the pathologic
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processes associated with diabetic nephropathy (gray boxes) are represented. ↑ means high expression and ↓ means low expression. 1. Lower expression of miR-93 promotes upregulation of VEGF-A. 2. Higher expression of miR-192 promotes downregulation of ZEB1 and ZEB2. 3. Higher expression of miR-21 promotes upregulation of TORC1, downregulation of PTEN and Smad7. 4. Higher expression of miR-377 promotes downregulation of superoxide dismutase and PAK1. 5. Lower expression of miR-29 promotes upregulation of collagen. 6. Higher expression of miR324-3p promotes downregulation of Prep. 7. Lower expression of the miR-200 family promotes downregulation of ZEB1 and ZEB2 zinc finger. All events from 1 to 7 are related to enhancement of fibrogenesis. 8. Lower expression of miR-21 promotes downregulation of PTEN. 9. Higher expression of miR-29c promotes downregulation of Spry1. 10. Lower expression of miR-451 promotes downregulation of Ywhaz. 11. Higher expression of miR-1207-3p promotes downregulation of G6PD, PMEPA1, PDPK1, and SMAD7 mRNA. All events from 8 to 11 are correlated with expansion of the mesangial extracellular matrix.
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Received Date : 30-Jan-2014
Revision Requested: 27-Feb-2014 Revised Date : 30-Apr-2014 Accepted Date : 12-May-2014 Article type
: Review Article
MicroRNAs: potential therapeutic targets in diabetic complications of the cardiovascular and renal systems
Miriam Frankenthal Figueira1,2,*, Gustavo Monnerat-Cahli1,*, Emiliano Medei1, Adriana Bastos Carvalho1, Marcelo Marcos Morales1, Marcelo Einicker Lamas1, Rodrigo Nunes da Fonseca2, Jackson Souza-Menezes1,2
1
Instituto de Biofísica Carlos Chagas Filho, Centro de Ciências da Saúde, Universidade Federal
do Rio de Janeiro, Rio de Janeiro, RJ, 21941-902, Brazil 2
Laboratório Integrado de Ciências Morfofuncionais, Núcleo em Ecologia e Desenvolvimento
Sócio-Ambiental de Macaé, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Macaé, RJ, 27965-045, Brazil
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/apha.12316 This article is protected by copyright. All rights reserved.
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*Both authors contributed equally to this work. Correspondence: Jackson de Souza Menezes, Laboratório Integrado de Ciências Morfofuncionais, Núcleo em Ecologia e Desenvolvimento Sócio-Ambiental de Macaé, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Avenida São José do Barreto, 764, Bairro Barreto, Macaé, RJ, 27965-045, Brazil. E-mail: [email protected] or [email protected]
Running head: MicroRNAs in diabetic cardiomyopathy and nephropathy
Abstract Diabetes mellitus is a serious health problem that can lead to several pathologic complications in numerous organs and tissues. The most important and most prevalent organs affected by this disease are the heart and the kidneys and these complications are the major causes of death in patients with diabetes. MicroRNAs (miRNAs), short non-coding RNAs, have been found to be functionally important in the regulation of several pathologic processes and they are emerging as an important therapeutic tool to avoid the complications of diabetes mellitus. This review summarizes the knowledge on the effects of miRNAs in diabetes. The use of miRNAs in diabetes from a clinical perspective is also discussed, focusing on their potential role to repair cardiovascular and renal complications.
Keywords: diabetes, nephropathy, cardiomyopathy, miRNAs, hyperglycaemia.
Introduction Diabetes mellitus is a metabolic disease; the main symptom is a high concentration of plasma glucose. The number of individuals developing this condition is increasing at an alarming rate
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worldwide. It is a serious health problem that can lead to several pathologic complications in many organs and tissues. In 2010, it was estimated that by 2030 there will be a 69% increase in the number of adults with diabetes in developing countries and a 20% increase in developed countries, which represents 439 million adults worldwide with diabetes (Shaw et al. 2010). During the development of the disease, the cardiovascular system is often damaged; at least 65% of patients with diabetes mellitus die of some form of heart disease or stroke (Schramm et al. 2008). The renal system is also severely impaired by diabetes. About 30–40% of patients with diabetes develop chronic renal disease, which is associated with increased mortality (Fig. 1) (Gross et al. 2002).
The pathophysiologic mechanisms of diabetes are not completely understood. In this context, several studies have attempted to understand the molecular basis of these events in order to create more effective treatments with fewer side effects (Li et al. 2009, Couzin 2008, Xiao et al. 2007). Recent studies have shown that microRNAs (miRNAs) could play an important role in different diseases, opening a new perspective for basic science and treatments. MiRNAs are small 19–23 nucleotide RNA molecules that act as regulators of protein expression in eukaryotic cells by inducing the translational arrest and degradation of messenger RNAs (Shantikumar et al. 2011, Cai et al. 2010, Feng et al. 2010, Lu et al. 2010, Zampetaki et al. 2010). Recently, this group of non-coding RNAs (ncRNAs) has received considerable attention in biomedical research as a possible alternative for treatment of disease. The basic features of miRNA biogenesis unveiled in recent years are described in the following.
In recent years, several bioinformatics tools have been developed to predict miRNA targets in human and mouse genomes (reviewed by Peterson et al. 2014). miRNA target prediction tools take into account several features of miRNA and mRNA target sequence including the seed match, the first 2–8 nucleotides starting at the 5′ end and counting towards the 3′ end, conservation across species, which is presumably higher at the seed region, and
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Gibbs free energy, where the binding of miRNA to a candidate target mRNA is predicted to be stable. These features have been used successfully to predict functional interactions (reviewed by Peterson et al. 2014). The putative miRNA regulators involved in kidney disease can be investigated using one of the following tools: DIANA-microT-CDS (Maragkakis et al. 2009), miRanda-mirSVR (Betel et al. 2010), and TargetScan (Lewis et al. 2005).
MiRNAs can be divided into two broad categories, canonical and non-canonical miRNAs, The canonical miRNA pathway involves step-wise processing of long genome-encoded primary miRNA (pri-miRNA) transcripts into short hairpin-like double-stranded miRNA duplexes of 19–21nt with 2-nt 3 overhangs. These double-stranded miRNAs are also referred to as short-interfering RNAs (siRNAs), which act as substrates for the gene silencing machinery (Chong et al. 2010, Westholm & Lai 2011). However, deviations from this paradigm have been observed: subclasses of miRNAs, non-canonical miRNA, undergo alternative biogenesis pathways that do not involve Drosha, thereby providing an additional level of complexity to miRNA-dependent regulation of gene expression (Fig. 2).
At the nucleus, canonical miRNAs biogenesis is characterized by a cleavage process catalysed by Drosha, an RNase III, and its essential cofactor known as DGCR8 (DiGeorge syndrome critical region) (Lee et al. 2004, Han et al. 2004, Gregory et al. 2004). After the cleavage process, an intermediate stem loop is released, known as the miRNA precursor or pre-miRNA. Pre-miRNA hairpins are then recognized by Exportin-5 for nuclear export. At the cytoplasm, the pre-miRNA is cleaved by Dicer, an RNase III, into the mature form of 20–22 nucleotides. The miRNA duplex is then unrolled and the functional strand is incorporated into the Argonaute (AGO) protein family and then loaded into the RNA-induced silencing complex (RISC). The RISC is guided by AGO and the mature miRNA to an almost complementary sequence, usually in the untranslated region (UTR) of a target messenger RNA (mRNA).
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Translational repression of imperfectly matched targets or target cleavage of perfectly matched targets takes place. Thus, miRNAs are generally negative regulators of gene expression (Lee et al. 2003, Yi et al. 2003, Farazi et al. 2013).
However, miRNAs have also been shown to be involved in the targeting of the promoter region, which leads to transcriptional gene silencing. In addition, miRNAs also have the ability to positively control global protein synthesis through ribosome biogenesis and ribosomal protein mRNA translation stimulation. Thus, miRNAs are now known to promote as well as inhibit gene expression (Du & Zamore 2005, Martinez & Tuschl 2004, Ørom et al. 2008, Schamberger et al. 2012).
An increasing number of non-canonical miRNA pathways have been described in recent years (Chong et al. 2010, Westholm & Lai 2011, Sand 2014). The first and most important alternative miRNA maturation pathway described is the mirtron pathway. Mirtrons are directly spliced out from host genes as pre-miRNAs with hairpin potential and do not depend on pri-miRNA Drosha/DGCR8 processing. Mirtron biogenesis is instead catalysed by the spliceosome. The product of intron splicing is not linear but mimics a transient lariat shape wherein the 3′ branch point is ligated to the 5′ end of the intron. After the lariat-shaped intron is processed by the lariat debranching enzyme (Ldbr), it refolds into a pre-miRNA shape, which is recognized and transferred to the cytoplasm by Exp-5, similar to the canonical pathway. Following regular Dicer cleavage, the mature miRNA can be loaded on the RISC and exerts its effect (Chong et al. 2010, Westholm & Lai 2011, Farazi et al. 2013, Sand 2014). Canonical and non-canonical miRNA biogenesis is summarized in Fig. 2.
The role of miRNAs has been demonstrated in many biological processes, including proliferation, differentiation, apoptosis, and development (Bartel 2004, Bentwich et al. 2005). Each miRNA has the capacity to regulate multiple genes, therefore deregulation of miRNAs can certainly lead to changes in several physiologic processes. This review summarizes the
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knowledge on the effects of miRNAs in diabetes. The use of miRNAs in diabetes from a clinical perspective is also discussed, focusing on their potential role in cardiovascular and renal complications.
Diabetic cardiomyopathy and miRNAs Cardiomyopathy caused by diabetes was first described in 1972 (Rubler et al. 1972). The pathology of the cardiac system induced by diabetes includes interstitial fibrosis, imbalance of cardiac ionic currents, apoptosis of cardiomyocytes, abnormal energy use, and cardiac neuropathy. Moreover, diabetes can cause severe vascular defects, leading to microand macroangiopathy (Aragno et al. 2008, McVeigh et al. 1992, Casis et al. 2000, TorresJacome et al. 2013). However, the mechanisms of this pathology are not entirely understood. Recent studies have shown an association between miRNAs and diabetic cardiomyopathy.
One of the most crucial clinical manifestations of diabetic cardiomyopathy is the high incidence of cardiac arrhythmias, including ventricular fibrillation and cardiac sudden death. Electrocardiograms of patients with diabetes show changes affecting the process of ventricular repolarization, such as QT prolongation (Kahn et al. 1987). The autonomic neuropathy caused by diabetes is also associated as a mechanism underlying the high incidence of arrhythmias (Ewing et al. 1991). An overview of miRNA expression and targets during the development of diabetic cardiomyopathy is presented in Fig. 3.
Xiao et al. (2007) showed remarkable upregulation of miR-133 expression in the heart in a rabbit model of diabetes. This study reported that this upregulation of miR-133 expression could decrease a rectifier K+ current by reducing the expression of this ion channel. Since potassium ionic currents are essential to physiologic repolarization, this change contributes to long QT syndromes, which are directly associated with cardiac arrhythmias (Xiao et al. 2007).
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During the progression of diabetes, a high percentage of apoptotic cardiomyocytes is observed, intensifying cardiac remodelling and leading to heart failure (HF) (Fiordaliso et al. 2000). A group of chaperones known as heat shock proteins (Hsp) can prevent several dysfunctional events that can lead to cell apoptosis. The increase in Hsp expression can be induced by different stress events such as depletion of oxygen or ATP, oxidative stress, or hypothermia. In the heart, Hsp60 is an important component of the defence mechanism against myocardial injury but its expression is reduced in the diabetic myocardium. Shan et al. (2010) demonstrated that the high expression of miR-1 and miR-206 in cardiomyocytes stimulated with high glucose concentrations was high. Moreover, this study proved that miR-1 and miR-206 could downregulate Hsp60 expression by directly targeting the 3′ UTR of Hsp60 mRNA, contributing to high glucose-mediated apoptosis in cardiomyocytes.
The risk of developing cardiovascular disease in diabetes is also related to myocardial insulin resistance, which promotes downregulation of the glucose transporter 4 (GLUT4) protein at the plasma membrane level. In 2009, Lu et al. (2010) showed that miR-223 plays an important role in the regulation of GLUT4 expression and in the glucose metabolism of cardiomyocytes. This study shows that miR-223 is consistently upregulated in the insulinresistant heart. They also evaluated the molecular mechanism of miR-223 and found that this miRNA could increase nuclear factor IA expression, but had no effect on myocyte enhancer factor 2c or insulin-like growth factor 1 receptor. They also examined insulin signalling and glucose transport proteins and found that phosphoinositide 3-kinase signalling and AMP kinase activity were not affected by miR-223 overexpression; however, GLUT4 protein expression was increased by miR-223 overexpression. The data obtained in this study show that miR-223 can upregulate target genes such as GLUT4 in adult cardiomyocytes, suggesting an important target for treatment in patients with type 2 diabetes. In diabetic human and mouse hearts,
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GLUT4 is markedly downregulated at the plasma membrane and the investigators suggested that the increased miR-223 levels observed in the diabetic heart are an adaptive/homeostatic response to restore GLUT4 expression and normal glucose uptake. This model is in agreement with the idea that miRNAs act as stress response genes and are important for maintaining the robustness of physiologic processes in the face of a pathophysiologic condition (Lu et al. 2010).
Ischaemic HF is one of the major causes of death in patients with diabetes. In a recent work (Greco et al. 2012), the expression profile of miRNAs was measured in the left ventricle of patients with HF; one group with type 2 diabetes (D-HF) and the other without diabetes (ND-HF). This study demonstrated that 17 miRNAs were modulated in both groups of patients compared with a control group (non-diabetic and no HF). This study revealed that the expression of 6 miRNAs was different in D-HF and ND-HF patients: miR-34b, miR-34c, miR199b, miR-210, miR-650, and miR-223. These observations are important to highlight the specific miRNAs involved in the diabetes disease mechanism during ischaemic cardiomyopathy. The authors also showed that miR-216a is the miRNA with the highest expression in both HF patient classes and that TGF-β may trigger miR-216a expression. For the first time, miR-216a overexpression has been found to be related to human HF. MiR-216a acts in many ways; one of them is to inhibit CAV2, a member of the caveolin family of scaffolding proteins, which regulates lipid metabolism and signal transduction pathways. The absence of caveolin expression leads to deleterious cardiac activity and progressive hypertrophy in cardiac myocytes (Greco et al. 2012).
Another complication of diabetes is mitochondrial dysfunction, which can lead to an increased oxidative stress state and a decrease in ATP production. Proteomic alterations in mitochondria resulting from diabetes have been reported, however, the mechanisms underlying the changes in the proteomic signatures are unknown. In this context, it was demonstrated that miR-141 upregulation can modulate Slc25a3 gene expression (the protein
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encoded by this gene catalyses the transport of phosphate into the mitochondrial matrix, either by proton cotransport or in exchange for hydroxyl ions) in type 1 diabetic hearts of mice, which could explain, at least in part, why diabetic cardiomyocytes have a low rate of ATP production and a decrease in ATP synthase activity (Baseler et al. 2012).
Diabetic nephropathy and miRNAs Diabetic nephropathy (DN) is an incurable kidney disease and one of the most important and disabling complications of diabetes. There are some therapies available for DN, but they are not effective enough to prevent many patients with diabetes reaching end-stage renal disease (Kato et al. 2009).
When DN is diagnosed by classic methods, such as the detection of microalbuminuria (urinary excretion of albumin between 20 and 200 μg min−1 in humans) or a reduction in the glomerular filtration rate (GFR), little can be done to prevent the progressive course towards renal failure. Research on markers and new therapeutic options for diabetic kidney disease is ongoing (Hong & Chia 1998, Swärd & Rippe 2012). Many studies have addressed miRNAs as a promising therapeutic target and potential diagnostic tool for this condition (Elmén et al. 2008, Zarjou et al. 2011, Long et al. 2010, Krupa et al. 2010). DN is classically characterized by albuminuria, thickening of the glomerular and tubular basal membrane, expansion of the mesangial extracellular matrix, microvascular damage, and tubulointerstitial fibrosis (Bohlender et al. 2005). The mechanisms by which these disease processes occur are not yet fully understood. Here, we summarize some features of this pathologic process in DN and the involvement of miRNA, emphasizing two main and correlated mechanisms underlying this disease: fibrogenesis and expansion of the mesangial extracellular matrix.
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Fibrogenesis and miRNA Tubulointerstitial fibrosis and fibrogenesis are the major pathologic problems in DN (Swärd & Rippe 2012). One of the most important characteristics of fibrogenesis is the production of collagen IV, collagen 1-α1, collagen 1-α2, and fibronectin. In this context, Long et al. (2010) showed a correlation between miR-93 and the fibrogenic process. They showed in vitro and in vivo that vascular endothelial growth factor (VEGF), which is a key factor involved in fibrogenesis and other microvascular complications of diabetes, is regulated by miR-93. Using diabetic mice, under hyperglycaemic conditions, the group demonstrated that miR-93 is downregulated in the glomeruli and this leads to upregulation of VEGF-A expression. These data highlight miR-193 and the VEGF signalling pathway as important targets in preventing the progression of DN.
Another miRNA associated with fibrogenesis is miR-192. In 2006, Kato et al. (2007) demonstrated that miR-192 is significantly increased in glomeruli isolated from diabetic db/db mice (an animal model of obesity, diabetes and dyslipidemia; the activity of the leptin receptor is deficient in these mice because they are homozygous for a point mutation in the leptin receptor gene) as well in mice with diabetes induced by streptozotocin (an established model of type 1 diabetes). High levels of collagen 1-α1, collagen-2, and TGF-β expression were also observed in both animal models. In mouse mesangial cells, the authors showed that high expression of miR-192 downregulated the expression of E-box repressors (ZEB1 and ZEB2). Conversely, in 2010, Krupa et al. (2010) showed that patients with DN have lower expression of miR-192 in the kidney, which is directly related to fibroneogenesis and a low GFR. The in vitro data showed that the lower expression of miR-192 occurs by the action of increased transforming growth factor β (TGF-β), which is a key cytokine involved in tubulointerstitial
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fibrosis. In addition, in accordance with Kato et al. (2006), they found that overexpression of miR-192 decreased the expression of the E-Box repressors ZEB1 and ZEB2. Despite these two seemingly contradictory reports, it can be assumed that both extremes, low and high levels of miR-192, may be associated with the progression of the complications associated with DN. The contradictory reports could be also due to the different models and methods used by the authors.
Dey et al. (2011) showed that a high glucose concentration enhances the expression of miR-21 in cultured proximal tubule cells and glomerular mesangial cells. In addition, the increased expression of miR-21 by a high glucose concentration generates an increase in TORC1 (target of rapamycin complex-1) activity, leading to fibronectin expression and renal cell hypertrophy. In addition, phosphatase and tensin homologue deleted in chromosome 10 (PTEN) is associated with this signalling pathway and is downregulated by miR-21. Thus, their findings identify a previously unknown action of miR-21, the reciprocal control of Akt/TORC1 and PTEN activity, that mediates critical pathologic processes in DN. Recently, Zhong et al. (2013) also showed a strong association between miR-21 and fibrogenesis. miR-21 was shown to be increased in the kidneys of mice with type 2 diabetes. In addition, the knockdown of miR-21 in diabetic mice restored Smad7 levels and repressed activation of TGF-β and NF-κB signalling pathways. The miR-21 knockdown also revealed a significant improvement in inflammation, microalbuminuria and renal fibrosis.
Wang et al. (2008) studied fibronectin production by exposing mouse and human mesangial cells to high glucose levels. Under these conditions, and in mouse models of DN, miR-377 displayed higher expression compared with controls. In addition, higher expression of miR-377 decreased the expression of superoxide dismutase and p21-activated kinase (PAK1), which increased fibronectin protein production. Thus, miR-377 upregulation in the diabetic kidney has a critical role in enhancing fibronectin protein production.
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Recent studies have shown that other miRNAs, such as miR-29, miR-324-3p, and miR200, are involved in renal fibrosis production (Macconi et al. 2012; Wang et al. 2012; Xiong et al. 2012). They have different targets and pathways that ultimately lead to the common point of fibrosis regulation. MiR-29 is negatively regulated by TGF-β1 and targets collagen gene leading to renal fibrosis (Wang et al. 2012). MiR-324-3p is involved in the modulation of a serine peptidase, prolyl endopeptidase (Prep), important in the synthesis of the antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) and the metabolism of angiotensin. In cultured tubular cells, overexpression of miR-324-3p is linked to a very low expression of Prep and increased deposition of collagen (Macconi et al. 2012). The miR-200 family was involved in inhibition of renal tubule dedifferentiation and renal fibrosis progression, which occur through the downregulation of E-cadherin transcriptional repressors, such as zinc finger and E-boxbinding proteins (ZEB1 and ZEB2) (Xiong et al. 2012).
Expansion of the mesangial extracellular matrix and miRNA Expansion of the mesangial extracellular matrix is also an important pathologic process typical of DN. Zhanga et al. (2009) examined miRNA expression involved in expansion of the mesangial extracellular matrix during the initial phase of DN. They showed that miR-21 expression is downregulated in the early stages of DN in mouse mesangial cells and in type 2 diabetic mice. In addition, overexpression of miR-21 was reported to downregulate PTEN in vivo and in vitro, prevent proliferation of mesangial cells and decrease the 24-hour urine albumin excretion in vivo.
Another miRNA involved in the expansion of the mesangial extracellular matrix is miR-29c. Long et al. (2011) examined the expression of miR-29c in microvascular endothelial cells from kidney glomeruli and podocytes treated with a high concentration of glucose in vitro and in
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db/db mice. Functionally, they found that overexpression of miR-29c downregulates Sprouty homologue 1 (Spry1), promotes Rho kinase activation, enhances extracellular matrix protein accumulation and induces cell apoptosis. In addition, knockdown of miR-29c by a specific antisense oligonucleotide significantly decreased kidney mesangial matrix accumulation and albuminuria in the db/db mice model.
A more recent study by Zhang et al. (2012) focused on miR-451. Using computational approaches, they identified some miR-451 targets. One of these targets is Ywhaz (tyrosine3monooxy-genase/tryptophan 5-monooxygenase activation protein, zeta), which is downregulated and is necessary for the miR-451-mediated negative regulation of MAP kinase kinase 3 (MKK3) and p38 mitogen-activated protein kinase (p38 MAPK). They also showed that miR-451 overexpression inhibits expansion of glomerular mesangial cells. These findings suggest that miR-451 has an important role in mediating the complex pathways involved in the early stages of DN.
Alvarez et al. (2013) showed a critical role of miR-1207-3p in the expansion of the mesangial extracellular matrix. The authors showed that miR-1207-3p is upregulated when exposed to high glucose levels using three different cell lines: podocytes, normal human renal proximal tubule epithelial cells (RPTEC), and normal human mesangial cells. MiR-1207-5p is a PVT1derived miRNA that was shown to be independent of its host gene, PCT1. In addition, miR1207-3p was shown to downregulate several targets such as G6PD, PMEPA1, PDPK1, and SMAD7 mRNA.
Thus, it is possible to suggest that some miRNAs are more correlated with fibrogenesis and others with the expansion of the mesangial extracellular matrix. Moreover, there is some
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overlap of miRNAs involved with both pathologic processes of DN. An overview of miRNA expressions and targets during DN is presented in Fig. 4
Clinical perspectives Many studies show extensive characterization of miRNA expression in malignant diseases such as cancer. MiRNA profiling is promising as a clinical tool for therapy development and diagnostic and prognostic evaluation (Bartel 2004). However, in non-malignant diseases such as diabetes, therapeutic targets are not well explored.
As mentioned previously, miRNA might be used as a clinical tool in different ways, one of which is its diagnostic value. Zampetaki et al. (2010) highlighted the value of miRNA as a potential biomarker. For the first time, the authors found 13 plasma miRNAs deregulated in patients with type 2 diabetes, including endothelial miR-126, using microarray screening and miRNA network inference. MiR-126 is known to maintain vascular integrity. The data demonstrated that in patients with diabetes, the loss of MiR-126 is correlated with subclinical and manifest peripheral artery disease. These results enhance the possibility of novel biomarkers for vascular complications associated with diabetes at an early stage. (Zampetaki et al. 2010).
Another clinical application of miRNA is the specific inhibition of deleterious miRNA. Different tools are used to block specific miRNAs such as the use of RNA interference (RNAi), a mechanism that controls gene expression using small double-stranded RNA (dsRNA) molecules (Aagaard and Rossi 2007). The use of vector-based miRNA inhibitors such as microRNA sponges and microRNA Decoy is a powerful tool; both can inhibit specifically an entire miRNA seed family (Bak et al. 2013). The use of locked nucleic acids (LNA) or LNA-antimiR is being
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widely studied. LNA-antimiR is an antisense chemically modified oligonucleotide capable of inhibiting binding of mature miRNA to its target sequence (Stenvang et al. 2012).
The new miRNA technologies can be exemplified, in the cardiovascular field, by the work of Caporali et al. (2011), in which adenovirus-mediated transfer of a miR-503 decoy (Ad.decoymiR-503) was used to inhibit miR-503 function in vivo. The authors demonstrated that the inhibition of miR-503 promotes benefits for ischaemic injury in a diabetic animal model. They concluded that miR-503 plays an important role in ischaemic disease, which is a major cause of morbidity and mortality in patients with diabetes. Although many mechanisms and pathways still have to be studied and better understood, their data demonstrate a possible and successful pathway for the clinical treatment of ischaemic disease, mainly for patients with diabetes.
Some important strategies have been studied for DN, such as the use of LNA or LNA-antimiR. LNA-antimiR is an antisense chemically modified oligonucleotide capable of inhibiting the binding of mature miRNA to its target sequence. Elmén et al. (2008) showed that LNA-antimiR effectively antagonizes liver-expressed miR-122 in non-human primates. Using three doses of LNA-antimiR (10 mg kg−1), they efficiently reached silencing of miR-122, which led to a reversible and long-lasting decrease in total plasma cholesterol. There was no evidence of histopathologic changes or LNA-associated toxicities in the animals used in the study. This study had the advantage of using systemically administered LNA-antimiR in primates to better understand the function of miRNA, which could lead to new strategies for treatment, establishing LNA-antimiR as a promising agent for treating disease-associated miRNAs.
Zarjou et al. (2011) also used LNA-antimiR technology. They found that miR-21 blockage, using LNA-antimiR-21, attenuated renal fibrosis induced by unilateral ureteral obstruction (UUO). This attenuation is probably due to decreased expression of profibrotic proteins and decreased infiltration of inflammatory macrophages in UUO kidneys. Their data demonstrate that
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targeting specific miRNAs could be an important new therapeutic approach to treat renal fibrosis.
In conclusion, miRNA therapy may help to prevent or stimulate the development of pathologic processes in both diabetic complications, cardiomyopathy and nephropathy. Thus, specific inhibition of one miRNA, using different tools, can contribute greatly to prevent worsening of the disease.
There is mounting evidence of an important role for miRNA in the development of human diseases, despite many fundamental questions remaining unanswered regarding its biology. Thus, the ability to restore and inhibit miRNA functions could be a useful approach in the treatment of many diseases such as diabetes and its complications in the heart and kidneys, but new strategies and studies in this area are still necessary.
Conflict of interest The authors have no conflicts of interest to declare.
Financial support FAPERJ, CNPq, CAPES, INCTEM
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Figure 1 Diabetes mellitus and its major complications. There are two forms of diabetes mellitus: type 1 and type 2. Hyperglycaemia is the main feature in both types. Hyperglycaemia is also the main factor responsible for diabetic complications such as cardiomyopathy and nephropathy, which increase the risk of morbidity in these patients. Figure 2 Steps of canonical and non-canonical miRNA biogenesis in the nucleus and cytoplasm. 1. miRNA biogenesis consists of cleavage of the primary miRNA, and then an intermediate stem loop is released, known as the miRNA precursor or pre-miRNA. 2. During canonical miRNA biogenesis, this process is catalysed by Drosha, an RNase III, and its essential cofactor known as DGCR8. Mirtron biogenesis is catalysed by the spliceosome. 3. The catalysts are responsible for cleaving the RNA duplex with the premiRNAs, which are carried from the nucleus to the cytosol by the export complex Exportin-5 and Ran-GTP. 4. When reaching the cytoplasm, they are cleaved by Dicer,
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another RNase III, into their mature form of 20–22 nucleotides. 5. Mature miRNAs (indicated in yellow) are then incorporated into the Argonaute (AGO) protein family and then loaded into the RNA-induced silencing complex (RISC). 6. RISC is guided by AGO and the mature miRNA to an almost complementary sequence, usually in the untranslated region (UTR) of a target messenger RNA (mRNA). 7. After ligation, RISC causes post-transcriptional silencing of genes by cleaving the target mRNA or blocking its translation. miRNAs may also positively control global protein synthesis through ribosome biogenesis and ribosomal protein mRNA translation stimulation.
Figure 3 Main miRNAs in diabetic heart complications. About 65% of patients with diabetes mellitus develop diabetic cardiomyopathy. Specific miRNAs are correlated with pathologic processes in diabetic cardiomyopathy through the modulation of target genes or proteins. MiRNA expression (yellow boxes), the miRNA target (blue boxes) and the pathologic processes associated with diabetic cardiomyopathy (gray boxes) are represented. ↑ means high expression and ↓ means low expression. 1. High expression of miR-133 reduces the expression of K+ channels and is correlated with arrhythmia. 2. High expression of miR-1 and miR-206 downregulates Hsp60 and is correlated with apoptotic cardiomyocytes. 3. High expression of miR-223 increases GLUT4 expression and is correlated with myocardial insulin resistance. 4. High expression of miR-216a inhibits CAV2 expression and is correlated with ischaemic heart failure. 5. High expression of miR-141 regulates Slc25a3 gene expression and is correlated with mitochondrial dysfunction.
Figure 4 Main miRNAs in diabetic kidney complications. About 40% of patients with diabetes mellitus develop diabetic nephropathy. Specific miRNAs are correlated with pathologic processes in diabetic nephropathy through the modulation of target genes or proteins. MiRNA expression (yellow boxes), the miRNA target (blue boxes) and the pathologic
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processes associated with diabetic nephropathy (gray boxes) are represented. ↑ means high expression and ↓ means low expression. 1. Lower expression of miR-93 promotes upregulation of VEGF-A. 2. Higher expression of miR-192 promotes downregulation of ZEB1 and ZEB2. 3. Higher expression of miR-21 promotes upregulation of TORC1, downregulation of PTEN and Smad7. 4. Higher expression of miR-377 promotes downregulation of superoxide dismutase and PAK1. 5. Lower expression of miR-29 promotes upregulation of collagen. 6. Higher expression of miR324-3p promotes downregulation of Prep. 7. Lower expression of the miR-200 family promotes downregulation of ZEB1 and ZEB2 zinc finger. All events from 1 to 7 are related to enhancement of fibrogenesis. 8. Lower expression of miR-21 promotes downregulation of PTEN. 9. Higher expression of miR-29c promotes downregulation of Spry1. 10. Lower expression of miR-451 promotes downregulation of Ywhaz. 11. Higher expression of miR-1207-3p promotes downregulation of G6PD, PMEPA1, PDPK1, and SMAD7 mRNA. All events from 8 to 11 are correlated with expansion of the mesangial extracellular matrix.
This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
Accepted Article This article is protected by copyright. All rights reserved.
