Translating MicroRNAs to the Clinic

Translating MicroRNAs to the Clinic

Editor

Jeffrey Laurence

Division of Hematology-Medical Oncology, Weill Cornell Medical College and New York Presbyterian Hospital, New York, NY, United States

Guest Editor

Mary Van Beusekom

Health Partners Institute for Education and Research and Synapse Writing and Editing, Exelsior, MN, United States

Table of Contents

Cover image

Title page

Copyright

List of Contributors

Chapter 1. MicroRNAs: Mirrors of Health and Disease

1. Introduction

2. Knocking Out MicroRNA: Small RNAs With Big Effects

3. Genetic Organization, Variation in MicroRNAs, and Tissue-Specific Expression of MicroRNAs

4. MicroRNA Regulatory Complexity: Feedback Loops and Environmental Sensing

5. Organ Diseases and MicroRNAs

6. Cancer and MicroRNAs

7. Aging and MicroRNAs

8. MicroRNA Therapeutics and Target Prediction: Successes and Challenges

9. Circulating MicroRNA as Biomarkers

10. Concluding Remarks

Chapter 2. Clinical and Therapeutic Applications of MicroRNA in Cancer

1. Introduction

2. MicroRNA Processing

3. Circulating MicroRNA as Biomarkers

4. MicroRNA Isolation and Amplification Methodologies

5. Packaging and Delivery Mechanisms

6. MicroRNA Dysregulation in Cancer

7. Role of MicroRNA in Cancer Diagnosis, Prognostication, and Treatment

8. MicroRNA in Clinical Trials

9. Future Directions for MicroRNA Research

List of Acronyms and Abbreviations

Chapter 3. MicroRNAs in Kidney Function and Disease

1. Introduction

2. Kidney Physiology

3. Blood Pressure Regulation

4. Kidney Disease

5. Kidney Transplantation

6. Biomarkers

7. Clinical Implications

8. Summary

Chapter 4. MicroRNAs in the Pathogenesis, Diagnosis, and Treatment of Liver Disease

1. Introduction

2. MicroRNA Biogenesis

3. MicroRNA Analysis and Bioinformatics

4. Hepatic MicroRNAs as Metabolic Modulators and Their Importance in NAFLD

5. MicroRNAs and Hepatitis C Virus

6. MicroRNAs and Hepatitis B Virus

7. MicroRNAs and Hepatocellular Carcinoma

8. MicroRNAs as Biomarkers of Liver Injury

List of Acronyms and Abbreviations

Chapter 5. Clinical Application of MicroRNAs in Liver Diseases

1. Introduction

2. Role of MicroRNAs in Liver Pathology and Pathophysiology

3. MicroRNAs as Diagnostic and Prognostic Markers in Liver Disease

4. MicroRNA-Based Therapeutics for Liver Disease

List of Acronyms and Abbreviations

Chapter 6. MicroRNAs in Inflammatory Lung Disease

1. Introduction

2. MicroRNAs in Asthma

3. Acute Respiratory Distress Syndrome

4. Chronic Obstructive Pulmonary Disease

5. Cystic Fibrosis

6. Sarcoidosis

7. Summary and Conclusion

Chapter 7. MicroRNAs in Idiopathic Pulmonary Fibrosis: Partners in Health and Disease

1. MicroRNAs

2. Idiopathic Pulmonary Fibrosis

3. MicroRNA Studies in Idiopathic Pulmonary Fibrosis

4. Laboratory Techniques

5. Limitations

6. MicroRNA Therapeutics

7. MicroRNA–Long Noncoding RNA Interaction

8. Conclusions and Future Directions

List of Acronyms and Abbreviations

Chapter 8. MicroRNA as Biomarkers of Malignant Mesothelioma

1. Clinical Problem: Mesothelioma

2. Molecular Pathology

3. MicroRNAs as Oncogenes and Tumor Suppressors

4. Translational Applications of MicroRNA in Mesothelioma

5. Conclusions and Future Directions

List of Acronyms and Abbreviations

Chapter 9. MicroRNA, an Important Epigenetic Regulator of Immunity and Autoimmunity

1. Introduction

2. MicroRNA Biogenesis

3. MicroRNA in Normal Immune System Development and Function

4. MicroRNA in the Development and Prevention of Autoimmunity

5. MicroRNA in Systemic Lupus Erythematosus

6. Cell-Free Circulating MicroRNAs as Biomarkers in Systemic Lupus Erythematosus

7. Regulation of MicroRNA Expression in Systemic Lupus Erythematosus

8. Conclusion and Perspective

Glossary

List of Acronyms and Abbreviations

Chapter 10. MicroRNA-Linked Heart Disease and Therapeutic Potential

1. Introduction

2. Cardiac Hypertrophy

3. Cardiac Fibrosis

4. Myocardial Ischemia and Cell Death

5. Vascular Diseases

6. Heart Failure

7. Circulating MicroRNAs

8. Therapeutic Strategies to Modulate the Functions of MicroRNAs

9. Summary and Conclusions

List of Acronyms and Abbreviations

Chapter 11. The Clinical Potential of Heart Failure–Related miRNAs

1. Introduction

2. MicroRNAs as Therapeutics

3. MicroRNAs in Cardiac Remodeling

4. Circulating MicroRNAs as Biomarker

5. Conclusions and Future Directions

Glossary

List of Acronyms and Abbreviations

Chapter 12. The Role of Noncoding RNAs in Prostate Cancer

1. Introduction

2. Prostate Cancer and MicroRNAs

3. MicroRNAs as Prostate Cancer Biomarkers

4. Conclusions and Future Directions for MicroRNAs in the Clinic

List of Acronyms and Abbreviations

Index

Copyright

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List of Contributors

S. Akkina,     University of Illinois at Chicago, Chicago, IL, United States

S. Ansar Ahmed,     Virginia Tech, Blacksburg, VA, United States

B.N. Becker,     University of Chicago, Chicago, IL, United States

C. Bime,     The University of Arizona, Tucson, AZ, United States

D.M. Brown,     Ohio State University Medical Center, Columbus, OH, United States

E.E. Creemers,     University of Amsterdam, Amsterdam, The Netherlands

C. Croce,     Ohio State University Medical Center, Columbus, OH, United States

R. Dai,     Virginia Tech, Blacksburg, VA, United States

A.A. Desai,     The University of Arizona, Tucson, AZ, United States

A. Esquela-Kerscher,     Eastern Virginia Medical School, Norfolk, VA, United states

J.G.N. Garcia,     The University of Arizona, Tucson, AZ, United States

C.I. Gurguis,     The University of Arizona, Tucson, AZ, United States

T. Hasegawa,     Eastern Virginia Medical School, Norfolk, VA, United states

L. Hecker,     The University of Arizona, Tucson, AZ, United States

C.D. Hoang,     National Institutes of Health, Bethesda, MD, United States

N. Kaminski,     Yale University, New Haven, CT, United States

T.A. Kerr,     University of Texas Southwestern Medical Center, Dallas, TX, United States

R.A. Kratzke,     University of Minnesota, Minneapolis, MN, United States

H. Lewis,     Eastern Virginia Medical School, Norfolk, VA, United states

B.B. Madison,     Washington University, Saint Louis, MO, United States

M.A. Montano,     Harvard Medical School, Boston, MA, United States

S.P. Nana-Sinkam,     Ohio State University Medical Center, Columbus, OH, United States

K. Ono,     Kyoto University, Kyoto, Japan

K.V. Pandit,     University of Pittsburgh, Pittsburgh, PA, United States

Y.M. Pinto,     University of Amsterdam, Amsterdam, The Netherlands

G. Song,     University of Minnesota, Minneapolis, MN, United States

A.J. Tijsen

Technion-Israel Institute of Technology, Haifa, Israel

University of Amsterdam, Amsterdam, The Netherlands

T.H. Tran,     Harvard Medical School, Boston, MA, United States

H. Vollbrecht,     University of Minnesota, Minneapolis, MN, United States

T. Wang,     The University of Arizona, Tucson, AZ, United States

Chapter 1

MicroRNAs

Mirrors of Health and Disease

T.H. Tran,  and M.A. Montano

Abstract

In this introductory chapter, we will discuss examples of tissue and cell type–specific knockdown of components of the microRNA machinery and their deleterious effects. We discuss the genetic organization of microRNAs and how that architecture is designed to regulate multiple targets within regulatory pathways, and their role in adaptive regulation in response to changes in the microenvironment. We discuss microRNA networks in specific tissue and organ diseases, including lung, heart, and skeletal muscle, as well as the role for microRNAs in modulating cancers in those tissues. We introduce newer research-linking shifts in microRNA expression with biological aging and diseases associated with aging. We explore the potential role for microRNAs as therapeutic tools to modulate target RNA sequences implicated in disease states. Finally, we discuss the utility of microRNAs as circulating biomarkers for disease risk and severity, as well as therapeutic response to candidate drug interventions.

Keywords

Aging; Biomarkers; Cancer; Fibrosis; Inflammation; MicroRNA; MyomiR; OncomiR

Key Concepts

This chapter gives examples of tissue and cell type-specific knockdown of components of the microRNA machinery and their deleterious effects. It discusses the genetic organization of microRNAs and how that architecture is designed to regulate multiple targets within regulatory pathways, and their role in adaptive regulation in response to changes in the microenvironment. It looks at the role of MicroRNA networks in specific tissue and organ diseases, including lung, heart, and skeletal muscle, as well as in modulating cancers in those tissues. Newer research is introduced linking shifts in microRNA expression with biological aging and diseases associated with aging. The potential role for MicroRNAs as therapeutic tools to modulate target RNA sequences implicated in disease states is explored. Finally, the utility of MicroRNAs as circulating biomarkers for disease risk and severity, as well as in the therapeutic response to candidate drug interventions is discussed.

1. Introduction

Since their discovery as a novel class of small noncoding RNAs capable of regulating protein translation and/or messenger RNA (mRNA) stability, microRNAs (miRNAs) have been implicated as key regulators in the homeostasis of multiple biological systems that include organs such as heart, lung, and skeletal muscle, as well as modulation of the pathobiology processes such as cancer and aging. Experimental loss of key regulators of miRNA biogenesis has revealed the crucial role for the control of posttranscriptional gene expression. The genetic organization, the variability of their loci, and the tissue specificity of their expression further illustrate an adaptive capacity of the miRNA machinery to modify and fine-tune the transcriptome and translated protein targets in response to physiologic environmental cues, as well as their vulnerability to becoming co-opted in diseases such as cancer, fibrosis, sepsis, aging, and autoimmune disease. Current efforts to leverage knowledge of the miRNA regulatory system to diagnose, track, and attenuate disease progression, represent a major new research opportunity, and challenge in this rapidly growing area of translational medicine.

2. Knocking Out MicroRNA: Small RNAs With Big Effects

As our understanding of miRNA biogenesis and downstream regulatory activities unfold, so too does our appreciation for the extent and scope of influence these small RNAs have on multiple biological processes across human health and disease. The endonuclease Dicer is an obligatory first step in the RNA interference pathway and formation of the RNA-inducing silencing complex (RISC). Without RISC formation, argonaute-mediated degradation of target mRNA is lost [1,2]. Therefore, Dicer knockdown, in effect, disables the processing of miRNA from precursors of miRNA, ie, premiRNA. This can have deleterious effects. In murine models of cancer, global gene-disruption of Dicer can enhance tumor susceptibility [3]. Tissue- and cell type–specific knockdown of Dicer can also lead to deleterious effects. For example, cardiomyocyte-specific ablation of Dicer can result in cardiomyopathy [4]. Kidney podocyte-specific knockdown of Dicer can lead to systemic proteinuria and glomeruli abnormalities in mice [5]. Moreover, conditional knockdown of Dicer in the kidney impairs its normal development, further emphasizing the normal and dysregulatory potential in miRNA processing. Dicer deletion in skeletal muscle can result in increased myocyte apoptosis, skeletal muscle hypoplasia, and eventually perinatal death [6]. Hepatocytes obtained from Dicer-null mice exhibit abnormal lipid buildup and eventual renal steatosis [7]. Liver-specific Dicer deletion at 3 weeks leads to progressive hepatic steatosis [8]. This phenotype, however, has not been universally observed. While unlikely to change the overall conclusion that miRNAs clearly play a significant role in regulating multiple pathways, the subtleties or disparities in observation of distinct phenotypes in these conditional knockdowns may hinge upon the buildup of precursor miRNAs that activate other RNA surveillance pathways, such as RNA editing, which would then introduce an additional variable [9,10]. Comparative transcriptome profiling with high throughput RNA sequencing will likely provide more insight into the global regulatory profile of these knockdowns.

3. Genetic Organization, Variation in MicroRNAs, and Tissue-Specific Expression of MicroRNAs

MicroRNAs (miRNAs) undergo multiple processing events to reach their functional 21–23 ribonucleotide RNA sequence. Canonical miRNAs are generated from protein-coding transcriptional units; whereas, other miRNAs (ie, noncanonical miRNAs) are generated from nonprotein-coding transcriptional units. In both cases, the miRNAs can be located either within intronic or exonic regions. A noteworthy mechanistic distinction in canonical versus noncanonical miRNAs is that canonical intronic miRNAs are Drosha dependent and are thus processed cotranscriptionally with protein-coding transcripts in the nucleus. The premiRNA then enters the miRNA pathway, whereas the rest of the transcript undergoes premRNA splicing to produce mature mRNA which will then direct protein synthesis. Noncanonical intronic small RNAs (also called mirtrons) can derive from small introns that resemble premiRNAs, and bypass the Drosha-processing step [11]. miRNAs tend to be organized in a related cluster and also tend to target multiple mRNA transcripts within common cellular response pathways (eg, proliferation, apoptosis). This organizational thematic provides miR clusters with a capacity for coordinate regulation of multiple steps within a pathway, providing an opportunity for complex and adaptive regulatory control of entire pathways. An interesting class of miRNAs is myomiRs—so-called because they are coded within myosin heavy chain (MYH) genes. myomiRs are transcribed in the same precursor mRNA as the parental MYH gene [4]. Of special note is the myomiR-499, which despite the absence of a parent mRNA, is one of the most highly expressed miRNAs in heart tissue. In an apparently novel evolutionary phenomenon, alternative splicing in the heart uncouples production of mature miR-499 from expression of parent MYH7b mRNA, meaning that the mRNA has perhaps evolved into a nonfunctional host mRNA for its intronic miR (ie, miR-499).

Comparative studies evaluating the organizational structure of the mammalian genome have identified a wealth of chromosomal insertion–deletions, copy number variants, and single nucleotide polymorphisms (SNPs) that, depending on the environmental context, contribute to the genetic variation that can underlay phenotypic diversity. This diversity is evident in nearly every aspect of human health and disease that has been investigated. Perhaps not surprisingly, there is now a growing recognition that variation in miRNAs and their target genes also contribute to this phenotypic variability. Several solid and hematologic malignancies can be linked to miRNAs located at amplified, deleted, or translocated chromosomal regions in the mammalian genome [12]. Variation in gene expression and regulation is likely influenced by genetic variants in cis- and trans-acting SNPs (also known as expression quantitative trait loci) [13]. An interesting observation of miRNA binding is their ability to recognize binding site polymorphism (miRSNPs) in transcribed functional genes. For example, miR-24 appears to be deregulated in human colorectal tumor through a target site polymorphism in the dihydrofolate reductase gene. In another example, a polymorphism within the myostatin gene creates a target site for miR-1 and miR-206, which are highly expressed in skeletal muscle. The binding of these miRs to the polymorphism in myostatin causes translational inhibition of myostatin transcripts and can phenocopy the observed muscle hypertrophy that is observed with genetic knockouts of the myostatin gene [14]. Given the significant differences in gene expression and genetic variation across human populations, analysis of the role of miRNAs in contributing to population differences in gene expression is likely to provide substantial insights in population based health disparities and physical functionalities [13]. Indeed, comparative genomic studies indicate that the target mRNA sequences for miRNAs: untranslated regions (UTRs) on mRNAs often display sequence diversity. This may suggest adaptive evolution of coexpressed miRNAs and cognate mRNAs with these UTR variants. Depending on whether the dampening of protein output is beneficial, inconsequential, or harmful, the UTR sites may be selectively conserved, neutral, or avoided during miRNA:mRNA coevolution [1].

Studies evaluating the tissue-specific expression of miRNAs illustrate a cross-regulation feature of miRNAs that contributes to cell fate specification by repressing alternate cell fates to facilitate commitment to one cell fate and to maintain stability of a differentiated phenotype [15]. For example the myomiRs miR-1, miR-133, miR-206, miR-208, miR-486, and miR-499 are enriched in skeletal muscle and play crucial roles in the development, growth, and maintenance of skeletal muscle [16]. Notably, miR-133 prevents osteogenic cell lineage differentiation by repressing Runx2, a factor essential for bone formation [15]. MiR-7, miR-24, miR-128, miR-134, miR-219, and others are highly expressed in the mammalian brain and regulate neurite outgrowth, neuronal differentiation, and dendritic spine size [17]. Regionally enriched miRNAs in specific tissues can exert specialized function. For example, miR-7 and miR-24 are highly expressed in the hypothalamus and fine-tuning expression of Fos and oxytocin which play vital roles in the control of water, lactation, and parturition [18].

4. MicroRNA Regulatory Complexity: Feedback Loops and Environmental Sensing

Perhaps one of the most salient and unexpected features of miRNA is the role for miRNA in amplifying or tempering cell signaling. The modulatory capacity provides an adaptive tool that allows the initiation of cellular signaling to be calibrated to accommodate cues from the microenvironment, as well as to potentially buffer signaling. This buffering function has been proposed to function as a network stabilizing effect in the context of dynamic and interlocking feedback loops [19]. In the lung, miR-21 promotes transforming growth factor (TGF-β) amplification and fibrosis in a feed-forward loop by relieving the repression of the inhibitor Smad 7 through targeting the mRNA [20]. Interestingly, TGF-β both inhibits miR let-7 and upregulates miR-21. These two miRNAs appear to be functionally opposed in lung tissue from human subjects with idiopathic pulmonary lungs (let-7 acts as a negative regulator and miR-21 as a positive regulator) that in effect can balance the fibrotic phenotype. In the context of sepsis, monocyte expression of miR-146 and miR-155 are rapidly upregulated during endotoxin/LPS exposure, representing an innate response regulatory role for these miRNAs [11,13]. MiR-146a appears to function as a negative feedback regulator by targeting IL-1 receptor-associated kinase 1(IRAK-1) and TNF receptor-associated factor 6 (TRAF6). A reciprocal negative feedback is achieved through MAP kinase phosphatase-1 (MKP-1)-mediated suppression of miR-155, which in turn targets suppressor of cytokine signaling-1. Thus, MKP-1 appears to function as a derepressor of miR-155-mediated suppression to modulate LPS response in monocyte/macrophages. In the context of skeletal muscle, C2C12, a skeletal muscle precursor cell line, miR-1 targets mRNAs for the insulin growth factor-1 (IGF-1) and the IGF-1 receptor. In a familiar theme of regulatory loops, IGF-1 reciprocally regulates miR-1 via the transcription factor Foxo3a, apparently through an enhancer-binding element within the miR-1 promoter. Interestingly, skeletal muscle response to endurance or resistance exercise stimuli has been linked to changes in miRNA levels [21,22]. However, such changes are depending on the age of the subjects, the mode of exercise, and the duration of exercise. For example, when anabolic response is dichotomized into so-called low responders versus high responders to resistance exercise-induced muscle hypertrophy, the former, but not the latter, exhibit a decrease in miR-26a, miR-29, and miR-378. Since miR-29 has been implicated as positive regulator of myogenesis, its reduction in low responders may contribute to the attenuated response in muscle hypertrophy. Interestingly, physical inactivity such as prolonged bed rest (10  days) leads to muscle atrophy and a reduction in the levels miR-206, miR23a, and several members of let-7 family of miRNAs [23]. These miRNAs contribute to a wide array of functions in that influence muscle function and maintenance, insulin response, growth and atrophy, cell cycle, differentiation, and glucose homeostasis. Other anabolic modulators such as estrogen and androgen also alter miRNA levels [11].

5. Organ Diseases and MicroRNAs

A potentially important and therapeutically exploitable distinction between disease and healthy states in tissue homeostasis may reside in the regulatory miRNA networks that reflect healthy and disease states. There is a growing recognition that miRNA networks are often associated with tissue dysfunction and are likely to be a key source of altered gene expression that underlays and distinguishes healthy versus disease states. A better understanding of the key perturbations that lead to these alternative regulatory network states may help to inform therapeutics. A reduction in Dicer expression has been implicated in cardiac disease. In skeletal muscle, a reduction of miR-29 expression has been associated with decreased muscle regeneration and Duchenne’s muscular dystrophy, but also chronic kidney disease, pointing to the complexity of miRNA control networks [21]. To add to this complexity, miR-29 expression increases with age and this induction may inhibit muscle regeneration by affecting overall protein translation via modulating IGF-1 and p85α. In the context of lung biology, profiling studies of bronchial airway epithelia reveal an array of changes, wherein multiple miRNAs were downregulated in smokers that could be correlated with mRNA target expression in vivo [24]. In the kidney, miR-155 is differentially expressed in different tissue compartments (ie, kidney cortex versus kidney medulla regions) [5]. In individuals with trisomy 21, the chromosomal disorder also known as Down’s syndrome, fibroblasts display higher levels of miR-155. In kidney, the expression of miR-192 is dysregulated in diabetic nephropathy, with a suggestive role in mediating TGF-β-induced collagen expression. In that study, deletion of the inhibitory Smad 7 promoted miR-192 expression, in a model for obstructive kidney disease. In diabetes, mRNA profiles are altered based on the observation that there is an apparent discordance between proteomic profiles and their respective mRNA levels, suggesting a potential role for miRNAs [7]. Interestingly, insulin-secreting cells exposed to proinflammatory cytokines display elevated miR-21 levels, and miR-146 levels are increased in a murine model for type 2 diabetes (db/db mice). Studies on insulin effects in skeletal muscle indicate that insulin expression is associated with a reduction in multiple miRNAs including miR-1, miR-206, and miR-133a (all canonical muscle regulatory miRNAs), in a process potentially mediated in part by the transcription factor sterol regulatory element-binding protein-1c (SREBp-1c) and myocyte enhancer factor 2C. In the liver, miR-122 is one of most abundant miRNAs and therapeutic knockdown of miR-122 can decrease hepatic lipogenesis, resulting in a protection from a high-fat diet–induced hepatic steatosis [8]. In the context of immune system regulatory networks, miR-155 was upregulated in T and B cells upon pathogen activation and required for the maintenance of lymphocyte homeostasis, notably in promoting Th17 cytokine expression [11]. Mice with an ablation of miR-146a specifically in regulatory T cells display elevated levels of the T helper-1 (Th1) cytokine interferon-gamma (IFN-γ) and a breakdown of immune tolerance with an increased risk for autoimmune disease. Several additional studies describe a dysregulated miRNA profile in blood cells from individuals with lupus, an autoimmune disease characterized by production of autoantibodies. The upregulation of miR-155 in lupus B and T cells may lead to the characteristic abnormal B-cell responses in germinal centers. Dysregulated expression of the miRNAs miR-146a and miR-155 in synovial fibroblasts is also implicated in rheumatoid arthritis (RA). Notably, although miR-146a was upregulated in active RA disease, the target genes, IRAK-1 and TRAF6, had no apparent change in their levels when compared to healthy controls [11]. This result may indicate that miRNAs may lose their capacity to function as negative regulators of inflammation in the context of RA, underscoring the contextually specific effects of miRNA networks and the need for global profiling approaches to define disease states and their contradistinction from healthy states.

6. Cancer and MicroRNAs

Perhaps one of the most critical developments in the pathobiology of miRNAs has been the recognition that miRNAs modulate the transcriptome and can reflect a cancerous state, and indeed promote or attenuate cancer risk. A reduction in miRNA expression has been associated with dedifferentiated tumors in lung epithelial cells [25] and downregulation of epithelial markers has been linked to an epithelial–mesenchymal transition [20], a key developmental program that is often activated during cancer invasion and metastasis, and in this case diagnostic of lung cancer. Global repression of miRNA expression may in fact be a common feature in cancer [12]. In lung tumors, as well as in advanced adenocarcinoma, Dicer expression is often reduced. In rhabdomyosarcoma, a muscle stem cell tumor affecting the pediatric population, myomiRs such as miR-1 and -133 tend to be significantly downregulated [26]. miRNAs may promote or attenuate the oncogenic phenotype by either decreasing expression of tumor-suppressor genes (oncomiRs) or alternatively by targeting oncogenic mRNA for silencing (tumor-suppressor miRNAs). Perhaps one of the most studies miRNAs is let-7, a tumor suppressor [12]. Let-7 overexpression has been shown to inhibit tumor development and growth. miRNAs (eg, miR-17-92, miR-21) can function as oncomiRs by modulating cell cycle regulators that in effect promote proliferation, eg, CDKN1A (p21), E2F family, and PTEN [12,20]. Diagnostically, low let-7a-2 levels and high miR-155 levels have been correlated with poor survival in adenocarcinoma of the lung [12]. Comparative miRNA expression profiling in adenocarcinoma and squamous cell carcinoma revealed a signature of miRNAs in male smokers and a small set of miRNAs that predict survival in a cohort of smokers with early stage squamous cell carcinoma. An additional complexity described in Liu et al. is that the cell and tissue context of miRNA expression can be critical to understanding the overall pathobiology of cancer. For example, while miR-31 appears to be prooncogenic in lung cancer, it can alternatively be associated with protective affects in breast cancer.

7. Aging and MicroRNAs

Recent studies have uncovered alterations in miRNA expression during the normal aging process, and also in extreme longevity [2], perhaps collectively providing more insights into mechanisms governing age-related regenerative disorders in lung, heart, skeletal muscle, liver, and in neurodegenerative disease. Deep sequencing analysis of miRNAs in mouse serum reveals an increase in multiple miRNAs and a decrease in some miRNAs with age [27]. Some of these miRNAs behave as functional clusters to putatively target regulatory networks, eg, the regulation of macromolecule biosynthesis (miR-134-5p, -148a-5p, -192-5p, -217-5p, -298-5p, -365-3p, and -434-3p), apoptosis (miR-3096b-5p, -376b-3p, -431-5p, and -138-5p), and the Wnt-signaling pathway (miR-592-5p, -667-3p, and -668-3p). In separate studies, increased expression miRNAs (miR-146a-5p, -148-5p, and -182-5p) and decreased miRNA (miR-144-3p) have been implicated in regulating the inflammatory pathway [28–31], known to be progressively deregulated during aging. Interestingly, three miRNAs (miR-151a-5p, -181a-5p, and -1248) associated with modulating the inflammatory pathway are significantly decreased in the serum of older individuals as compared to those of young individuals [32]. However, whether the change in the circulating miRNA abundance is the cause or the consequence of aging requires more comprehensive studies to establish the causal pathways that miRNAs engage during the aging process. There is also evidence of age-related miRNA alterations that are tissue specific. For example, in the liver, levels for miRNAs targeting the oxidative stress defense such as miR-93, -214, -669c, and -709 increase with age [33]. Similarly, in the brain, miRNAs (miR-101a, -720, and -721) regulating oxidative phosphorylation are upregulated during aging. In mouse skeletal muscle, among the 57 differentially expressed miRNAs during aging, miR-7, -486, -542, and -698 were significantly increased, whereas miR-124a, -181a, -221, -382, -434, and -455 were substantially decreased [33]. In human skeletal muscle, let-7, known to regulate cell cycle and perhaps satellite cell turnover, is among the 18 miRNAs that are upregulated during aging [34]. Cellular senescence is one of the hallmarks of cellular aging that hinders stem cell regenerative capacity. Signaling pathways regulating cellular senescence include the insulin/IGF-1 (IIS), reactive oxygen species, mitogen-activated protein kinase, p53, retinoblastoma, and inflammatory pathways [33]. As such, changes in the abundance of miRNAs targeting these pathways profoundly affect life span based on studies in the nematode Caenorhabditis elegans. For example, lin-4, miR-71, miR-238, and miR-246 promote longevity, whereas, miR-239 reduces life span [32,35]. In humans, the deregulation of miR-126 which targets the IGF/IIS pathway is proposed as one of the possible contributors to the observed blunted response of aging muscle following anabolic stimulation, in a phenomenon described as anabolic resistance [36]. Furthermore, miRNAs known to regulate IGF/IIS pathway have been associated with heart disease (miR-1, miR-122, and miR-375), skeletal muscle growth (miR-1 and -206), and neurodegenerative disease (let-7 and miR-320) [35]. As high-throughput technologies for identifying functional miRNA targets such as HITS-CLIP (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation) and PAR-CLIP (photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation) become more advanced and affordable, so will our understanding of the regulatory network of miRNAs in modulating aging and age-related disorders [35].

8. MicroRNA Therapeutics and Target Prediction: Successes and Challenges

The prospect of leveraging the growing knowledge of miRNAs and their respective targets to achieve therapeutic outcomes has prompted an explosion of research to identify miRNA modulators with applications to a broad range of disease conditions. miRNAs can be chemically modified to act either as miRNA antagonists or agonists to counteract or reduce miR expression associated with specific disease states. miRNA antagonist approaches include locked nucleic acid (LNA), antimiRs, antagomirs, morpholinos, and miRNA sponges—with each there is the promise of efficient inhibition of miRNA function and evidence for effective activity in vivo [7]. A particularly intriguing example of their use to date involves the use of miRNA sponges as decoys against specific miRNAs linked to type 1 and type 2 diabetes. In other studies the use of an in vivo antagomir antisense oligonucleotide for miR-122 (one of the most abundant miRs in liver that has been associated with cholesterol regulation) resulted in almost compete depletion of miR-122 in hepatocytes and a decrease in hepatic fatty acids, cholesterol synthesis, and plasma cholesterol levels. In their review, Nana-Sinkham and colleagues [12] have described additional successes in the use of miR-based therapeutics; wherein the achieved systemic delivery of an oligonucleotide-LNA of miR-122 in a nonhuman primate model for chronic HCV. In those experiments there was evidence for early success, with a potent reduction in the HCV burden. Nevertheless, an important caveat and challenge in the design of miRNA therapeutics will be estimating the likelihood for off-target binding or quenching (in the case of miRNA sponges) and designing safeguards against those undesirable off-target effects [4]. Targeted delivery approaches for miRNA therapeutics include both passive and active delivery systems that have been developed to minimize off-target effects [37]. Passive targeted delivery includes utilizing the cell-penetrating peptide (penetratin) in the biodegradable poly(lactide-co-glycolide) nanoparticles, the enhanced permeability and retention of solid tumors, and solid lipid nanoparticles. Active targeted delivery includes utilizing peptide or protein ligands, antibodies, aptamers, other ligands (ie, hyaluronic acid, folate acid), and magnetic nanoparticles. Currently, the antimiR-122 for HCV and the miR-34 mimic to treat liver cancer are in clinical trials [38].

With the increasing sophistication of bioinformatics tools for probing genome-wide sequence features, and the wide accessibility of reference genomes for several organisms, most notably human, mouse, and nematode, there has been an explosion of Web-accessible algorithms designed to predict genomic interactions between miRNA and putative mRNA. This has been coupled with substantial progress in the in vitro validation of sequence recognition criteria for miRNA:mRNA target interaction. Balancing the many algorithmic successes, miRNA-binding predictions based on the use of the most popular computational tools (ie, miRanda, TargetScan, PicTar) have not generally been correlated to each other and their predictions for interaction are often not supported by experimental evidence [13]. Clearly refinements, based on more empirical evidence and theoretical considerations, will be needed to expand the utility of these tools to interrogate complex rules for genomic interaction. One level of improved sophistication will be the implementation of additional limitations in these bioinformatics tools to incorporate critical information obtained from empirical data for miRNA and mRNA expression differences [4]. In the context of different states, eg, healthy and disease tissue, weighted differences in miRNA recognition will likely influence global target recognition and overall predicted expression profiles. One approach to this concern of contextual expression of miRNA is discussed by Dorn and colleagues [4] who have championed the use of high-throughput sequencing procedures coupled to immunoprecipitation of miRNA:mRNA complexes bound within the RISC complex. In a technique termed RISC-Seq, RISC complexes are immunoprecipitated (eg, using anti-Argonaute 2 antibodies), followed by RNA sequencing. This approach allows for precise mapping of target sequences in the context of overexpressed miRNAs and will likely be instrumental in profiling tissue and disease specific miRNA expression.

9. Circulating MicroRNA as Biomarkers

There is substantial interest in identifying circulating biomarkers that are useful in classifying disease severity and in gauging therapeutic response to candidate drug interventions. With the surprising observation that miRNAs are detectable in serum and are relatively stable—due to protection in extracellular vesicular structures called exosomes, there has been a substantial new growth and enthusiasm for this line of research. In skeletal muscle, serum level of myomiRs (miR-1, miR-133, and miR-206) are increased in the muscular dystrophic mouse and dog (canine X-linked muscular dystrophy in Japan) compared to expression of these miRs in control animals [39]. Notably, the myomiR miR-206 is significantly increased not only in rhabdomyosarcoma (RMS) cell lines, but also in serum of patients with RMS tumors [40]. Interestingly, miR-206 is observed in serum of patients with RMS, but not in other types of cancer [41]. However, miR-206 is increased in serum of patients with chronic obstructive pulmonary disease [42]. Furthermore, physical activity also alters circulating myomiR levels. In non-small cell–lung cancer, a four-miRNA serum signature has been identified [3]. In the context of heart disease, serum levels of miR-208b and miR-499 were strikingly increased (∼1000-fold) after myocardial infarction [4]. Despite these early successes, there are many validation studies required [25]. Foremost among these validation studies is the need for RT-PCR as a validation for miR profiling as well as novel solutions to the difficulty of normalizing biomarker levels in serum. In the immune system, miRNAs in plasma and serum including miR-150, miR-146a, and miR-223 have been proposed as potential biomarkers for sepsis [13,20]. MiR-150 is correlated with aggressiveness of sepsis, and as such, may be a prognostic marker in patients with sepsis. In diabetes, serum miRNA profiles from diabetic and healthy subjects identified 65 common and 42 differently expressed miRNAs [7]. Studies using pooled sera have identified 13 miRNAs in diabetic patients compared with healthy subjects, further highlighting the promise of miRNAs as serum and plasma biomarkers. In the mouse, circulating miRNAs targeting macromolecule biosynthetic process, apoptosis regulators, and the Wnt signaling pathway are increased with age and intriguingly are decreased by calorie restriction [27]. Furthermore, miRNAs regulating the inflammatory pathways in detectable in serum of young and old individuals are altered with age [32]. However, whether changes in circulating miRNAs could be used as biomarkers for disease diagnosis and/or prognosis is a work still in progress. One of the technical challenges involves standardize approaches for sample processing to minimize variations, analytically and biologically. Analytical variation includes blood draw technique (ie, epithelial cell contamination due to skin plug), specimen processing (ie, hemolysis, lipemia, drugs, and antibodies), and normalization techniques [43,44]. For example, Wang et al. [45] have shown discrepancies in miRNA levels in serum and plasma of corresponding subjects in which miRNA levels in serum are consistently higher than those in plasma, possibly due to contamination of miRNAs from platelets. Biological variation can consists of extrinsic variation among individuals at the time of sampling (ie, diurnal, fasting/fed, health state) and intrinsic differences between individual (ie, age/gender, genetic/ethnicity, chronic disease) [44]. Another challenge involves establishing the source(s) of circulating miRNAs and their role in disease. Blood cells, endothelium lining the blood, and cells from other tissues all are likely contribute to circulating miRNAs [44]; therefore, determining tissue-specific miRNAs in the plasma and serum would further improve biomarker specificity and diagnostic utility. Collectively, in the coming years, it will be interesting to see how sensitive and stable these miRNA circulating biomarkers are and to what extent these peripheral markers can reflect organ health and disease interventions.

10. Concluding Remarks

In summary, miRNA is an essential feature of lineage commitment and regulatory guidance during tissue development such that when absent or hampered, often leads to disease states. In the coming years, there is much to be learned about adaptive (and maladaptive) states by examining how expression of miRNAs is influenced by the genetic architecture of miRNA genes, clusters, and mirtrons, as well as miRNA polymorphism and polymorphism in their mRNA targets. There are manifold modes of miRNA regulation (negative feedback, positive feedback, cross-regulatory) that monitor, modulate, or resolve signaling pathways in a variety of biological processes that include sepsis response, fibrosis, acute exercise, aging, and steroid biology. Perhaps the homeostasis and micromanagement of these miRNA regulatory systems, when perturbed, can arrive at new quasistable state of networked interactions that have an undesired effect of promoting or antagonizing disease severity and cancer progression. Clearly, a better understanding of these miRNA regulatory networks, as well as improved therapeutic tools for guiding miRNA expression and their targets toward desired outcomes, and healthy regulatory states, as a form of pathway therapeutics, will be the subject of many advances in miRNA biology over the coming years. Undoubtedly, recent advance of miRNA-based medicine to various phases of clinical trials will provide more insights into the bioavailability, biosafety, and efficacy of antimiRs and miRNA mimetics.

References

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[2] Montano M. Detecting disease in blood: what miRNA biomarkers can tell us. In: Science technology webinar series. Science/AAAS Custom Publishing Office; 2012.

[3] Liu X, et al. Involvement of microRNAs in