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MicroRNAs and inflammation biomarkers in obesity
Chapter 17
MicroRNAs and inflammation biomarkers in obesity Bruna Jardim Quintanilha1, 2, Bruna Zavarize Reis3, Telma A. Faraldo Correˆa2, 3, Graziela Biude da Silva Duarte3 and Marcelo Macedo Rogero1, 2 1
Department of Nutrition, School of Public Health, University of São Paulo, São Paulo, Brazil; 2Food Research Center (FoRC), CEPID-FAPESP,
Research Innovation and Dissemination Centers São Paulo Research Foundation, São Paulo, Brazil; 3Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Science, University of São Paulo, São Paulo, Brazil
Introduction
Micro RNAs
Obesity, a multifactorial and polygenic condition, is a public health issue of great concern in both developed and developing countries. It is estimated that in 2016 alone there were more than 1.9 billion overweight adults worldwide, that is, 39% of the world’s adult population; of these, more than 650 million (approximately 13% of the world’s adult population) were obese. Obesity also affects children: an estimated 41 million people under 5 years of age, as well as 340 million people aged 5e19 years, were either overweight or obese in 2016 [1]. Obesity results from the interaction of a series of genetic, metabolic, behavioral, and environmental factors. It leads to metabolic inflammation or meta-inflammation, a chronic form of low-grade inflammation in white adipose tissue (WAT) that differs from a classic inflammatory response [2]. Two proteins play a key role in metainflammation: inhibitor of nuclear factor kappa-B kinase (IKK-b), and c-Jun N-terminal kinase 1 (JNK-1), whose activation in turn activates nuclear factor kappa B (NF-kB) and activator protein 1 (AP-1), respectively. NF-kB and AP-1 then translocate to the cell nucleus and activate the transcription of genes related to inflammation, such as tumor necrosis factor alpha (TNF-a), interleukin 6 (IL-6), and interleukin 1 beta (IL-1b) [3]. Adipocytes and infiltrating inflammatory cellsdmainly macrophagesdsecrete cytokines such as TNF-a, IL-6, and IL-1b, and inflammatory modulators such as leptin, resistin, and adiponectin [2,4]. Obesity is strongly associated with metabolic diseases such as type 2 diabetes (T2D), atherosclerosis, cardiovascular disease (CVD), nonalcoholic fatty liver disease (NAFLD), and some cancers [5].
MicroRNAs (miRNAs) play a key role in obesity, since they can posttranscriptionally regulate a large number of genes. They may also be important in maintaining metabolic homeostasis; if so, their regulation could serve as potential therapeutics for metabolic diseases [6]. MicroRNAs are noncoding RNAs that modulate gene expression. Mature miRNAs regulate the expression of proteins, by cleaving mRNA or repressing its translation, depending on the level of complementarity between the miRNA and the target mRNA [7]. Changes in miRNA levels have been shown to affect gene expression and thereby cell function, in several pathophysiological disorders related to obesity, including inflammation, oxidative stress, impaired adipogenesis, insulin signaling, apoptosis, and angiogenesis [8e12]. MicroRNAs are present in tissues or body fluids in stable form, protected from endogenous RNAse activity [6]. They may play a role in communication between adipocytes, as well as between adipose tissue and other tissues. Notably, miRNAs can act as potential diagnostic biomarkers, as they fulfill most of the necessary criteria: they can be rapidly and accurately detected through noninvasive methods, have high sensitivity and specificity to the disease in question, allow for early detection, and have a long halflife in the sample [13e15]. The identification and characterization of miRNAs associated with obesity could yield a new generation of therapeutic targets for antiobesity treatments. Additionally, identifying miRNAs that are dysregulated during the development of obesity could provide early obesity biomarkers for clinical diagnosis.
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MicroRNAs concepts MicroRNAs are a class of small, noncoding endogenous RNA molecules (w18e25 nucleotides) involved in posttranscriptional gene regulation, by binding to a target messenger RNA (mRNA), which results in the degradation or inhibition of translation [16]. To date, 2654 mature human miRNAs have been identified (MiRBase, release 16, December 2018; http://www.mirbase.org). MicroRNAs can act directly on the target genes or indirectly, by regulating transcription factors that in turn control the expression of genes [4]. One miRNA can regulate several mRNAs, but not all miRNAs incur translational repression. Some of them have the ability to activate the translation of proteins, alter the structure of chromatin by regulating histone modification, and even target genes with low DNA methylation directly [3,16].
Biogenesis Biogenesis of miRNAs is a sequential process involving a variety of enzymes and proteins [16]. MicroRNAs are usually transcribed by RNA polymerase II from miRNA genes, first forming the primary miRNA transcript (pri-miRNA). This transcript is then cleaved by the DROSHA-DiGeorge syndrome critical region gene 8 (DGCR8) microprocessor complex, creating a shorter sequence called the miRNA precursor (pre-miRNA) that displays a hairpinlike secondary structure. The pre-miRNA is exported to the cytoplasm and processed by DICER, a ribonuclease III enzyme that produces mature miRNA, which is incorporated into an RNA-protein complex (i.e., the RNA-induced silencing complex, RISC). Under most conditions, mature RISC represses gene expression posttranscriptionally, by binding the three prime untranslated regions (30 -UTRs) of specific mRNAs, and mediating mRNA degradation, destabilization, or translational inhibition, according to sequence complementarity to the target [3,17e19]. Evidence shows that, besides intracellular function, miRNAs are present in extracellular fluids in the human body, including plasma, serum, urine, and saliva; recently, they have also been associated with diseases such as obesity, cancer, and cardiovascular disease (CVD) [20e22]. MicroRNAs also play an important role in cell-tocell communication in peripheral blood, either through membrane-enclosed vesicles such as exosomes (extracellular vesicles of endosomal origin), or by binding to lipoproteins (LDL or HDL), proteins, apoptotic bodies, and ribonucleoprotein complexes (linked to Argonaut) [21,22]. In addition to their stability, it should be noted that circulating miRNAs are conserved across species, have expression patterns that are tissue- and biological-stage specific, and are easily determined through real-time
polymerase chain reaction (RT-PCR) [16]. Thus, these molecules are promising noninvasive biomarkers of certain diseases, and even of nutritional status [23].
Inflammation and miRNAs: a role in chronic diseases Obesity is related to endocrine and metabolic changes in WAT, which is the main source of systemic inflammatory response. White adipose tissue produces a variety of proinflammatory cytokines and chemokines known as adipokines, which include TNF-á, IL-1â, IL-6, and monocyte chemotactic protein (MCP-1). Consequently, obese individuals present an increase in proinflammatory biomarkers such as TNF-á, IL-1â, IL-6, and MCP-1, as well as a decrease in antiinflammatory adipokines such as adiponectin [24e26]. In addition, activation of endothelial cells and oxidative stress exacerbate the inflammatory response [24,27,28]. Two important signaling inflammatory pathways are involved in these processes: the NF-êB and JNK pathways. The former can be activated by TNF-á, lipopolysaccharides (LPSs), and saturated fatty acids; it involves the enzymatic complex IKK, which induces the phosphorylation of inhibitor-êB (IêB). This phosphorylation results in IêB polyubiquitination, which, in turn, leads to IêB degradation, as mediated by the 26S proteasome. This degradation allows NF-êB to translocate to the nucleus, and activate the transcription of several êB-dependent genes such as those encoding proinflammatory cytokines, adhesion molecules, and chemokines. The JNK signaling pathway can be activated by cytokines, fatty acids, and reactive oxygen species (ROS), among others. The active JNK pathway promotes the activation of transcription factor AP-1, which is related to gene expression of proinflammatory cytokines, and may act directly on the insulin signaling pathway. In this context, an increase in inflammatory response may trigger resistance to insulin action due to an increase in Ser307 phosphorylation of insulin receptor substrate 1 (IRS-1), and lower activity of phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K) in skeletal muscle. It should be noted that Ser307 phosphorylation of IRS-1 is associated with activation of JNK and IKK-â. In addition, activation of IL-6 signaling can increase the expression of cytokine signaling suppressor proteins SOCS-1 and SOCS3, in the three major insulin-sensitive peripheral tissues: WAT, liver tissue, and muscle tissue. SOCS-1 and SOCS-3 impair insulin action, by binding to IRS-1 and IRS-2, which leads to IRS-1 and IRS-2 ubiquitination and degradation [2,29e32]. There is substantial evidence of miRNAs’ transcriptionallevel regulation of genes encoding proteins related to the
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inflammatory process, in metabolic diseases [33]. Regulation may occur on many levels. Inflammation may induce the transcriptional process; if so, miRNAs will become rapidly active, since they do not need to be translated or translocated back into the nucleus, to repress their target. The expression of miRNAs occurs in different cell types with different functions, and some target proteinsd for example, proteins regulating miRNA processingd may be linked to the inflammatory response. MicroRNAs can also regulate several mechanisms related to inflammation, such as adipogenesis, macrophage activation, and oxidative stress [8].
MicroRNAs as biomarkers in obesity MicroRNAs found in human body fluids such as urine and plasma, are called circulating miRNAs; they act not only within cells but also in distant tissues, as hormones controlling gene expression. The physiological function of circulating miRNAs is mostly unknown, but different studies have shown that these molecules have essential roles, including immune cell modulation [34e36] (Fig. 17.1).
The miR-221/222 family The miR-221/222 gene cluster in humans is located in chromosome Xp11.3; genes in this cluster share an almost identical seed sequence. In terms of biogenesis, they are first transcribed as a single long noncoding RNA precursor;
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then, in the nucleus, the common pri-miR-221/222 transcript is spliced and split by the Drosha-DGCR8 complex, resulting in the formation of two individual precursors: premiR-221 and pre-miR-222. After the final processing steps, mature transcripts are formed: in particular, miR-221-5p or miR-221-3p (23 nucleotides), and possibly also miR-2225p and miR-222-3p (21 nucleotides) [37,38]. These miRNAs are upregulated in obese individuals, and related to metabolic processes involved in obesity [39]. Both miR-221 and miR-222 are involved in adipogenesis, a process through which preadipocytes differentiate into mature adipocytes. In primary human bone marrow stromal cells (BMSCs), these miRNAs downregulate adipogenesis, by reducing expression of peroxisome proliferator-activated receptor gamma (PPARã) and CCAAT/enhancer-binding protein alpha (CEBPá) [40]. Expression of miR-221 in WAT is different in obese and lean people [41]. miR-221 expression evaluated in abdominal subcutaneous adipose tissue was shown to positively correlate with body mass index (BMI), in a nondiabetic Indian population. This miRNA was found to be highly expressed in abdominal subcutaneous adipose tissue, and upregulated in individuals with obesity (BMI >37 kg/m2) [42]. In addition, miR-221 expression was found to be upregulated in samples of human subcutaneous fat, from obese women with T2D [43]. Both miR-221 and miR-222 play an important role in the process of chronic low-grade inflammation, through inhibition of endothelial cell proliferation and angiogenesis, which
FIGURE 17.1 Mechanisms of some microRNAs in (A) adipogenesis, visceral WAT expansion, and hypoxia; (B) molecular pathways of insulin signaling and NF-kB activation. (C) Plasma profile of miRNAs in obese people: Y low expression, [ high expression. AKT, Protein kinase B; GLUT4, glucose transporter member 4; IR, insulin receptor; IRS-1, insulin receptor substrate 1; NF-êB, nuclear factor-kappa B; PI3K, activation of phosphoinositide 3-kinase; TNF-á, tumor necrosis factor.
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contributes to tissue hypoxia. This can impact the production of proinflammatory adipokines and other miRNAs such as miR-27, involved in insulin resistance (IR) [39]. It has been demonstrated that miR-221 plays a relevant role in TNF-á signaling pathway, since it can bind directly to the cytokine’s 30 -UTR, and result in its mRNA degradation. In human adipose-derived stem cells (hASCs), hASC-differentiated adipocytes transfected with pre-miR221 showed 50% reduction in mRNA TNF-á. In addition, pre-miR-221 treatment resulted in a small reduction (w10%) in MCP-1 mRNA levels, and had no effect on IL6 mRNA levels. These results indicate that if miR-221 is overexpressed, TNF-á expression in adipocytes decreases. miR-221 might be helpful in attenuating chronic inflammation in obese women [44]. Some evidence suggests that miR-222 can also affect glucose metabolism. Plasma miR-222 levels have been shown to be higher in patients with T2D, than in those with normal glucose tolerance. When only obese individuals were evaluated, miR-222 plasma concentration increased in the group with T2D. Plasma miR-222 levels were also found to positively correlate with fasting glycemia, and glycated hemoglobin (HBA1c) levels [45]. In women diagnosed with metabolic syndrome, high miR-221 serum levels were observed, and no correlation was found between them and cardiometabolic risk factors. However, upregulation of miR-221/222 in blood vessels of individuals with metabolic syndrome, obesity, or T2D, for example, increases cardiovascular risk, and contributes to the development of atherosclerosis by endothelial dysfunction and neointimal hyperplasia [37]. In samples of human atherosclerotic vessels obtained from patients undergoing coronary bypass graft procedure, miR-221/222 levels were elevated in the intima, and posed a risk of endothelial dysfunction, by suppressing peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1á), thus contributing to progression of atherosclerosis [46].
Multifunctional miR-155 miR-155 is a multifunctional miRNA: it has been associated with the regulation of different immune-related processes, such as hematopoiesis [47], innate immunity [48], B-cell and T-cell differentiation [49], and cancer [50]. It is one of the most studied miRNAs involved in obesity, since it plays a role in adipogenesis, adipocyte function, and inflammation [51e54]. The induction of miR-155 expression is mediated by TNF-a in adipocytes and in WAT, which explains the role of this miRNA in obesity-mediated inflammation [52]. It was recently demonstrated that adipose tissue macrophages can secrete exosomal miR-155 molecules, which are then efficiently transported into adipocytes [55]. Similarly, in vivo study has shown that transgenic mice overexpressing
miR-155 in the B cell lineage produced more TNF-a when challenged with LPSs [56]. In a culture of human adipocytes, miR-155 was found to be substantially upregulated by TNF-a and induced inflammation, chemokine expression, and macrophage migration [52]. These results corroborate findings, whereby miR-155 levels in the adipose tissue of TNF-a knockout mice were lower, than in wild-type mice [52]. Overexpression of miR-155 has been found to significantly reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes and L6 muscle cells, as well as expression of miR-155 target gene PPARg. Furthermore, miR-155 overexpression appears to lead to a decrease in insulininduced phosphorylation of protein kinase B (AKT) in adipocytes, myocytes, and hepatocytes [55]. In addition, miR-155 knockout mice on high-fat diet, exhibit significant weight gain compared to wild-type mice, suggesting that there is a potential miRNA-based mechanism, contributing to the development of diet-induced obesity [57]. Obese subjects display upregulation of miR-155 in adipose tissue, presenting a significant positive correlation between miR-155 and BMI, and between miR-155 and mRNA coding for TNF-a [52]. miR-155 deletion may be promising in protecting against obesity, since it may regulate the development and persistence of obesity via several signaling pathways, including adipogenesis and inflammation. miR-155 has been implicated in adipocyte differentiation toward a white, rather than a brown/beige, phenotype. Brown adipocytes are key sites of energy expenditure; therefore, a brown adipocytelike phenotype may increase energetic efficiency in mammals. Finally, miR-155 may influence adipose tissue accumulation, by activating proinflammatory pathways [58].
miR-145 miR-145 is a member of the miR-143/145 cluster, and the most abundant miRNA in the vascular wall. Due to its positive correlation with high-sensitive C-reactive protein (hs-CRP) levels in acute ischemic stroke (AIS) patients, it is suggested that this miRNA might be a possible biomarker of the beginning and severity of AIS [59,60]. Furthermore, miR-145 is involved in lipid metabolism and inflammatory pathways in a tissue-specific manner. In WAT, higher expression of both TNF-a and IL-6 was observed, when this miR was downregulated. Decrease in miR-145 levels upregulated expression of ADP-ribosylation factor 6 (ARF6), a small GTPase responsible for activating the NF-kB-mediated inflammatory pathway in macrophages [61]. However, certain studies have yielded different results: in particular, overexpression of miR-145 has been found to increase proinflammatory mediators such as TNF-alpha, IL1-beta,
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CCL-2, CCL-3, and CCL-4 in macrophages, by upregulating NF-kB expression and activity [62]. A negative correlation between miR-145 and leptin receptor (LEPR) expression has been found in morbidly obese patients [63]. Obese individuals have lower miR-145 levels than lean individuals, which could be related to the seemingly paradoxical increase in plasma concentration of leptin (an anorexigenic hormone), and LEPR resistance in this phenotype [64].
miR-27a/b The miR-27 family includes two isoforms: miR-27a, an intergenic miRNA, and miR27b, an intronic miRNA, located within the 14th intron of the human C9orf3 host gene. These miRNAs are homologous and conserved in mammals during evolution [65]. miR-27 has been found to be upregulated in the omental adipose multipotent stem cells of obese human adults [66]. Evidence has shown that miR-27 is a negative regulator of adipocyte differentiation and obesity [67,68]. miR-27a is highly expressed in the stromal vascular fraction (SVF) of murine adipose tissues, and it is involved in adipocyte differentiation, by targeting PPARã 30 -UTR. Overexpression of miR-27a has been found to suppress PPARã expression, and adipocyte differentiation in 3T3-L1 preadipocyte cells [69]. miR-27a and miR-27b are downregulated in response to hydrogen peroxide (H2O2) [65], a reactive oxygen species that contributes to oxidative stress, and could also contribute to persistence of inflammation, and development of atherosclerosis. In RAW 264.7 cells transfected with miR-27b, translocation of the p65 subunit of NF-êB appears to be affected. However, miR-27b overexpression does not appear to modify mRNA expression of its target proteins, such as IL-1â, IL-6, and TNF-á. A reduction in CCl-2 mRNA expression has been observed in cells, transfected with miR-27b mimics [65,70]. A study performed in vivo with adipose tissue samples of hyperglycemic rats, and in vitro with 3T3-L1 adipocyte cells exposed to high glucose concentrations, verified an upregulation of miR-27a. Results suggest that this miRNA plays an important role in pathogenesis of T2D [71]. Moreover, miR-27a/b has been related to lipid metabolism through the repression of certain genes, such as sterol regulatory element-binding transcription factor 1c (SREBP1c), retinoid X receptor alpha (RXR-á), adiponectin, PPARã, glucose transporter member 4 (GLUT-4), fatty acid-binding protein 4 (FABP-4), and FASN [68]. miR-27b also targets peroxisome proliferator-activated receptor a (PPARá), which is involved in lipid metabolism through the regulation of genes such as ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette subfamily G member 1 (ABCG1). Overexpression
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of miR-27 has been shown to result in downregulation of PPARá [65]. miR-27b expression in human hepatic Huh7 cells can affect ABCA1 protein levels and cholesterol efflux to apolipoprotein A1 (ApoA1). In vivo, overexpression of pre-miR-27b in mice liver samples was found to reduce hepatic expression of ABCA1 by 50%, and low-density lipoprotein receptor (LDLR) by 20% without changing blood cholesterol and triglyceride levels [72].
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MicroRNAs and inflammation biomarkers in obesity Bruna Jardim Quintanilha1, 2, Bruna Zavarize Reis3, Telma A. Faraldo Correˆa2, 3, Graziela Biude da Silva Duarte3 and Marcelo Macedo Rogero1, 2 1
Department of Nutrition, School of Public Health, University of São Paulo, São Paulo, Brazil; 2Food Research Center (FoRC), CEPID-FAPESP,
Research Innovation and Dissemination Centers São Paulo Research Foundation, São Paulo, Brazil; 3Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Science, University of São Paulo, São Paulo, Brazil
Introduction
Micro RNAs
Obesity, a multifactorial and polygenic condition, is a public health issue of great concern in both developed and developing countries. It is estimated that in 2016 alone there were more than 1.9 billion overweight adults worldwide, that is, 39% of the world’s adult population; of these, more than 650 million (approximately 13% of the world’s adult population) were obese. Obesity also affects children: an estimated 41 million people under 5 years of age, as well as 340 million people aged 5e19 years, were either overweight or obese in 2016 [1]. Obesity results from the interaction of a series of genetic, metabolic, behavioral, and environmental factors. It leads to metabolic inflammation or meta-inflammation, a chronic form of low-grade inflammation in white adipose tissue (WAT) that differs from a classic inflammatory response [2]. Two proteins play a key role in metainflammation: inhibitor of nuclear factor kappa-B kinase (IKK-b), and c-Jun N-terminal kinase 1 (JNK-1), whose activation in turn activates nuclear factor kappa B (NF-kB) and activator protein 1 (AP-1), respectively. NF-kB and AP-1 then translocate to the cell nucleus and activate the transcription of genes related to inflammation, such as tumor necrosis factor alpha (TNF-a), interleukin 6 (IL-6), and interleukin 1 beta (IL-1b) [3]. Adipocytes and infiltrating inflammatory cellsdmainly macrophagesdsecrete cytokines such as TNF-a, IL-6, and IL-1b, and inflammatory modulators such as leptin, resistin, and adiponectin [2,4]. Obesity is strongly associated with metabolic diseases such as type 2 diabetes (T2D), atherosclerosis, cardiovascular disease (CVD), nonalcoholic fatty liver disease (NAFLD), and some cancers [5].
MicroRNAs (miRNAs) play a key role in obesity, since they can posttranscriptionally regulate a large number of genes. They may also be important in maintaining metabolic homeostasis; if so, their regulation could serve as potential therapeutics for metabolic diseases [6]. MicroRNAs are noncoding RNAs that modulate gene expression. Mature miRNAs regulate the expression of proteins, by cleaving mRNA or repressing its translation, depending on the level of complementarity between the miRNA and the target mRNA [7]. Changes in miRNA levels have been shown to affect gene expression and thereby cell function, in several pathophysiological disorders related to obesity, including inflammation, oxidative stress, impaired adipogenesis, insulin signaling, apoptosis, and angiogenesis [8e12]. MicroRNAs are present in tissues or body fluids in stable form, protected from endogenous RNAse activity [6]. They may play a role in communication between adipocytes, as well as between adipose tissue and other tissues. Notably, miRNAs can act as potential diagnostic biomarkers, as they fulfill most of the necessary criteria: they can be rapidly and accurately detected through noninvasive methods, have high sensitivity and specificity to the disease in question, allow for early detection, and have a long halflife in the sample [13e15]. The identification and characterization of miRNAs associated with obesity could yield a new generation of therapeutic targets for antiobesity treatments. Additionally, identifying miRNAs that are dysregulated during the development of obesity could provide early obesity biomarkers for clinical diagnosis.
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MicroRNAs concepts MicroRNAs are a class of small, noncoding endogenous RNA molecules (w18e25 nucleotides) involved in posttranscriptional gene regulation, by binding to a target messenger RNA (mRNA), which results in the degradation or inhibition of translation [16]. To date, 2654 mature human miRNAs have been identified (MiRBase, release 16, December 2018; http://www.mirbase.org). MicroRNAs can act directly on the target genes or indirectly, by regulating transcription factors that in turn control the expression of genes [4]. One miRNA can regulate several mRNAs, but not all miRNAs incur translational repression. Some of them have the ability to activate the translation of proteins, alter the structure of chromatin by regulating histone modification, and even target genes with low DNA methylation directly [3,16].
Biogenesis Biogenesis of miRNAs is a sequential process involving a variety of enzymes and proteins [16]. MicroRNAs are usually transcribed by RNA polymerase II from miRNA genes, first forming the primary miRNA transcript (pri-miRNA). This transcript is then cleaved by the DROSHA-DiGeorge syndrome critical region gene 8 (DGCR8) microprocessor complex, creating a shorter sequence called the miRNA precursor (pre-miRNA) that displays a hairpinlike secondary structure. The pre-miRNA is exported to the cytoplasm and processed by DICER, a ribonuclease III enzyme that produces mature miRNA, which is incorporated into an RNA-protein complex (i.e., the RNA-induced silencing complex, RISC). Under most conditions, mature RISC represses gene expression posttranscriptionally, by binding the three prime untranslated regions (30 -UTRs) of specific mRNAs, and mediating mRNA degradation, destabilization, or translational inhibition, according to sequence complementarity to the target [3,17e19]. Evidence shows that, besides intracellular function, miRNAs are present in extracellular fluids in the human body, including plasma, serum, urine, and saliva; recently, they have also been associated with diseases such as obesity, cancer, and cardiovascular disease (CVD) [20e22]. MicroRNAs also play an important role in cell-tocell communication in peripheral blood, either through membrane-enclosed vesicles such as exosomes (extracellular vesicles of endosomal origin), or by binding to lipoproteins (LDL or HDL), proteins, apoptotic bodies, and ribonucleoprotein complexes (linked to Argonaut) [21,22]. In addition to their stability, it should be noted that circulating miRNAs are conserved across species, have expression patterns that are tissue- and biological-stage specific, and are easily determined through real-time
polymerase chain reaction (RT-PCR) [16]. Thus, these molecules are promising noninvasive biomarkers of certain diseases, and even of nutritional status [23].
Inflammation and miRNAs: a role in chronic diseases Obesity is related to endocrine and metabolic changes in WAT, which is the main source of systemic inflammatory response. White adipose tissue produces a variety of proinflammatory cytokines and chemokines known as adipokines, which include TNF-á, IL-1â, IL-6, and monocyte chemotactic protein (MCP-1). Consequently, obese individuals present an increase in proinflammatory biomarkers such as TNF-á, IL-1â, IL-6, and MCP-1, as well as a decrease in antiinflammatory adipokines such as adiponectin [24e26]. In addition, activation of endothelial cells and oxidative stress exacerbate the inflammatory response [24,27,28]. Two important signaling inflammatory pathways are involved in these processes: the NF-êB and JNK pathways. The former can be activated by TNF-á, lipopolysaccharides (LPSs), and saturated fatty acids; it involves the enzymatic complex IKK, which induces the phosphorylation of inhibitor-êB (IêB). This phosphorylation results in IêB polyubiquitination, which, in turn, leads to IêB degradation, as mediated by the 26S proteasome. This degradation allows NF-êB to translocate to the nucleus, and activate the transcription of several êB-dependent genes such as those encoding proinflammatory cytokines, adhesion molecules, and chemokines. The JNK signaling pathway can be activated by cytokines, fatty acids, and reactive oxygen species (ROS), among others. The active JNK pathway promotes the activation of transcription factor AP-1, which is related to gene expression of proinflammatory cytokines, and may act directly on the insulin signaling pathway. In this context, an increase in inflammatory response may trigger resistance to insulin action due to an increase in Ser307 phosphorylation of insulin receptor substrate 1 (IRS-1), and lower activity of phosphatidylinositol-4,5-bisphosphate-3-kinase (PI3K) in skeletal muscle. It should be noted that Ser307 phosphorylation of IRS-1 is associated with activation of JNK and IKK-â. In addition, activation of IL-6 signaling can increase the expression of cytokine signaling suppressor proteins SOCS-1 and SOCS3, in the three major insulin-sensitive peripheral tissues: WAT, liver tissue, and muscle tissue. SOCS-1 and SOCS-3 impair insulin action, by binding to IRS-1 and IRS-2, which leads to IRS-1 and IRS-2 ubiquitination and degradation [2,29e32]. There is substantial evidence of miRNAs’ transcriptionallevel regulation of genes encoding proteins related to the
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inflammatory process, in metabolic diseases [33]. Regulation may occur on many levels. Inflammation may induce the transcriptional process; if so, miRNAs will become rapidly active, since they do not need to be translated or translocated back into the nucleus, to repress their target. The expression of miRNAs occurs in different cell types with different functions, and some target proteinsd for example, proteins regulating miRNA processingd may be linked to the inflammatory response. MicroRNAs can also regulate several mechanisms related to inflammation, such as adipogenesis, macrophage activation, and oxidative stress [8].
MicroRNAs as biomarkers in obesity MicroRNAs found in human body fluids such as urine and plasma, are called circulating miRNAs; they act not only within cells but also in distant tissues, as hormones controlling gene expression. The physiological function of circulating miRNAs is mostly unknown, but different studies have shown that these molecules have essential roles, including immune cell modulation [34e36] (Fig. 17.1).
The miR-221/222 family The miR-221/222 gene cluster in humans is located in chromosome Xp11.3; genes in this cluster share an almost identical seed sequence. In terms of biogenesis, they are first transcribed as a single long noncoding RNA precursor;
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then, in the nucleus, the common pri-miR-221/222 transcript is spliced and split by the Drosha-DGCR8 complex, resulting in the formation of two individual precursors: premiR-221 and pre-miR-222. After the final processing steps, mature transcripts are formed: in particular, miR-221-5p or miR-221-3p (23 nucleotides), and possibly also miR-2225p and miR-222-3p (21 nucleotides) [37,38]. These miRNAs are upregulated in obese individuals, and related to metabolic processes involved in obesity [39]. Both miR-221 and miR-222 are involved in adipogenesis, a process through which preadipocytes differentiate into mature adipocytes. In primary human bone marrow stromal cells (BMSCs), these miRNAs downregulate adipogenesis, by reducing expression of peroxisome proliferator-activated receptor gamma (PPARã) and CCAAT/enhancer-binding protein alpha (CEBPá) [40]. Expression of miR-221 in WAT is different in obese and lean people [41]. miR-221 expression evaluated in abdominal subcutaneous adipose tissue was shown to positively correlate with body mass index (BMI), in a nondiabetic Indian population. This miRNA was found to be highly expressed in abdominal subcutaneous adipose tissue, and upregulated in individuals with obesity (BMI >37 kg/m2) [42]. In addition, miR-221 expression was found to be upregulated in samples of human subcutaneous fat, from obese women with T2D [43]. Both miR-221 and miR-222 play an important role in the process of chronic low-grade inflammation, through inhibition of endothelial cell proliferation and angiogenesis, which
FIGURE 17.1 Mechanisms of some microRNAs in (A) adipogenesis, visceral WAT expansion, and hypoxia; (B) molecular pathways of insulin signaling and NF-kB activation. (C) Plasma profile of miRNAs in obese people: Y low expression, [ high expression. AKT, Protein kinase B; GLUT4, glucose transporter member 4; IR, insulin receptor; IRS-1, insulin receptor substrate 1; NF-êB, nuclear factor-kappa B; PI3K, activation of phosphoinositide 3-kinase; TNF-á, tumor necrosis factor.
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contributes to tissue hypoxia. This can impact the production of proinflammatory adipokines and other miRNAs such as miR-27, involved in insulin resistance (IR) [39]. It has been demonstrated that miR-221 plays a relevant role in TNF-á signaling pathway, since it can bind directly to the cytokine’s 30 -UTR, and result in its mRNA degradation. In human adipose-derived stem cells (hASCs), hASC-differentiated adipocytes transfected with pre-miR221 showed 50% reduction in mRNA TNF-á. In addition, pre-miR-221 treatment resulted in a small reduction (w10%) in MCP-1 mRNA levels, and had no effect on IL6 mRNA levels. These results indicate that if miR-221 is overexpressed, TNF-á expression in adipocytes decreases. miR-221 might be helpful in attenuating chronic inflammation in obese women [44]. Some evidence suggests that miR-222 can also affect glucose metabolism. Plasma miR-222 levels have been shown to be higher in patients with T2D, than in those with normal glucose tolerance. When only obese individuals were evaluated, miR-222 plasma concentration increased in the group with T2D. Plasma miR-222 levels were also found to positively correlate with fasting glycemia, and glycated hemoglobin (HBA1c) levels [45]. In women diagnosed with metabolic syndrome, high miR-221 serum levels were observed, and no correlation was found between them and cardiometabolic risk factors. However, upregulation of miR-221/222 in blood vessels of individuals with metabolic syndrome, obesity, or T2D, for example, increases cardiovascular risk, and contributes to the development of atherosclerosis by endothelial dysfunction and neointimal hyperplasia [37]. In samples of human atherosclerotic vessels obtained from patients undergoing coronary bypass graft procedure, miR-221/222 levels were elevated in the intima, and posed a risk of endothelial dysfunction, by suppressing peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1á), thus contributing to progression of atherosclerosis [46].
Multifunctional miR-155 miR-155 is a multifunctional miRNA: it has been associated with the regulation of different immune-related processes, such as hematopoiesis [47], innate immunity [48], B-cell and T-cell differentiation [49], and cancer [50]. It is one of the most studied miRNAs involved in obesity, since it plays a role in adipogenesis, adipocyte function, and inflammation [51e54]. The induction of miR-155 expression is mediated by TNF-a in adipocytes and in WAT, which explains the role of this miRNA in obesity-mediated inflammation [52]. It was recently demonstrated that adipose tissue macrophages can secrete exosomal miR-155 molecules, which are then efficiently transported into adipocytes [55]. Similarly, in vivo study has shown that transgenic mice overexpressing
miR-155 in the B cell lineage produced more TNF-a when challenged with LPSs [56]. In a culture of human adipocytes, miR-155 was found to be substantially upregulated by TNF-a and induced inflammation, chemokine expression, and macrophage migration [52]. These results corroborate findings, whereby miR-155 levels in the adipose tissue of TNF-a knockout mice were lower, than in wild-type mice [52]. Overexpression of miR-155 has been found to significantly reduce insulin-stimulated glucose uptake in 3T3-L1 adipocytes and L6 muscle cells, as well as expression of miR-155 target gene PPARg. Furthermore, miR-155 overexpression appears to lead to a decrease in insulininduced phosphorylation of protein kinase B (AKT) in adipocytes, myocytes, and hepatocytes [55]. In addition, miR-155 knockout mice on high-fat diet, exhibit significant weight gain compared to wild-type mice, suggesting that there is a potential miRNA-based mechanism, contributing to the development of diet-induced obesity [57]. Obese subjects display upregulation of miR-155 in adipose tissue, presenting a significant positive correlation between miR-155 and BMI, and between miR-155 and mRNA coding for TNF-a [52]. miR-155 deletion may be promising in protecting against obesity, since it may regulate the development and persistence of obesity via several signaling pathways, including adipogenesis and inflammation. miR-155 has been implicated in adipocyte differentiation toward a white, rather than a brown/beige, phenotype. Brown adipocytes are key sites of energy expenditure; therefore, a brown adipocytelike phenotype may increase energetic efficiency in mammals. Finally, miR-155 may influence adipose tissue accumulation, by activating proinflammatory pathways [58].
miR-145 miR-145 is a member of the miR-143/145 cluster, and the most abundant miRNA in the vascular wall. Due to its positive correlation with high-sensitive C-reactive protein (hs-CRP) levels in acute ischemic stroke (AIS) patients, it is suggested that this miRNA might be a possible biomarker of the beginning and severity of AIS [59,60]. Furthermore, miR-145 is involved in lipid metabolism and inflammatory pathways in a tissue-specific manner. In WAT, higher expression of both TNF-a and IL-6 was observed, when this miR was downregulated. Decrease in miR-145 levels upregulated expression of ADP-ribosylation factor 6 (ARF6), a small GTPase responsible for activating the NF-kB-mediated inflammatory pathway in macrophages [61]. However, certain studies have yielded different results: in particular, overexpression of miR-145 has been found to increase proinflammatory mediators such as TNF-alpha, IL1-beta,
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CCL-2, CCL-3, and CCL-4 in macrophages, by upregulating NF-kB expression and activity [62]. A negative correlation between miR-145 and leptin receptor (LEPR) expression has been found in morbidly obese patients [63]. Obese individuals have lower miR-145 levels than lean individuals, which could be related to the seemingly paradoxical increase in plasma concentration of leptin (an anorexigenic hormone), and LEPR resistance in this phenotype [64].
miR-27a/b The miR-27 family includes two isoforms: miR-27a, an intergenic miRNA, and miR27b, an intronic miRNA, located within the 14th intron of the human C9orf3 host gene. These miRNAs are homologous and conserved in mammals during evolution [65]. miR-27 has been found to be upregulated in the omental adipose multipotent stem cells of obese human adults [66]. Evidence has shown that miR-27 is a negative regulator of adipocyte differentiation and obesity [67,68]. miR-27a is highly expressed in the stromal vascular fraction (SVF) of murine adipose tissues, and it is involved in adipocyte differentiation, by targeting PPARã 30 -UTR. Overexpression of miR-27a has been found to suppress PPARã expression, and adipocyte differentiation in 3T3-L1 preadipocyte cells [69]. miR-27a and miR-27b are downregulated in response to hydrogen peroxide (H2O2) [65], a reactive oxygen species that contributes to oxidative stress, and could also contribute to persistence of inflammation, and development of atherosclerosis. In RAW 264.7 cells transfected with miR-27b, translocation of the p65 subunit of NF-êB appears to be affected. However, miR-27b overexpression does not appear to modify mRNA expression of its target proteins, such as IL-1â, IL-6, and TNF-á. A reduction in CCl-2 mRNA expression has been observed in cells, transfected with miR-27b mimics [65,70]. A study performed in vivo with adipose tissue samples of hyperglycemic rats, and in vitro with 3T3-L1 adipocyte cells exposed to high glucose concentrations, verified an upregulation of miR-27a. Results suggest that this miRNA plays an important role in pathogenesis of T2D [71]. Moreover, miR-27a/b has been related to lipid metabolism through the repression of certain genes, such as sterol regulatory element-binding transcription factor 1c (SREBP1c), retinoid X receptor alpha (RXR-á), adiponectin, PPARã, glucose transporter member 4 (GLUT-4), fatty acid-binding protein 4 (FABP-4), and FASN [68]. miR-27b also targets peroxisome proliferator-activated receptor a (PPARá), which is involved in lipid metabolism through the regulation of genes such as ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette subfamily G member 1 (ABCG1). Overexpression
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of miR-27 has been shown to result in downregulation of PPARá [65]. miR-27b expression in human hepatic Huh7 cells can affect ABCA1 protein levels and cholesterol efflux to apolipoprotein A1 (ApoA1). In vivo, overexpression of pre-miR-27b in mice liver samples was found to reduce hepatic expression of ABCA1 by 50%, and low-density lipoprotein receptor (LDLR) by 20% without changing blood cholesterol and triglyceride levels [72].
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