# MOLECULAR FUNCTION AND REGULATION OF NON-CODING RNAs IN MULTIFACTORIAL DISEASES

EDITED BY: Mohammadreza Hajjari, Seyed Javad Mowla and Mohammad Ali Faghihi PUBLISHED IN: Frontiers in Genetics

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ISSN 1664-8714 ISBN 978-2-88945-002-2 DOI 10.3389/978-2-88945-002-2

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# **MOLECULAR FUNCTION AND REGULATION OF NON-CODING RNAs IN MULTIFACTORIAL DISEASES**

Topic Editors:

**Mohammadreza Hajjari,** Shahid Chamran University of Ahvaz, Iran **Seyed Javad Mowla,** Tarbiat Modares University, Iran **Mohammad Ali Faghihi,** University of Miami Miller School of Medicine, USA

Our understanding of the mechanisms underlying the development of multifactorial diseases such as diabetes, autism, Alzheimer's disease, and cancer has been greatly advanced. Non-coding RNAs (ncRNAs), generally including microRNAs and long non-coding RNAs, have recently been found to have potential roles in these diseases, and provide new opportunities for developing both specific biomarkers and therapeutic targets. However, the molecular function and regulation of these RNAs still remains challenging. Numerous studies are focusing on this field in order to fully appreciate the role and regulation of these molecules in human medicine and biology.

This e-book aims to bring together new findings on Non-coding RNAs in different complex diseases. It will highlight the characterization, roles, mechanism, and mode of action of these RNAs in complex disorders. We believe that the publications on this topic would be exponentially extended in future. The improved approaches at multiple levels may pave the way for designing and applying new biomarker and therapeutic targets for specific diseases based on these attractive molecules.

**Citation:** Hajjari, M., Mowla, S. J., Faghihi, M. A., eds. (2016). Molecular Function and Regulation of Non-coding RNAs in Multifactorial Diseases. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-002-2

# Table of Contents

## **Chapter 2: Long non-coding RNAs in complex diseases**

Alireza Shahryari, Marie Saghaeian Jazi, Nader M. Samaei and Seyed J. Mowla

*67 Exosomal lncRNA-p21 levels may help to distinguish prostate cancer from benign disease*

Mustafa Is¸ ın, Ege Uysaler, Emre Özgür, Hikmet Köseog˘lu, Öner S¸ anlı, Ömer B. Yücel, Ug˘ur Gezer and Nejat Dalay

# Editorial: Molecular Function and Regulation of Non-coding RNAs in Multifactorial Diseases

#### Mohammadreza Hajjari 1 \*, Seyed Javad Mowla2 \* and Mohammad Ali Faghihi 3 \*

1 Department of Genetics, Shahid Chamran University of Ahvaz, Ahvaz, Iran, 2 Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran, 3 Department of Psychiatry and Behavioral Sciences, Center for Therapeutic Innovation, University of Miami Miller School of Medicine, Miami, FL, USA

Keywords: long non-coding RNA (lncRNA), microRNA (miRNA), multifactorial disease, biomarker discovery, non-coding RNA (ncRNA)

**The Editorial on the Research Topic**

#### Edited by:

Subbaya Subramanian, University of Minnesota, USA

#### Reviewed by:

Venugopal Thayanithy, University of Minnesota, USA Mainá Bitar, Universidade Federal de Minas Gerais, Brazil Anne Elizabeth Sarver, University of Minnesota, USA

#### \*Correspondence:

Mohammadreza Hajjari<EMAIL_ADDRESS><EMAIL_ADDRESS>Seyed Javad Mowla<EMAIL_ADDRESS>Mohammad Ali Faghihi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to RNA, a section of the journal Frontiers in Genetics

Received: 31 October 2015 Accepted: 22 January 2016 Published: 19 February 2016

#### Citation:

Hajjari M, Mowla SJ and Faghihi MA (2016) Editorial: Molecular Function and Regulation of Non-coding RNAs in Multifactorial Diseases. Front. Genet. 7:9. doi: 10.3389/fgene.2016.00009 **Molecular Function and Regulation of Non-coding RNAs in Multifactorial Diseases**

Most of the parts in the human genomic DNA do not produce any proteins. They encode functional RNA molecules, which are not translated, and are thus named as non-coding RNAs (ncRNAs; Cammaerts et al.; Hajjari et al., 2014). Depending on the length and the function of these RNAs, they are categorized into different types such as microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Different studies have shown that these RNAs have crucial roles in cellular and molecular mechanisms. Considering their function, it is believed that the dysregulation of ncRNAs is involved in different diseases, especially such complex ones as cancers, neurological disorders, and cardiovascular diseases (Nouraee and Mowla; Merelo et al.; Hajjari et al., 2014). Accordingly, some researchers believe that these molecules can help with the diagnosis and the treatment of complex diseases. However, we have still a limited understanding of the functions of these molecules, and the different aspects of ncRNAs are still to be discovered in future. The above consideration was the motivation for the production of the topic entitled "Molecular function and regulation of non-coding RNAs in multi-factorial diseases." We believe that the articles in this issue and the attributed e-book can provide the interesting viewpoints for the researchers interested in this topic.

miRNAs are small RNAs with about 20–24 nucleotides length and function in post transcriptional regulation of gene expression. They usually destabilize and repress target RNAs via binding to 3′UTRs, 5′UTRs or the coding sequences of the transcripts (Lytle et al., 2007; Qin et al., 2010). miRNAs have been demonstrated to play major roles in a wide range of developmental processes as well as diseases. To understand the role of miRNA pathogenesis in different diseases such as cancers, two different strategies including the expression analysis and the genetic approaches are proposed (Reviewed in Cammaerts et al.). As such, the miRNAs are currently considered as new potential biomarkers for different diseases (Angelini and Emanueli). Furthermore, some reports have interestingly shown the plasma and saliva miRNAs as potentially sensitive and specific biomarkers (Lin et al.; Khoo et al., 2012). Nonetheless, there are still some concerns with the functionality of miRNAs that need to be addressed in future.

By modulating different targets or pathways, miRNAs have been quickly considered as potential therapeutic molecules for some diseases such as cancer (reviewed in Naidu et al., 2015) and cardiovascular ones (Nouraee and Mowla). miRNA-based therapeutics mainly focuses on modulation of the miRNA expression levels (Misso et al., 2014). MRX34, an agent mimicking miR-34 and developed by Mirna Therapeutics, has progressed into phase I clinical trials (identifier: NCT01829971, Currently recruiting participants, Estimated study completion date: Dec 2016). The results may hopefully provide more insights for the researchers. In spite of different researches, there are still some challenges in designing a suitable carrier for targeting miRNAs into the desired cells. Bakhshinejad reports the studies proposing the nanocarriers as the potential tools to overcome delivery problems concerned with miRNA-based pharmaceutical tools (Bakhshinejad).

lncRNAs, the RNAs longer that 200 nucleotides, comprise the largest proportion of the human transcriptome. The biology of lncRNAs seems more complicated in comparison to miRNAs. These RNAs act through more different pathways and modes of action within the cell. They function at transcriptional and post-transcriptional levels, in the cytosol and nucleus through cis and trans-regulatory mechanisms (Hajjari et al., 2014; Angrand et al.). Different lncRNAs such as HOTAIR, H19, MEG3, ANRIL, HULC, and XIST are transcribed within the genome and play important roles in different cellular pathways (reviewed in Hajjari et al., 2014). Multiple lines of observation increasingly associate the mutations and the dysregulations of lncRNAs to some human diseases such as cancer (reviewed in Angrand et al.; Hajjari et al., 2014). On the current subject matter, Shahryari et al. have provided some evidence showing that some lncRNAs such as SOX2OT are also involved in pluoripotency (Shahryari et al.). Peschanscky et al. have also described the novel facets of FMR4 lncRNA functionality and its relation to neurodevelopment (Peschansky et al.).

The growing evidence of the dysregulated lncRNAs not only introduces a new layer of complexity in the molecular mechanism of human diseases, but also opens up the opportunity to use

## REFERENCES

lncRNAs as therapeutic targets and biomarkers. The prominent example of lncRNAs application in clinical practices is PCA3 (Prostate cancer gene 3), an FDA-approved biomarker for the prostate cancer. According to this test, the PSA and PCA3 RNA molecules are measured, and then the PCA3 Score (a ratio of PCA3 RNA to PSA RNA) is calculated (http://www.fda. gov). Circulating lncRNAs have been also suggested as potential biomarkers for cancer and some other diseases. Exomal lncRNAs have been suggested as potential biomarkers to diagnose the malignant state of patients with prostate cancer (I¸sin et al.).

The rapidly growing list of ncRNAs holds promises that miRNAs and lncRNAs will become ever more important in illness management in future. The elucidation of the mechanisms by which lncRNAs act will help us to design suitable biomarkers or therapeutic agents for some diseases, especially cancer. The deciphering of the genetic networks and pathways regulated by the abnormally expressing lncRNAs, the characterization of miRNA-mRNA-lncRNA interaction, and the genetic/epigenetic mechanisms regulating the lncRNA expression provide more insights into the application of lncRNA research to human health. Considering the different molecular functions of ncRNAs, these RNAs can be the subject of a large diversity of further studies on complex diseases. We trust that the publications and the novelties on this subject will be exponentially extended in future.

# AUTHOR CONTRIBUTIONS

All authors listed, have made substantial, direct, and intellectual contribution to the work, and approved it for publication.

Naidu, S., Magee, P., Michela Garofalo, M. (2015). MiRNA-based therapeutic intervention of cancer. J. Hematol. Oncol. 8:68. doi: 10.1186/s13045-015-0162-0

Qin, W., Shi, Y., Zhao, B., Yao, C., Jin, L., Ma, J., et al. (2010). MiR-24 regulates apoptosis by targeting the open reading frame (ORF) region of FAF1 in cancer cells. PLoS ONE 5:e9429. doi: 10.1371/journal.pone.0009429

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2016 Hajjari, Mowla and Faghihi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# MicroRNAs as clinical biomarkers?

Timothy G. Angelini 1 and Costanza Emanueli 2, 3 \*

*1 Royal Surrey County Hospital, Guildford, UK, 2 Bristol Heart Institute, School of Clinical Sciences, University of Bristol, Bristol, UK, 3 National Heart and Lung Institute, Imperial College London, London, UK*

Keywords: microRNAs, clinical biomarkers, exosomes, cardiac disease, myocardial infarction

A clinical biomarker has been defined as "any cellular, biochemical, molecular, or genetic alterations by which a normal, abnormal, or simple biological process can be recognized or monitored" (Drucker and Krapfenbauer, 2003; Rahim et al., 2015). Biomarkers are widely employed throughout medicine on a day to day basis to diagnose, prognosticate, and predict outcomes of disease and illness or even as guides of treatment. They can either be used as a standalone, or more commonly, in conjunction with other test results. Moreover, biomarkers are often chosen as "surrogate endpoints" in clinical trials. Biomarkers must fulfill certain criteria in order to make it in to regular use in clinical practice. They must be specific to the condition that they are indicated in, sensitive, and must be practical in terms of accessibility of the sample, ease of testing method and provide information in which to guide clinical decisions. However, many biomarkers have unsatisfactory specificity or are not as sensitive as they are made out to be. In addition, biomarkers for some conditions are not yet available. Consequently, research in to novel biomarkers should be encouraged.

#### Edited by:

*Seyed Javad Mowla, Tarbiat Modares University, Iran*

#### Reviewed by:

*Alessio Paone, Sapienza University of Rome, Italy Vijay Kumar Prajapati, Central University of Rajasthan, India*

> \*Correspondence: *Costanza Emanueli<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to RNA, a section of the journal Frontiers in Genetics*

Received: *08 May 2015* Accepted: *29 June 2015* Published: *14 July 2015*

#### Citation:

*Angelini TG and Emanueli C (2015) MicroRNAs as clinical biomarkers? Front. Genet. 6:240. doi: 10.3389/fgene.2015.00240*

Biomarkers can be classified in to different classes, one being the sources where they measured from. In this respect, there are two distinct kinds of biomarker; intracellular, and extracellular (which can be broken down into three further groups by means of sampling; invasive, minimally invasive, and non-invasive). Examples of intracellular biomarkers include many of those used in oncology, such as BRCA1/2 (Drooger et al., 2015), and estrogen receptor and hormone receptor status in breast cancers (Yang et al., 2014), and BRAF genes in melanoma (Dar et al., 2015). For these tests, the cells from the suspected tumor must be invasively sampled, often by core sampling, or tissue scrapings. Extracellular biomarkers are often but not always linked with less invasive sampling, for example the withdrawal of a peripheral venous blood sample or urine collection. Examples are urine test for beta-human chorionic gonadotrophin (Chen et al., 2015) as an indicator of pregnancy or prostate specific antigen (PSA) (Ravery, 1999) in a suspected prostate cancer case.

An exciting, but not yet conclusive, new area of research with the potential to derive new clinical biomarkers has been into microRNAs (miRs). These short RNA molecules were discovered in 1993 (Lee et al., 1993) in C. elegans and found present in human samples in 2000 (van Rooij, 2011). From there, their love story with the biomedical community has seen a crescendo of enthusiasm and they have been highly regarded as both novel therapeutic targets, and possible intracellular and extracellular biomarkers. We might speculate that the reason behind this popularity is two-folds:

(1) The mode of action of miRs is relatively straightforward. MiRs do not contain any coding for protein production (and hence they are classified as noncoding RNAs–ncRNAs) and each miR is capable of post-translational regulation of the expression of a plethora of target messenger RNAs (mRNAs), which it recognizes through the semi complementary nucleotide base-pairing between its "seed sequence" (of just eight nucleotides) and one or more miR binding sites in the 3′ -untranslated region of the mRNA targeted (Jackson and Standart, 2007). The biology of miR appears much simpler in comparison to, as an example, the long ncRNA that act via multiple, with still largely un-clarified mechanisms, at transcriptional and post-transcriptional levels, in the nucleus and the cytosol, and in cis and trans (Rinn and Chang, 2012). Moreover, miRs exist in a limited number, thought to be around 2000 in humans. However, miRs variants are still being discovered through RNA-sequencing approaches.

(2) MiR are released by their producing cells in protected forms allowing them to remain for long periods in biological fluids. Moreover, miRs were initially believed to be tissue- and even cell-specific. This led to the assumption that what was found in the blood could sense the altered status of the cell types or tissues that were supposed to produce such miR. Now the concept of tissue/cell-specificity has been largely dismissed and it is becoming clear that at best we can talk of cell/tissue type enrichment for most, if not all, the known miRs. Hence, it is quite unrealistic to think that, under most clinical scenarios (with exceptions discussed later), the level of a single miR measured in the whole plasma or serum can be informative of a local slowly or relatively slowly evolving condition, such as, a developing cancer (Washam et al., 2013).

Circulating individual miRs, such as miR-21 (suggested to be useful for the detection of various carcinomas) (Wang et al., 2014; Wu et al., 2015), and circulating groups of miRs (such as a serum miR classifier encompassing miR-29a, miR-29c, miR-133a, miR-143, miR-145, miR-192, and miR-505, that has been proposed to detect hepatocellular carcinoma) (Lin et al., 2015) have been suggested as potential biomarkers that could be used in cancer detection and staging, and to follow-up already diagnosed cancer patients. However, in a recent article reporting the results of the comparison among 15 previous reports on potential new breast cancer biomarkers (Leinder et al., 2013) there was a scarce overlap between results: "Of the 143 circulating miRNAs reported to be differently regulated, 100 were supported by just 1 reference; 25 others had discordant results across publications and of the remaining 18 miRs, 8 had fold changes too low to be confirmed. Of the 10 concordant results, 9 were supported entirely by publications from the same institution and had authors in common" (Leinder et al., 2013; Witwer, 2015). This suggests that further efforts are needed before miR-based biomarkers can benefit cancer patients and that this could also apply to other disease conditions. Cancer has been the first translational area for miR work, closely followed by heart failure. Looking at both clinical scenarios, we find a typical example of unspecificity: circulating miR-21 has been proposed as a biomarker of both prostate cancer (Egidi et al., 2013) and myocardial fibrosis (in heart failure) (Thum et al., 2008). miR-21 is also the most expressed miR by vascular endothelial cells (Greco and Martelli, 2014), which are the cells directly lining the circulating blood and for this reason supposed to be the highest contributors to miRs circulating in the peripheral blood (Greco and Martelli, 2014; Witwer, 2015). A miR such as this, amongst others that are widely and highly expressed, might not be an ideal circulating candidate biomarker, suggesting that miRs that are usually under expressed, but upregulated under a particular condition could be better suited to be employed in a diagnostic test. For example, we recently found that miR-503 appears in the blood of diabetic patients at the last stage of critical limb ischemia, i.e., when they need an amputation (Caporali et al., 2011). It is possible that circulating miR-503 could have a diagnostic/prognostic value when measured at the earlier stages of the disease, however, measuring miR-503 in a small leg muscle biopsy could provide more reliable information.

There are areas where we believe that circulating miRs show more promise and this is in the recognition of acute events, such as myocardial infarct (MI), as well as in the surgical setting, where time-restricted changes in miR expression have been reported consistently. For examples, the heart (and skeletal muscle)—enriched miR-1 has been noted to increase in patients after open heart surgery, after MI or transcoronary ablation of septal hypertrophy, an interventional procedure that mimics MI (D'Alessandra et al., 2010; Widera et al., 2011; Liebetrau et al., 2013; Nabialek et al., 2013). Diagnostic biomarkers are a key part in emergency service provision and the rapid diagnosis, and therefore treatment of patients with life threatening conditions. One of the most widely used in the emergency department are cardiac troponins (cTns: cTn-T and cTn-I) for the diagnosis of MI. CTns are used in conjunction with other investigations, such as electrocardiogram (ECG) changes, allowing, for example, to determine whether a patient has had a "STEMI" type MI (with ST segment Elevation by ECG). ECG cannot pick up non-STEMI cases and here laboratory biomarkers are highly important (Alpert et al., 2000). However, cTns are not always specific to MI, they can be raised in patients with other cardiac conditions and also after infection. In light of this, research into miRs as potentially better biomarkers has been carried out showing timedependent increases in cardiac-enriched, ischemia-responsive miRs in the blood of MI patients (D'Alessandra et al., 2010; Nabialek et al., 2013). There has also been claim that miRs can help differentiating the diagnosis of a STEMI compared with other myocardial conditions, such as stable angina, non-STEMI, and Takotsubo cardiomyopathy (Nabialek et al., 2013; Ward et al., 2013; Jaguszewski et al., 2014). However, it is yet to be demonstrated that miRs can replace cTns as routine biomarkers used in the intensive coronary care unit, in depth investigations of specificity and sensitivity in different cohorts or patients are needed. Additionally, in the realm of emergency medicine, sensitivity and specificity of miRs response in terms of circulating changes are not the only issues, because the time necessary by this putative biomarker to appear elevated in the blood or another biological fluid is critical. Classically, cTns take a few hours to increase in the blood after an MI. MiR-1 has been proposed to go up earlier than cTns (Liebetrau et al., 2013). However, for these comparisons, the time to obtain the test results and the test reproducibility are big obstacles yet to be overcome. Differently from high sensitive cTns that today are measured by immune-enzymatic reactions allowing for results to be obtained in around 20 min, PCRbased miR analyses are still quite time-consuming. Alternative techniques for miR quantification have been proposed (Arata et al., 2012) but they are far away from being commonly employed by the scientific community, let alone the clinical diagnostic lab. Additionally, in a clinical laboratory staff cannot reason as in observational studies, where everybody is often quite satisfied with saying "miR-1 is increased in the blood of MI patients in comparisons to a control group." Diagnostic use of miRs needs a different rigor and first of all a definition of what the "normal" threshold of miR concentration is, above which we can suspect in a patient who is experiencing a heart attack. Scientists working in the miR field are aware that this is not an easy task and by using PCR-based methods quantitative differences between different studies are common. In addition, the data normalization approaches are still debated and interference by heparin (used in interventional procedures) with the PCR reaction has been reported (Mayr et al., 2013), even if protocol to nullify the "heparin effect" are adopted. Alternative technologies can be developed, but this will require further investment, time and validation efforts (Arata et al., 2012).

In conclusion, We believe that miRs hold potential value as clinical biomarkers, but, their journey to the diagnostic lab is still long and needs improved approaches at multiple levels, starting with technical refinement in the miR concentration

#### References

evaluation, the use of RNA-sequencing to possibly recognize new miRs that are better candidates (higher tissue/cell-specificity, lower expression under healthy conditions etc.) and the use of blood fractions potentially enriched in miRs (like exosomes and microparticles) for diagnostic tests. It is also possible that miR clusters have more specificity than single miRs. Moreover, miR could be associated to other biomarkers to improve the diagnostic power.

#### Funding and Acknowledgments

This work was funded by the National Institute of Health Research (NIHR) Bristol Cardiovascular Biomedical Research Unit (BRU) The views expressed are those of the Authors and not necessarily those of the NHS, the NIHR or the Department of Health. CE is a PI in the Leducq transatlantic network in vascular microRNAs (MIRVAD).

cardiomyopathy from acute myocardial infarction. Eur. Heart. 35, 999–1006. doi: 10.1093/eurheartj/eht392

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Angelini and Emanueli. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# miRNA therapeutics in cardiovascular diseases: promises and problems

#### *Nazila Nouraee and Seyed J. Mowla\**

*Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran*

microRNAs (miRNAs) are a novel class of non-coding RNAs which found their way into the clinic due to their fundamental roles in cellular processes such as differentiation, proliferation, and apoptosis. Recently, miRNAs have been known as micromodulators in cellular communications being involved in cell signaling and microenvironment remodeling. In this review, we will focus on the role of miRNAs in cardiovascular diseases (CVDs) and their reliability as diagnostic and therapeutic biomarkers in these conditions. CVDs comprise a variety of blood vessels and heart disorders with a high rate of morbidity and mortality worldwide. This necessitates introduction of novel molecular biomarkers for early detection, prevention, or treatment of these diseases. miRNAs, due to their stability, tissue-specific expression pattern and secretion to the corresponding body fluids, are attractive targets for cardiovascular-associated therapeutics. Explaining the challenges ahead of miRNA-based therapies, we will discuss the exosomes as delivery packages for miRNA drugs and promising novel strategies for the future of miRNA-based therapeutics. These approaches provide insights to the future of personalized medicine for the treatment of CVDs.

#### *Edited by:*

*Narasaiah Kolliputi, University of South Florida, USA*

#### *Reviewed by:*

*Gaetano Santulli, University of Naples Federico II, Italy Michael Teng, University of South Florida, USA*

#### *\*Correspondence:*

*Seyed J. Mowla, Department of Molecular Genetics, Faculty of Biological Sciences, Tarbiat Modares University, P.O.Box 14115-175, Tehran, Iran<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to RNA, a section of the journal Frontiers in Genetics*

*Received: 27 February 2015 Accepted: 17 June 2015 Published: 30 June 2015*

#### *Citation:*

*Nouraee N and Mowla SJ (2015) miRNA therapeutics in cardiovascular diseases: promises and problems. Front. Genet. 6:232. doi: 10.3389/fgene.2015.00232*

Frontiers in Genetics | www.frontiersin.org June 2015 | Volume 6 | Article 232 |

Keywords: miRNA, cardiovascular disease, cardio-miR, exosome, cell communication, delivery vehicle, secretory miRNA, miRNA therapeutics

# Introduction

Among multifactorial diseases, cardiovascular diseases (CVDs) are significant due to their variable symptoms and high mortality rate accounting for one third of global deaths (Santulli, 2013). They include a wide range of disorders connected to blood vessels and heart ranging from coronary artery disease (CAD), pulmonary arterial hypertension (PAH), and congenital heart disease to deep vein thrombosis and cerebrovascular disease. Major CVD risk factors include family history, obesity, hypertension, diabetes mellitus, and hypercholesterolemia (Mendis et al., 2011). Since CVDs are hard-to-cure, several investigations have focused on different mechanisms underlying CVD in order to manage the symptoms. microRNAs have emerged as one of the most favorable molecular targets in this regard. microRNAs are a class of non-coding RNAs with a short length of 18–24 nucleotides. They mainly act as post-translational repressors of gene expression. By regulating the fundamental cellular mechanisms such as cell differentiation, proliferation, growth, and apoptosis, miRNAs have received enormous attention for therapeutic applications. Huge investigations and investments have been made to bring these molecules into the clinic. In this review, we will explain the novel diagnostic, therapeutic and prognostic approaches based on miRNAs in the field of CVDs (cardio-miRs) and the problems ahead of this research area; where we are and where we expect to be in the future.

# miRNA Mechanisms of Action

Calin et al. (2002) were first to link miRNAs with cancer progression. Since then several studies have focused on the role of these small molecules in the pathogenesis of different human diseases. The primary findings emphasized on the posttranscriptional regulatory role of miRNAs through base pairing with the 3 untranslated region (UTR) of their target mRNAs. This leads to the mRNA degradation or translation inhibition and in both cases the miRNA binding results in the suppression of their target mRNAs. This is the main mechanism reported for the miRNAs regulatory role; however, many variations have also been reported (Ha and Kim, 2014; Lin and Gregory, 2015). miRNA binding to the 5- UTR of transcripts has also been demonstrated to be able to activate or suppress target genes (Lee et al., 2009). Some miRNAs can also bind to the open reading frame (ORF) of mRNA transcripts and repress translation. This mechanism was first reported by Tay et al. (2008). They demonstrated that miRNA binding to the coding region of pluripotency genes can regulate the embryonic stem cell differentiation. miRNA binding might also happen at the promoter of target gene which causes repression of gene translation. On the other hand, some miRNAs modulate their target expression by binding to RNA-binding proteins that regulate the expression of mRNA transcripts (Eiring et al., 2010). Salmena et al. (2011) proposed the competing endogenous RNA (ceRNA) hypothesis according to which, miRNAs, long non-coding RNAs (lncRNAs) and target mRNAs are in a finely tuned interaction through miRNA response elements (MREs) and their competition for target binding based on their total concentration in the cytoplasm defines a higher level of regulation. Recent studies have indicated another fascinating aspect of miRNA regulatory network. Fabbri et al. (2012) proved that miRNAs excreted from cancer cells, can directly bind the toll-like receptors (TLRs) at the surface of neighboring immune cells and activate the relevant signaling pathways in the recipient cells. The complexity of miRNAmediated regulatory systems highlights the importance of these small molecules in the clinic and needs further proceeding technologies to get closer to clinical therapies.

# miRNA Diagnostics in CVD

microRNAs show tissue-specific and time-dependent expression patterns turning them into a leading fact in using these small molecules as diagnostic and therapeutic targets (Lu et al., 2005). Similar to other developmental phenomena in embryogenesis, miRNAs play critical roles in cardiovascular development and also their expression profile changes according to different pathological conditions. Cardiac resynchronization therapy (CRT) after heart failure helps improve the heart arrhythmia and it also affects the myocardial miRNA expression. In responder patients, CRT affects cardiac processes including cardiac fibrosis, apoptosis, angiogenesis, and channel alterations and consequently alters the expression levels of miR-29 (implicated in cardiac fibrosis), miR-30, miR-92, and miR-145 (involved with cardiac angiogenesis), miR-30 (modulated in cardiac apoptosis), and miR-26 (affected by modified ionic channel function; Sardu et al., 2014).

Recently circulatory miRNA expression profiling has provided strong molecular markers for detection of various diseases including CVD such as myocardial hyperthrophy, infarction, angiogenesis and fibrosis (Charan Reddy, 2014; Wronska et al., 2015). van Rooij et al. (2006) defined a miRNA signature for cardiac hypertrophy. They showed that miR-195 overexpression is sufficient to drive cardiac hypertrophy. miR-1 family, miR-133a/b and miR-208 are well-known for their implication in myogenesis both in skeletal muscles and cardiac development and their dysregulation has been detected in several CVDs including myocardial infarction, hypertrophy, and arrhythmias (Thum et al., 2008a). While miR-208 showed significant upregulation (pro-hypertrophic), miR-1 and miR-133a expression levels significantly decreased (anti-hypertrophic) in acute myocardial infarction (AMI) compared with normal individuals heart (Bostjancic et al., 2010). In CAD, miR-1 is upregulated in the left ventricular endocardium (Yang et al., 2007) and also plasma levels of miR-1, miR-133, and miR-208b have been reported to be elevated after AMI (Widera et al., 2011; Devaux et al., 2015). However, the circulatory miR-1 is not sensitive or specific enough to be known as an AMI specific biomarker since it is also affected by factors other than AMI. Moreover, by targeting Protein phosphatase 2A regulatory subunit B56 alpha (*PP2A*), miR-1 has been linked to the arrhythmia mainly through hyperphosphorylation of ryanodine receptor (RyR2; Terentyev et al., 2009). Following hypertension or cardiac infarction, cardiac hyperthrophy, and cardiac fibrosis are common diseases in which the excess amounts of extracellular matrix (ECM) proteins accumulate in cardiac tissue in order to adapt the system to the pathological conditions. Overexpression of miR-29 and miR-21 and downregulation of miR-133 and miR-30 have been linked to ECM remodeling and fibrosis by regulating several components of the ECM including collagen type I alpha 1 and 2 (*Col1A1* and *Col1A2*) as targets of miR-29 (van Rooij et al., 2008), sprouty homolog 1 (*SPRY1*) as a target of miR-21 (Thum et al., 2008b) and connective tissue growth factor (*CTGF*) as target of miR-133 and miR-30c (Duisters et al., 2009). miR-133 is specifically expressed in cardiomyocytes while miR-30 is also detectable in cardiac fibroblasts as well as cardiomyocytes. These and other miRNAs (including miR-328) have been linked to atrial fibrillation which is mainly resulted from structural remodeling and fibrosis (Santulli et al., 2014a). **Table 1** summarizes a list of miRNAs involved in different CVDs (cardio-miRs).

# miRNA-Bearing Exosomes as Communicators of CVD

Recent miRNA-based studies have focused on the role of exosomes as natural delivery vehicles for some proteins, mRNA, and miRNAs which facilitate communication between cells and their neighboring stroma. Exosomes are small vesicles (40–100 nm) originating from the plasma membrane or

*AMI, acute myocardial infarction; AP, angina pectoris; NYHA, New York Heart Association.*

multivesicular bodies (MVBs) and are present in almost all biological fluids. They shuttle between neighboring cells and transfer their cargoes which can be cellular components including proteins, mRNA, and non-coding RNAs. These cargoes can perform regulatory effects and control gene expression in the recipient cells (Pegtel et al., 2010). Exosomes are famous for being the communicators of microenvironment. Exosomal membrane proteins as well as other components are usually similar to their originating cell. Their membranes usually contain higher levels of sphingomyelin, cholesterol and phosphatidylserine compared to their originating cells. Their cargo also might represent the cellular composition of RNAs and proteins or they might show a separate profile. Methodologies including immunoblotting, affinity extraction into magnetic beads and flow cytometry have been used to identify the protein components of exosomes. Profiling of RNAs – miRNAs in particular – is usually performed by means of microarrays, qPCR-based arrays and most recently by next generation sequencing techniques which analyze the transcriptome – miRnome – of each sample.

Recent studies have proved targeted packaging of miRNAs and their biogenesis components including Dicer and AGO2 in exosomes (Melo et al., 2014). Exosome shuttling is implicated in a variety of disease including cancer, CVDs or viral infections. In CVD, exosomal transfer of miRNAs is a well-known mechanism through which, cells educate their adjacent environment in order to confront the pathological condition. In heart ischemia or

fibrosis, paracrine or endocrine secretion of miRNA-bearing exosomes into cardiomyocytes or active fibroblasts of the heart have been demonstrated. This leads to trans-differentiation of fibroblasts into an active state with a different rate of growth factors secretion (van Rooij and Olson, 2009; Yoo et al., 2011). Bang et al. (2014) proved that following heart infarction, exosome-secretion from cardiomyocytes with a new miRNA profile, can reprogram the cardiac fibroblasts leading to cardiac hypertrophy. These mechanisms can introduce potential novel therapeutic targets. As well, recognizing tissue-specific CAF markers, the mechanisms underlying their reactivation and the miRNA signals in these processes, will potentially provide promising targets for CVD therapies. In addition to the fibroblasts in the microenvironment, exosomal transfer of signals is also used for cardiovascular protection after a disease. For example in heart ischemia, secretory exosomes with miRNA cargos from cardiomyocytes have been shown to activate and induce the bone marrow-derived stem cells. These latter cells release the second subset of exosomes that lead to myocardial regeneration or protection (Sahoo and Losordo, 2014). Several investigations have demonstrated the selective packaging of miRNAs in exosomes and their secretion to the stroma of cells by means of signal molecules. This is beyond shedding the vesicles by plasma membrane (Yang et al., 2011; Montecalvo et al., 2012; Stoorvogel, 2012). These disease-specific expression patterns of exosomal miRNAs – which can be non-invasively detected in the body fluids – provides potential molecular markers and promising therapeutic targets for treatment of CVDs. Several circulating miRNAs have been linked to some CVDs. In patients with AMI and myocardial injury, higher levels of miR-208a have been detected in the plasma of patients and they showed higher specificity and sensitivity than conventional biomarkers such as cardiac troponin I (TnI) for diagnosis of the disease (Ji et al., 2009; Wang et al., 2010). Another potential biomarker for AMI is reported by Adachi et al. (2010) who detected higher plasma levels of miR-499 in patients with AMI compared with normal individuals. Using microarray analysis of exosomes obtained from cardiac progenitor cells, Gray et al. (2015) introduced 11 miRNAs significantly up-regulated in response to hypoxic conditions while miR-292 showed the highest variations in these exosomes. Moreover, exosome profiling from cardiac fibroblasts was shown to be enriched with miR-21-3p (miR-21∗). These fibroblasts affect cardiomyocytes and secrete the mediators of cardiac hypertrophy (Bang et al., 2014). Knowing the mechanisms underlying these communications and recognizing potential strong biomarkers for diagnosis and treatment of CVDs will help us in development of future molecular therapeutics.

# miRNA Therapeutics in CVD

In the past decade, outstanding researches in the field of miRNA drugs have changed the face of molecular medicine. miRNA-based therapeutics mainly focus on reinstating the miRNA expression levels. Two main approaches include overexpression of downregulated miRNAs and suppression of overexpressed ones. These approaches have been subject to different modifications in order to improve the efficiency of delivery and less off-target effects. Promising tools in this regard are the oligonucleotides that mimic the endogenous miRNA or suppress the mature miRNA by sequence complementarity. Addition of locked nucleic acids (LNAs) or 2- -*O*-methylation of the antisense oligonucleotides increases the binding specificity while cholesterol conjugation enhances the circulation time, serum stability, and cellular uptake. Expression vectors bearing the pre-miRNA sequences or tandem miRNA target sites (sponges) sound promising for *in vitro* overexpression or suppression of miRNAs, respectively. Another issue to overcome is the efficient delivery of each of the miRNA drugs. Naked oligonucleotides are less efficient due to their instability *in vitro* or *in vivo* which subject them to different nucleases. Lipid-based vehicles, viral systems, and cationic polymers are among the main delivery tools for miRNA-based therapeutics. Each of these strategies has its own challenges and still needs improvements to address problems such as cytotoxicity, immunogenicity, and low efficiency (van Rooij and Olson, 2012). Fiedler et al. (2011) compared the effect of different doses of cholesterol-based anti-miR-24 (antagomirs) on miR-24 expression in cardiomyocytes and cardiac endothelial cells. They showed a cell-type specific tendency or mechanism for antagomir uptake by these tissues. Cholesterol-based antagomirs for silencing miR-21 were also proved to inhibit cardiac fibrosis and dysfunction (Kumarswamy et al., 2012). van Rooij et al. (2008) used anti-miR-29b oligonucleotides (cholesterol modified) after myocardial infarction and observed upregulation of ECM proteins leading to cardiac fibrosis. Also, Bonauer et al. (2009) demonstrated that intravenous injection of miR-92a antagomir, improved the function of damaged tissue in models of myocardial infarction. Inhibition of miR-92a results in neoangiogenic effects and functional recovery of ischemic tissues (Bonauer et al., 2009). Another promising miRNA in CVD therapeutics is miR-208 which is implicated in cardiac remodeling. LNA-modified anti-miR-208 oligonucleotides have successfully prevented pathologically associated cardiac remodeling during diastolic heart disease (van Rooij et al., 2007). A common issue in these approaches is *in vivo* instability and the low homing efficiency of oligonucleotides which results in modest changes in the expression levels of the target protein. Accordingly, improved stabilization or efficient delivery vehicles are required. Examples of such vehicles are adeno-associated viruses (AAVs). AAV9, a specific serotype of AAVs, has tropism for myocardiocytes and enriches in the heart (Bish et al., 2008). In another study Santulli et al. (2014b). proved that miR-126-3p down-regulation using an adeno-viral vector containing miR-126-3p target sites inhibits proliferative vascular smooth muscle cells and prevents restenosis in animal models. miR-126 is an endothelial specific miRNA that regulates vascular integrity and angiogenesis. These strategies provide an efficient tool for cardiac gene transfer and targeted delivery of miRNA-based therapeutics.

Due to their natural role in miRNA secretion and shuttling between different cells, exosomes are of great interest in miRNA therapeutics. Exosomes are flexible in size and cargo type and their non-synthetic nature potentiates them for more efficient and non-immunogenic delivery of cargo while they maintain the cargo integrity and stability. Exosomal membranes contain certain proteins which have binding affinity to specific receptors on the surface of recipient cells. So, they can selectively target cell types of interest and manipulating their miRNA components – as well as other molecular cargoes – will provide promising tools for the future of personalized medicine. In CVD, exosomes can be used as therapeutic agents, as protein delivery carriers or as gene therapy devices. Mesenchymal stem cell- derived exosomes have been investigated in the field of cardiac regenerative medicine especially in myocardial ischemia/reperfusion injuries (Lai et al., 2010). Martinez et al. (2006) have demonstrated that exosomes bearing differentiation signals can affect neovascularization and they are promising for treatment of angiogenic defects. Moreover, several exosomal miRNAs are implicated in angiogenesis and vascular repair. Zhang et al. (2010) showed that exosomes enriched with pre-miR-150 enhance endothelial cell migration. They transfected pre-miR-150/anti-miR-150 oligonucleotides to the THP-1 cell line and collected the conditioned media of these cells which was enriched with the exogenous oligonucleotidesbearing exosomes (Zhang et al., 2010). This conditioned medium affects the migration ability of endothelial cells. Exosome are novel promising elements for the future of CVD treatment due to their targeted delivery capacity and their microenvironmentdependent nature. The latter property triggers their activation in relation to the pathological microenvironment such as pH or substrate concentration. However, further studies would be necessary to overcome obstacles such as engineering and purifying exosomes, cargo loading into them and optimizing their quality and characterization for targeted delivery.

## Conclusion

Cardiovascular diseases are the one of the leading causes of death worldwide. Most of CVDs can be prevented by controlling behavioral and environmental risk factors and early diagnosis and management play an important role in this regard. In this review, we focused on miRNAs as small non-coding RNAs involved in a variety of key cellular processes. Several miRNA biomarkers have been introduced for different CV situations. miRNAs have emerged as attractive novel therapeutics and they have several advantages over other molecular therapeutics due to their small size, conserved sequences and their stability in the body fluids. First anti-cancer miRNA-based drug, MRX-34 (a liposomebased miR-34 mimic) developed by Mirna Therapeutics came to the clinic in 2013 for the treatment of hepatocellular

# References

carcinoma (mirnarx.com; NCT01829971). Current achievements have portrayed a promising future for miRNA-based therapeutics although still several obstacles including their stability, renal clearance, off-target effects, inefficient endocytosis by target cells or the immunogenicity of delivery vehicles, need to be overcome. In CVD, some miRNA-based strategies have resulted in promising findings, including anti-miR-126 approaches that resulted in vascular smooth muscle cells and restenosis inhibition (Santulli et al., 2014b). Also, novel methodologies such as exosome-based delivery of miRNA drugs provide reliable evidence to overcome impediments such as inefficient, unspecific delivery, and immunogenic reactions. But these drugs still need investigations on different aspects of miRNA biology, their long term effects, subsequent biochemical and off-target effects and miRNA pathway analysis.

# Acknowledgment

NN is supported by a grant (#92002177) from Iran National Research Foundation (INSF).

implications for a role of microRNAs in myocardial matrix remodeling. *Circ. Res.* 104, 170–178. doi: 10.1161/CIRCRESAHA.108.182535

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Nouraee and Mowla. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Genetic variants in microRNA genes: impact on microRNA expression, function, and disease

*Sophia Cammaerts1, Mojca Strazisar1, Peter De Rijk1 and Jurgen Del Favero1,2\**

*1 Applied Molecular Genomics Unit, Department of Molecular Genetics, VIB, University of Antwerp, Antwerp, Belgium, 2 Multiplicom N.V., Niel, Belgium*

MicroRNAs (miRNAs) are important regulators of gene expression and like any other gene, their coding sequences are subject to genetic variation. Variants in miRNA genes can have profound effects on miRNA functionality at all levels, including miRNA transcription, maturation, and target specificity, and as such they can also contribute to disease. The impact of variants in miRNA genes is the focus of the present review. To put these effects into context, we first discuss the requirements of miRNA transcripts for maturation. In the last part an overview of available databases and tools and experimental approaches to investigate miRNA variants related to human disease is presented.

#### *Edited by:*

*Mohammadreza Hajjari, Shahid Chamran University of Ahvaz, Iran*

#### *Reviewed by:*

*David Langenberger, ecSeq Bioinformatics, Germany Jun Yasuda, Tohoku Medical Megabank Organization, Japan*

#### *\*Correspondence:*

*Jurgen Del Favero, Applied Molecular Genomics Unit, Department of Molecular Genetics, VIB, University of Antwerp, Universiteitsplein 1, Antwerp 2610, Belgium<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to RNA, a section of the journal Frontiers in Genetics*

*Received: 30 March 2015 Accepted: 05 May 2015 Published: 21 May 2015*

#### *Citation:*

*Cammaerts S, Strazisar M, De Rijk P and Del Favero J (2015) Genetic variants in microRNA genes: impact on microRNA expression, function, and disease. Front. Genet. 6:186. doi: 10.3389/fgene.2015.00186* Keywords: microRNA, genetic variants, expression, function, disease

### Introduction

miRNAs are short, non-protein-coding RNA molecules that mediate post-transcriptional regulation by affecting mRNA stability and translational repression or activation (Vasudevan et al., 2007; Filipowicz et al., 2008).

miRNAs were first discovered in 1993 in *Caenorhabditis elegans* (Lee et al., 1993). In 2000– 2001 many other miRNA genes were identified in *C. elegans* and miRNAs were shown to be widely present in other species (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001). Since then, given their impact on expression, miRNAs have been extensively studied and the miRBase database, the central miRNA sequence repository, continues to expand, collecting novel miRNA genes in a wide variety of species (Kozomara and Griffiths-Jones, 2014). Even in the human genome, with 1881 precursors and 2588 mature miRNA sequences deposited in miRBase version 21, many novel miRNA genes are being identified and published continuously (Friedländer et al., 2014; Cheng et al., 2015; Londin et al., 2015). Despite the extensive research, we are still uncovering aspects of the miRNA maturation process and we have to date only a limited understanding of the functions of specific miRNAs.

miRNAs add a layer of complexity to gene regulation. They target transcripts mainly by complementarity to the seed region, nucleotides (nt) 2–7 of the mature miRNA molecule (Bartel, 2009). This minimal requirement for complementarity results in targeting of many mRNAs by a single miRNA and targeting of one mRNA by several miRNAs (Lim et al., 2005; Peter, 2010; Wu et al., 2010). In addition, miRNAs can have sequence and length variability (isomiRs), potentially resulting in altered targeting capacity and/or specificity. One of the aspects that further influences the complexity of the miRNA repertoire are genetic variants.

This review provides an overview of the current knowledge of the influence of genetic variants on miRNA biogenesis and function. First, miRNA biogenesis and requirements of the miRNA processing enzymes are described, followed by a discussion of isomiRs and their functional implications. In the second part, we highlight the effect of genetic variants on the expression and functioning of miRNAs. In the last part, approaches that are used to identify miRNAs involved in diseases are discussed, with a focus on genetic strategies. Furthermore, we present available tools, databases, and experimental approaches to aid functional characterization studies of disease-associated miRNA variants.

# MicroRNA Biogenesis and Generation of IsomiRs

miRNA genes can be classified in several categories according to their location in the genome. A majority of the currently known human miRNA genes deposited in miRBase are intergenic (68%). Of the intragenic miRNAs, most are intronic (12% of all genes). The remaining genes are located in repeats, lncRNAs, UTRs, or coding regions of host genes (Londin et al., 2015). miRNA genes are often located close to other miRNA genes in so-called clusters. miRNAs located in host genes or clustered with other miRNAs can be cotranscribed, which is supported by good correlation of their expression patterns for several genes (Baskerville and Bartel, 2005). Alternatively, intronic miRNAs can be transcribed independently from their host gene and polycistronic miRNA transcripts can undergo alternative splicing to yield specific miRNA expression (Ramalingam et al., 2014).

In the canonical biogenesis pathway, primary miRNA transcripts (pri-miRNAs) are cleaved in the nucleus by the Microprocessor complex, a complex consisting of ribonuclease Drosha and its cofactor DGCR8, to release the shorter precursor miRNA (pre-miRNA). This precursor molecule is transported to the cytosol by the nucleocytoplasmic transport protein Exportin-5, where it is cleaved by the ribonuclease Dicer to result in a mature miRNA duplex consisting of the mature 5p and 3p strands. The duplex is loaded onto an Argonaute protein, the core component of the RNA induced silencing complex (RISC). One of the strands is discarded, while the other mediates posttranscriptional regulation by base pairing to target mRNAs.

There are different types of target sites. Canonical sites for miRNA targeting are seed matches (nt 2–7 of the mature miRNA) with an adenine opposite from the first nt of the mature miRNA and/or complementarity to the eighth nt of the miRNA. This seed match can be supplemented with complementarity to nt 13–16 (3- supplementary sites). Extended sites of complementarity at the 3 end of the miRNA can also compensate for seed mismatches (3 compensatory sites) (Bartel, 2009). Though a majority of the miRNA-target interactions include seed matches, many non-seed interactions are also observed (Helwak et al., 2013). Similarly, though mRNAs are the predominant miRNA targets, 30% of all miRNA targets are other classes of RNA, such as rRNA, tRNA, snRNA, miRNA, lincRNA, and pseudogenes (Helwak et al., 2013).

Mirtrons, miRNAs encoded within short introns of host genes, bypass processing by the Microprocessor complex. By splicing the mirtron is released and can be, if needed after 5 or 3 tail trimming, transported to the cytosol to be further processed by Dicer. In the study of Ladewig et al. (2012), 240 human mirtrons were identified, indicating that bypassing Drosha is not a rare phenomenon. Other non-canonical miRNA biogenesis pathways have also been discovered and were recently reviewed by Abdelfattah et al. (2014). In the next sections, we will focus on the general miRNA characteristics and substrate requirements of the key enzymes of the canonical miRNA maturation pathway.

# Maturation Enzyme Requirements

Microprocessor Complex Drosha, a nuclear ribonuclease, is the enzyme that cleaves the miRNA hairpin from the primary transcript, whereby it generates a precursor with 3 end overhangs of 2 nt (Lee et al., 2003). An essential cofactor for its pri-miRNA processing activity is the protein DGRC8. The complex containing Drosha and DGCR8, also called the Microprocessor complex, is sufficient for primiRNA processing (Denli et al., 2004; Gregory et al., 2004; Han et al., 2004; Landthaler et al., 2004). The capacity of DGCR8 to bind pri-miRNAs is necessary for Drosha cleavage (Yeom et al., 2006). DCGR8 trimerizes upon binding of primiRNAs and formation of this protein-pri-miRNA complex may allow specific recognition of pri-miRNAs as opposed to other types of RNA and trigger subsequent cleavage by Drosha (Faller et al., 2010).

Several common features of pri-miRNAs were determined by thermodynamic profiling of secondary structure predictions on a set of human and fly pri-miRNAs: the transcript contains a local hairpin structure with an imperfectly paired stem of ∼33 base pairs (bp), consisting of an upper stem (∼22 bp, region of the mature miRNA duplex) and a lower stem (∼11 bp), a terminal loop region and flanking segments that are usually singlestranded or have large bulges or internal loops (Han et al., 2006). Essential requirements of pri-miRNAs for Drosha processing were experimentally determined. The stem-loop structure within the pri-miRNA needs to be larger than the pre-miRNA: it needs an extended stem (i.e., the lower stem, ∼11 bp) and an unstructured region flanking the hairpin (Lee et al., 2003; Chen et al., 2004; Zeng and Cullen, 2005; Zeng et al., 2005; Han et al., 2006; Auyeung et al., 2013). *In vitro* cleavage assays showed that Drosha was able to process pri-miR-30a and pri-miR-16-1 with as little as ∼20–25 nt of flanking regions (Lee et al., 2003; Han et al., 2004). However, for correct *in vivo* processing longer flanking regions are required (Chen et al., 2004). The presence of a large unstructured terminal loop region is beneficial for Drosha processing, as reduction of the size of the predicted terminal loop results in reduced processing (Zeng et al., 2005; Han et al., 2006; Zhang and Zeng, 2010). These properties correspond with the *in silico* determined characteristics of known miRNA hairpins.

Two different models have been proposed to explain how Drosha determines where to cleave the pri-miRNA. One study proposed that the Microprocessor mainly determines the distance from the terminal loop region of the hairpin and cleaves ∼22 bp upstream (Zeng et al., 2005). Another hypothesis proposed that Drosha predominantly cleaves ∼11 bp away from the junction between the lower hairpin stem and the flanking regions (Han et al., 2006). A recent study showed that the complex determines the distance to both ssRNA–dsRNA junctions and that both distances need to be optimal in order to result in precise cleavage (Ma et al., 2013).

Next to the structural prerequisites of the hairpin, positionspecific sequence motifs present in the hairpin flanking segments of the primary transcript (UG motif, CNNC motif) and in the loop region (UGU or GUG motif) can enhance processing efficiency in human cells. Nearly 80% of the human pri-miRNAs that are conserved between human and mice contain at least one of these motifs (Auyeung et al., 2013).

Exportin-5 The transport of pre-miRNAs from the nucleus to the cytosol is mediated by Exportin-5 in the presence of cofactor RanGTP (Yi et al., 2003; Bohnsack et al., 2004; Lund et al., 2004). The RNA structure is the main determinant for precursor binding: a stem of at least 18 bp and a blunt end or a 3 overhang, such as the overhang created by Drosha (Lee et al., 2003), is preferential (Zeng and Cullen, 2004). Upon binding, the 3- 2 nt overhang of the precursor and a large part of the stem is bound by Exportin-5- RanGTP in a sequence-independent manner, which protects the precursor from degradation by nucleases (Bohnsack et al., 2004; Zeng and Cullen, 2004; Okada et al., 2009).

Dicer Dicer, a ribonuclease that processes dsRNA into duplexes with a length of ∼22 nt, processes pre-miRNAs into mature miRNA duplexes (Bernstein et al., 2001; Grishok et al., 2001; Hutvágner et al., 2001; Ketting et al., 2001; Knight and Bass, 2001). Dicer cleaves its substrates gradually with a preference to start at the termini of the RNA duplex and generates 2 nt 3 overhangs (Zhang et al., 2002, 2004). The structure of the termini contributes to the size of the end product, with 2 nt 3 overhangs resulting in products less than 24 nt, while blunt ends result in longer fragments (Vermeulen et al., 2005). Dicer efficiency is affected by several substrate parameters, such as the size of the loop (in case of pre-miRNAs) and the sequence and the size of the overhangs, with 3 overhangs of 2 nt being the most efficient substrate for human Dicer (Vermeulen et al., 2005; Lund and Dahlberg, 2006; Zhang and Zeng, 2010; Park et al., 2011). Dicer binds both the 5 and 3 end of the precursor molecule and mainly cleaves at a distance of 22 nt from the 5 end (Park et al., 2011). The accuracy of the cleavage seems to depend on the distance between this canonical cleavage site and the nearest bulge or the terminal loop (Gu et al., 2012). The Drosha-mediated generation of pre-miRNAs with a 3- overhang of 2 nt and a stem of ∼22 bp thus provides suitable substrates for Dicer. This results in the removal of the loop by Dicer and acquirement of mature miRNA duplexes of ∼22 nt bearing 2 nt overhangs at the 3 end of both strands, on one side generated by Drosha and at the other side generated by Dicer.

Argonaute The mature miRNA duplex is loaded onto an Argonaute protein by the RISC loading complex. This complex matures to RISC when one of the strands, termed the passenger strand, is removed (Czech and Hannon, 2011). The other strand, termed the guide strand, can then, in complex with Argonaute, target RNAs via sequence complementarity for posttranscriptional regulation. The process of strand selection is asymmetric: for many miRNAs one of the strands is preferentially retained. This bias is in part explained by the relative stability of the 5 end of both strands, where the strand with the lowest stability is preferentially retained within RISC (Khvorova et al., 2003; Schwarz et al., 2003). Another contributing factor is the sequence composition of the strands: for human miRNA duplexes with a large difference in expression between both strands, the highest expressed strand has a bias toward a uracil at position 1 and a high purine content, while the lower expressed strand has a bias toward a cytosine at position 1 and a high pyrimidine content (Hu et al., 2009). However, as the dominant strand can vary between tissues (Cloonan et al., 2011), other, yet unknown, factors than sequence and structure of the miRNA duplex itself must be contributing to this variable selection bias.

### Proteins Involved in Regulation of Biogenesis

In addition to the biogenesis key proteins described above, a number of other proteins are known to be involved in the regulation of miRNA biogenesis. The following examples highlight the relevance of the terminal loop region in this regulation. The first example is the protein KSRP. This protein binds directly to the terminal loop of a set of miRNAs and enhances their processing by Drosha and Dicer (Trabucchi et al., 2009). Another example is the regulation by Lin28 and terminal uridyl transferase TUTase4. Lin28 binds a GGAG sequence motif in the terminal loop of specific pre-miRNAs, including let-7 family members, upon which TUTase4 binds to the Lin28-premiRNA complex and uridylates the 3 end of the pre-miRNA. This oligo-uridylation prevents processing by Dicer (Heo et al., 2008, 2009; Hagan et al., 2009). In addition to the Lin28-mediated inhibitory role of TUTase4, this enzyme (and other terminal uridyl transferases) also mono-uridylates a subset of pre-let-7 family members and other miRNAs with a 3- 1 nt overhang, promoting Dicer processing (Heo et al., 2012). An overview of all proteins involved in the regulation of miRNA biogenesis can be found in Siomi and Siomi (2010).

#### IsomiR Repertoire

Mature miRNAs originating from one arm of a pre-miRNA can have sequence and length heterogeneity *in vivo*, with sequences and lengths closely related to, but different from the canonical miRNA sequence reported in miRBase. Such alternative sequences are termed isomiRs (Morin et al., 2008). Different types of isomiRs have been observed: templated additions or deletions at the 5 and/or 3 end of the miRNA, non-templated additions at the 3 end and substitutions within the sequence (Landgraf et al., 2007; Morin et al., 2008; Martí et al., 2010). Alternative processing by Drosha or Dicer, Dicer-independent Argonaute2-mediated cleavage and exonuclease degradation of the 3 terminus of miRNAs can be a source of templated isomiR genesis. Non-templated changes can be established by nucleotidyl transferases, posttranscriptional editing or due to the presence of genetic variants within the transcript (Morin et al., 2008; Cloonan et al., 2011; Neilsen et al., 2012; Lee et al., 2013; Ma et al., 2013).

Since miRNAs target transcripts via imperfect sequence complementarity, isomiRs with a changed sequence for the nucleotides contributing to the target specificity are expected to affect the target spectrum of the miRNA. Given that the majority of miRNA-target interactions include matches to the miRNA seed (Helwak et al., 2013), isomiRs with an altered seed sequence, such as 5 isomiRs, may have a large influence on target specificity. Sets of isomiRs with an altered seed sequence were predicted to gain the capacity to regulate many additional genes and/or lose the capacity to regulate a subset of genes compared to the canonical miRNA (Gong et al., 2012; Tan et al., 2014). Gain or (partial) loss of function of isomiRs with altered seed sequence and even of isomiRs with a preserved seed has been confirmed experimentally (Kawahara et al., 2007; Gong et al., 2012; Chan et al., 2013; Tan et al., 2014). The size of the effect will, however, be largely determined by the absolute and relative expression and stability of the isomiR compared to the canonical miRNA *in vivo* and its binding efficiency to RISC, which can also be affected by the sequence variability (Chan et al., 2013; Llorens et al., 2013). Alternatively, it has been suggested that isomiRs, which often show a high expression correlation with their canonical miRNAs, may function to regulate the same pathways as the canonical miRNA and that the sequence heterogeneity within isomiR populations may act to reduce offtarget effects (Cloonan et al., 2011). The broader functional implications of isomiRs, their contribution to gene expression regulation and how their expression is controlled remains to be further elucidated.

# Impact of Genetic Variants on miRNA Expression and Function

As presented above, miRNA transcripts need to fulfill structural and sequence prerequisites in order to result in expression of the correct mature miRNA sequence(s). Sequence variability in miRNA genes can therefore influence both the expression level as the functionality of the miRNA and consequently will result in differential regulation of their target genes.

Large-scale *in silico* analyses of single nucleotide polymorphisms (SNPs) in human miRNA genes have demonstrated that miRNA genes have lower SNP densities than their flanking regions or the human genome (Saunders et al., 2007; Gong et al., 2012; Han and Zheng, 2013). Within the miRNA gene the mature sequence has a lower SNP density than the precursor, with the seed having the lowest SNP density, reflecting their functional importance. In addition, it was shown that there is a negative correlation between the number of SNPs a miRNA gene harbors and the number of diseases the miRNA is associated with (Han and Zheng, 2013). This again highlights the importance of genetic variants in miRNA genes related to their function and their involvement in human diseases.

Genetic variants can affect miRNAs on several levels. Variants in miRNA promoter regions and other regulatory regions may result in an altered transcription rate. Variants in splice sites of the host gene (for intronic miRNAs) or of the polycistron (clustered miRNAs) could result in aberrant expression patterns. Next, variants within the miRNA transcript can have an effect on miRNA maturation in multiple aspects. They can change the binding affinity of the miRNA hairpin to biogenesis enzymes or accessory proteins (sequence motifs or structural motifs that are changed due to the modified underlying sequence). Variants can lead to altered processing accuracy or to changed frequency of alternative cleavage sites of biogenesis enzymes. They can also lead to altered strand loading bias into RISC. These changes in maturation can all result in altered expression of the canonical miRNA and its existing isomiRs, resulting in deregulation of target genes. It may also result in production of novel isomiRs, which can lead to altered functionality of the miRNA. Lastly, genetic variants within the mature sequence can affect target specificity by generating isomiRs.

Given the potentially huge impact of genetic variants on the tightly regulated miRNA repertoire, and the importance of miRNA-mediated gene regulation, it is not surprising that genetic variants have been found to be causal for or associated with human diseases (Mencía et al., 2009; Hughes et al., 2011). Variants in miRNA biogenesis enzymes and in miRNA binding sites can also lead to impaired miRNA regulation. However, the latter category of variants would likely have a smaller impact than the former two categories, because only the targeting of one gene by one miRNA would be disrupted in case the target does not contain multiple binding sites for that miRNA. Genetic variants in miRNA genes, biogenesis genes and target binding sites associated with human diseases were recently reviewed by Kawahara (2014). Here, we will focus on illustrating the different effects genetic variants can have on miRNA transcription, maturation, and targeting with examples from human miRNA variants studied in disease where possible.

### Variants Influencing miRNA Transcription or Splicing

Though bio-informatics approaches have been used to predict miRNA gene promoters (Saini et al., 2007; Marsico et al., 2013), many miRNA gene promoters have not yet been experimentally validated. Below we describe an example of a variant in a miRNA promoter, which was experimentally delineated. The second example is a case of a variant in a host gene promoter for an intronic miRNA cluster. However, experimental investigation of this type of variants can be complicated by the presence of an independent miRNA promoter in addition to the host gene promoter.

SNP rs57095329 is located in the promoter region of MIR146A, 17 kb upstream of the pre-miR-146a sequence. This variant was found to be associated with systemic lupus erythematosus (SLE). The risk allele reduces the binding to transcription factor Ets-1, another SLE susceptibility gene, and results in decreased promoter activity. Consistent with this mode of action, risk allele carriers have lower expression of miR-146a-5p (Luo et al., 2011).

SNP rs999885 is located in the promoter region of the protein-coding gene MCM7. This variant was associated with a decreased risk of chronic hepatitis B infection, but also with an increased risk of hepatocellular carcinoma (HCC) in individuals with chronic hepatitis B virus infection. The miR-106b-25 cluster, coding for miR-106b, miR-93, and miR-25, is located in the 13th intron of MCM7. HCC patients that carry the risk allele have higher expression of the miRNA transcript in non-tumor liver tissue than non-carriers (Liu et al., 2012). Recently it was shown that this cluster can be transcribed independently of MCM7 via different promoters and that the miRNA polycistronic transcript can undergo alternative splicing (Ramalingam et al., 2014). Therefore it would be very interesting to uncover whether and how this variant influences the relative and absolute expression of the different mature miRNAs in this cluster.

To the best of our knowledge, variants in splice sites that affect miRNA expression in human disease have not been published yet. Nevertheless, the effect of host gene splice variants on mature miRNA expression has been demonstrated by mutagenesis experiments (Janas et al., 2011).

#### Variants Influencing miRNA Maturation

Variants within the pri-miRNA can affect miRNA expression levels by inducing changes in the maturation process, as described above. This effect can take place at several steps in the maturation pathway: Drosha processing, Dicer processing, and/or altering strand preference.

An example of a variant with and effect on the Drosha processing step is rs2910164. This variant is located in the seed sequence of miR-146a-3p and was predicted to lead to a mismatch in the hairpin stem. The heterozygous variant genotype was associated with increased risk of papillary thyroid carcinoma (PTC). After overexpression of pri-miR-146a with the G allele or the C allele in cells, the C allele resulted in nearly twofold reduction of pre-miR-146a compared to the G allele. The reduced production of pre-miR-146a from primiR-146a with a C allele was also confirmed by an *in vitro* processing assay (Jazdzewski et al., 2008). An example of a primiRNA variant affecting the first processing step is a variant located four bases upstream of the miR-510-5p sequence. This variant was identified in a study screening X-linked miRNA genes in male schizophrenia patients and control individuals. The variant was predicted to alter the secondary structure around the Drosha processing site. Functional validation showed that the variant results in increased expression of pre-miR-510, miR-510-5p, and miR-510-3p (Feng et al., 2009; Sun et al., 2009).

A variant affecting Dicer processing is rs546098287, located within the seed sequence of miR-96-3p. This variant was identified as the causative mutation in an Italian family with non-syndromic hearing loss (Soldà et al., 2012). Two segregating point mutations in miR-96-5p were previously identified in two Spanish families with non-syndromic hearing loss, demonstrating the importance of this gene in the disease pathogenesis (Mencía et al., 2009). The mutation in miR-96- 3p reduces the processing from pre-miR-96 to both miR-96-5p and miR-96-3p. This is likely induced by a structural change, because the variant was predicted to enlarge an internal loop in the hairpin stem and when a second variant was introduced to restore the original stem structure, the expression was largely brought back to normal levels (Soldà et al., 2012).

An example of a variant influencing the relative strand abundance is a variant in MIR133A2, identified in a patient with atrial fibrillation. The variant is located at the 3 end of the miR-133a-3p sequence and was predicted to alter the secondary structure of the hairpin at the base of the miR-133a duplex. miR-133a was found to be highly expressed in atrial tissue, with the 3p strand being the dominant one, while miR-133a-5p represented less than 1% of all the miR-133a reads. Functional validation experiments demonstrated that the variant resulted in increased miR-133a-5p expression, but had no significant effect on miR-133a-3p expression. The variant thus increases the relative abundance of the 5p strand compared to the 3p strand (Ohanian et al., 2013).

The cases described above are all variants located either within the precursor sequence or close to it. A study by Diederichs and Haber (2006) reported the functional validation of 15 primiRNA variants ranging from 5 to 133 nt outside the pre-miRNA sequence, identified in cancer cell lines. Despite some of these variants had predicted structural changes at or near the base of the miRNA hairpin, none of the variants displayed detectable differences in mature miRNA expression. On the other hand, for the variant rs11134527, which is located 96 nt outside the MIR218-2 hairpin sequence, it was shown that it does change the expression of miR-218-5p significantly (Gao et al., 2013). Therefore variants located further outside the hairpin can still affect miRNA biogenesis, although variants closer to the hairpin are more likely to have a higher impact.

#### miRNA Variants Influencing Targeting

Beside resulting in altered expression, miRNA variants can also result in altered target specificity by creating isomiRs.

An interesting example of a variant creating a polymorphic isomiR is rs2910164 in miR-146a-3p. This variant also affects Drosha processing as described above (Jazdzewski et al., 2008). The isomiR and canonical miR-146a-3p are predicted to share only a small set of target genes. Transcriptome analysis in healthy and in tumor tissue of PTC patients with GG or GC genotype showed that hundreds of genes (358 in unaffected tissue, 575 genes in tumor tissue) were differentially expressed (Jazdzewski et al., 2009). Even though this does not provide direct evidence that all those genes are direct targets of the isomiR, it does give insight in the large-scale downstream changes isomiRs may induce.

Direct evidence of altered targeting was shown in a study investigating the effect of eight seed SNPs and three mature sequence SNPs (among which one deletion) identified in dbSNP in nine miRNA genes. Four of the tested miRNAs (miR-627-5p, miR-379-5p, miR-499-3p, and miR-124-3p) partially or completely lost their potential to suppress their original target due to a SNP in their seed sequence. The seed sequence variant rs2620381 in miR-627-5p not only resulted in loss of targeting of SEMA3F, but also in gained capacity to suppress ATP6V0E1 (Gong et al., 2012). In addition, miR-379-5p, miR-940, and miR-34a-5p containing variants in the mature sequence also displayed partial or total loss of function of their original mRNA targets tested.

# Studying miRNAs Involved in Disease

#### miRNA Profiling and Genetic Studies

To study if and which miRNAs play a role in the pathogenesis of a disease, two main strategies can be used: a miRNA profiling approach and a genetic approach.

In the miRNA profiling approach, the affected tissue of a group of patients is subjected to miRNA expression profiling and compared to the expression profile of either the same tissue in healthy individuals, tissue of patients with a different disease or subphenotype, or to adjacent non-affected tissue of the same patients (Calin et al., 2005; Kan et al., 2012; Feliciano et al., 2013; Huang et al., 2014). The tested miRNAs can range from selected candidate miRNAs to a genome-wide approach and different technologies are available, such as RT-qPCR, microarray based methods or RNA sequencing (Pritchard et al., 2012). The miRNA expression profiles are compared between the disease phenotype and the controls in order to determine whether and which miRNAs are differentially expressed. Resulting signatures may be used as diagnostic or prognostic biomarkers (Calin and Croce, 2006) and can provide clues about the role of miRNAs in the disease mechanism. Additionally, interesting miRNAs can be further investigated to dissect their mode of action.

In the genetic approach, the starting point is a linkage or an association analysis (genome-wide or candidate gene approach) including but not necessarily limited to miRNA genes between patients and control individuals (Mencía et al., 2009; Schizophrenia Psychiatric Genome-Wide Association Study (GWAS) Consortium, 2011; Zhang et al., 2015). When a genetic variant in or near a miRNA gene is associated with or is causal for the disease, functional validation can be initiated to assess its effect and its role in the pathogenesis of the disease. Variants in miRNA genes linked to the disease could also be used for disease diagnosis, as exemplified by the patent on rare miRNA gene variants identified in schizophrenia patients (Sommer and Rossi, 2012). The genetic screening approach focused on miRNA genes is not yet as frequently applied on a large scale as genome-wide miRNA profiling.

Both strategies have limitations and benefits. General considerations regarding miRNA profiling were reviewed by Pritchard et al. (2012). In the study of disease, genome-wide miRNA profiling can provide a global insight in which miRNAs may play a role in the disease by generating disease-specific miRNA expression signatures. An advantage of this approach is that the deregulated miRNA provides a first indication of its possible functional role in the disease. The main limitation, however, is that the differential expression of a miRNA in itself does not provide an indication of causality: the altered expression can be a causal factor, a consequence, or could even be unrelated to the disease pathogenesis (Kan et al., 2012). Another constraint is that the affected tissue has to be accessible. This is especially problematic in the study of neurological diseases. Alternatively, brain tissue of deceased patients and healthy individuals, if available, can be used. However, though many miRNAs seem to be relatively stable (Bail et al., 2010; Winter and Diederichs, 2011), the half-lives of some miRNAs are less than 1 hour (Sethi and Lukiw, 2009). This implicates that when brain samples with different postmortem delay from death to tissue processing are compared, variability may arise due to differential instability of the miRNAs in the samples. The restricted access to postmortem brain samples can also result in suboptimal sample sizes. A much more accessible proxy for unavailable affected tissues is blood. While this may provide a good avenue for determining diagnostic or prognostic biomarkers, it is not clear whether the miRNAs deregulated in blood would also be deregulated in the affected tissue. In addition, several factors can influence miRNA expression in blood, such as age, drug use, and other diseases (Margis et al., 2011; de Boer et al., 2013; Meder et al., 2014).

The main advantage of a genetic approach is that when a variant is associated with disease risk, it gives an indication that the miRNA involved has a primary effect in the disease pathomechanism (large or small contributing effect), which cannot be determined by expression profiling. This advantage also has a downside: while the association or linkage analysis may indicate a miRNA variant is likely to be involved in the disease, it is not at all clear whether the identified variant has a functional effect and whether the expression of that miRNA is altered in the disease state. Another asset of the genetic approach is that diseases for which the main affected tissue is not accessible can still be studied because genomic DNA can be isolated from more accessible patient material, such as blood or saliva. There is, however, still a chance of missing a mutation, if the patient is mosaic for the mutation and the load of the mutation is lower in the examined tissue than in the affected tissue.

Both approaches are complementary and should both be used to determine the involvement of a miRNA in relation to a disease: genetic approaches can pinpoint the causal or risk factors involved in the disease, while expression studies reveal whether and how the expression of the disease-associated miRNAs is altered in the affected tissue. Neither approach is sufficient to determine both. An example of a study combining both approaches is the study of Calin et al. (2005) where miRNA profiling in affected tissue of patients was performed, followed by variant screening in a set of relevant miRNA genes. An expression signature of thirteen miRNA genes was found to be able to distinguish between patient subgroups. In two of these genes, patient-specific variants were found. One of the variants was a functional germline mutation in pri-miR-15a/16-1. On the other hand, when a genetic variant is associated with a disease, usually only the expression of that particular miRNA is assessed in patient material. The profiling and genetic approach has also been combined on a large scale by miRNA profiling in blood samples of patients with different diseases and investigating whether the deregulated miRNA genes were located within 250 kb of published GWAS significant SNPs (Keller et al., 2011). While on average 103 deregulated miRNAs were identified in each disease, of all the deregulated miRNAs they found only six miRNA genes in proximity to a GWAS hit associated with the same disease. Combining the genetic and the profiling approach on the same set of patients could thus provide an invaluable amount of information on both DNA and RNA level from which the diseasecontributing miRNAs can be separated from the miRNAs that are probably deregulated due to secondary disease processes.

#### Databases and Tools for Genetic Approaches

To provide direct evidence that a genetic variant in or near a miRNA gene and the deregulated miRNA expression in disease tissue are linked, detailed functional validation is required. Functional validation is labor-intensive, costly, and time-consuming, therefore researchers usually prioritize the potential candidate variants using *in silico* approaches. In this section, we will focus on available databases and tools for the impact prediction of genetic variants on miRNA function (**Tables 1** and **2**). Different types of resources for miRNA research have been reviewed by Vlachos and Hatzigeorgiou (2013).

A first step in the assessment of the relevance of a genetic variant or a set of genetic variants is determining whether the variant is located near or in a specific region of a miRNA gene. For a single variant of interest, the variant location may be assessed in a genome browser to determine the position of the variant compared to neighboring miRNA genes. To evaluate the exact location of the variant relative to the different miRNA gene regions (e.g., seed, mature, terminal loop), one also has to compare this location with the information provided about the gene in miRBase (Kozomara and Griffiths-Jones, 2014). This procedure is tedious and error-prone, especially when investigating multiple miRNA variants. For variant lists such as those derived from massively parallel sequencing projects, the first annotation step can be done with any of the annotation tools available to determine which (miRNA) genes, if any, are located near the variants.

Online repositories compiling information about known SNPs in miRNA genes, such as miRNASNP (Gong et al., 2012), miRNA SNiPer (Zorc et al., 2015), and miRvar (Bhartiya et al., 2011), can be used to extract exact location information for these variants relative to the miRNA hairpin. A limitation for the last two databases is that the search needs to be initiated by using the miRNA gene name instead of the SNP ID. In addition, databases cannot be used when investigating novel variants and are less suitable when investigating multiple variants in several miRNA genes. To eliminate the time-consuming processes related to assessing the position of variants in the context of miRNA genes and what their predicted structural impact is, we recently developed the freely available software miRVaS1 . Required input is the genomic location of a (known or novel) variant or of a list of variants.

The next possible step in determining the potential relevance of a genetic variant is predicting its effect on the secondary structure of the miRNA. For known SNPs within miRNA genes, structure prediction results of the miRNA hairpin with and without the variant can be found in the miRNASNP database (Gong et al., 2012). The difference in free energy is calculated and a predicted effect on the miRNA expression is provided, based on the assumption that variants destabilizing the hairpin

1http://mirvas*.*bioinf*.*be/

TABLE 2 | Overview of described software.

will reduce the expression and stabilizing variants will increase the expression of the mature miRNA (Gong et al., 2012). For novel variants the RNA secondary structures can be predicted for wild type and variant sequences using web-based RNA structure prediction tools, such as the Mfold or RNAfold web servers (Zuker, 2003; Gruber et al., 2008), and compared. Again, this process is time-consuming and not suitable for the analysis of large variant sets. miRVaS1 automates this type of analysis, predicting structural impact for lists of variants, needing only the genomic location and alternative allele of the variant.

If a variant is located in a miRNA seed sequence, the effect on target binding can also be assessed *in silico*. Both PolymiRTS database (Bhattacharya et al., 2014) and miRNASNP (Gong et al., 2012) contain lists of predicted gain and loss of target binding sites for seed sequence variants that can be browsed. To assess the impact of variants in miRNA promoter regions on transcription factor binding, the dPORE-miRNA database can be queried (Schmeier et al., 2011). Algorithms to predict Dicer cleavage patterns and preferential strand loading into RISC, such as PHDcleav and RISC binder (Ahmed et al., 2009, 2013) could also be useful to assess the impact of variants. The predicted effect on Dicer cleavage and RISC binding induced by variants is incorporated in the miRvar database (Bhartiya et al., 2011).

#### Functional Characterization of a Disease-Associated miRNA Variant

Using tools and databases, a set of genetic variants can be prioritized based on their potential functional relevance. The most promising candidates can then be chosen for direct functional validation in cell-based assays. The assays used depend on the location of the variant and the hypothesis.

For variants in miRNA promoter regions, the wild type and variant promoter can be cloned into a promoter-less reporter gene vector and transfected (transiently or stably) into cell lines to assess whether there is a difference in promoter activity due to the variant and whether the variant promoter has different binding affinity to transcription factors (Luo et al., 2011).

To assess the impact of a genetic variant on miRNA biogenesis and/or targeting, cell lines overexpressing wild type and mutant miRNAs are usually established. The constructs should include the full precursor gene and upstream and downstream flanking sequences. The inclusion of flanking regions is crucial: the biogenesis machinery in the cell may not efficiently recognize or process the transcript when the flanking regions are absent or too small, as evidenced by the study of Chen et al. (2004) where only constructs for miR-223 with at least 40 nt of flanking region resulted in detectable miR-223 expression. Using upand downstream flanks of 125 nt resulted in mature miRNA expression for all 13 tested miRNAs in this study, showing that the incorporation of longer flanks in miRNA constructs is generally a good strategy. To assess the effect on biogenesis, miRNA expression analysis is performed. Comparison of wild type and variant miRNA levels can be done at the level of the mature miRNA (Shen et al., 2009), assessing the total effect of the variant on the whole biogenesis process, or it can be done on several maturation levels of the miRNA, to deduce the exact step of the biogenesis that is affected by the variant (Duan et al., 2007).

If a variant is located in the mature miRNA sequence, targeting may be affected. Investigating which genes and pathways are affected due to the variant may provide insight in the disease mechanism by identifying new pathways and/or by confirming pathways previously hypothesized to be involved in the disease. Investigation and validation of direct interactions between miRNA and target RNAs can be done using reporter assays. Hereto, the potential binding site is cloned downstream of a reporter gene and cells are cotransfected with the miRNA and the reporter vector to assess whether the miRNA (or the variant miRNA) can target the site and thus reduce the reporter gene activity (Duan et al., 2007; Gong et al., 2012). While this allows confirmation of direct interactions, overexpression of the binding site and the miRNA may also lead to validation of interactions that may not take place endogenously in the cell type or tissue of interest, due to, for instance, absence of colocalization, or co-expression.

Investigation of variant induced targeting alterations can also be performed on a large scale by subjecting the wild type and variant miRNA expressing cells to transcriptome and/or proteome profiling. Single genes or proteins can be analyzed for differential expression. Pathway analysis can also identify which set of genes are influenced by the variant miRNA and may lead to biological meaningful findings about the variant miRNA and the pathways in the cell it affects in the disease state (Jazdzewski et al., 2009). If a variant is located outside the mature miRNA sequence, this approach can still be used if the variant affects mature miRNA expression, as the altered expression of the miRNA will also deregulate its target genes (Strazisar et al., 2015). However, in contrast to the reporter gene assays, these large-scale approaches cannot distinguish between direct and indirect targets of the miRNA, so target prediction algorithms need to be used to determine which of the deregulated genes can potentially be targeted directly by the miRNA.

Different crosslinking and immunoprecipitation (CLIP) approaches were developed and applied for global miRNA–RNA target interaction identification by Argonaute precipitation, such as HITS-CLIP and PAR-CLIP (Ule et al., 2005; Chi et al., 2009; Hafner et al., 2010). In these methods, RNA and proteins are crosslinked in cells by UV irradiation (in case of PAR-CLIP after incorporation of photoactivatable nucleosides). After cell lysis and partial RNA digestion, Argonaute-RNA complexes are pulled down and purified stringently. Subsequently, coprecipitated RNA is sequenced to identify which miRNAs and which target RNA regions were bound to Argonaute. Because miRNA and target RNAs are not physically linked to each other during the procedure, *in silico* target prediction is required to assess the interactions between the identified RNA sequences. These approaches thus provide enrichment for actual target RNA sites bound to Argonaute and limits the target prediction to these sites and to the detected miRNAs.

A method that can identify direct miRNA-target interactions on a large scale is the crosslinking, ligation, and sequencing of hybrids (CLASH) technology (Kudla et al., 2011; Helwak et al., 2013). Similar to the CLIP techniques, cells are UV irradiated to mediate crosslinking, after which Argonaute-RNA complexes are pulled down and RNA is partially digested. Then a ligation step is performed to ligate miRNAs to their target RNAs. The RNA is sequenced to identify chimeric RNAs. The non-chimeric reads provide the same information as the CLIP experiments, while the chimeric RNAs contain linked miRNA and target RNA fragments and hence provide direct proof of miRNA-target interactions. This technology may thus be used to directly identify whether mature miRNA variants alter targeting capacity of a disease-associated miRNA.

Together, these approaches can be used to assess what the effect of the genetic variant is on the expression and the functionality of the miRNA.

# Concluding Remarks

In this review we summarized the current knowledge of the miRNA maturation process, focusing on the prerequisites of the miRNA transcript to be processed. From these requirements it is clear that the RNA structure, and thus also the underlying sequence determining the structure, are of paramount importance for maturation into mature miRNAs. Thus genetic variants in miRNA genes can have large effects, not only on the miRNA gene itself, but due to its role as a fine-tuner of gene expression and translation, also on its downstream targets. In line with this, different miRNA variants have been found to be involved or hypothesized to be involved in human disease. We also highlighted the biological relevance of genetic variants located within or near miRNA genes and provided an overview of *in silico* and experimental approaches to investigate the effect of these variants on miRNA expression and function. This in turn could then be correlated to changes in miRNA and target

# References

gene abundance in the affected tissue of patients, as a first step in the understanding of the role of the miRNA in the disease pathomechanism.

As we are now coming to realize that the miRNA repertoire is much more complex than previously appreciated with the recognition of the wide-spread presence of isomiRs and their functional relevance, the possibilities of intronic miRNAs being transcribed independently from their host genes and even alternative splicing, many more questions arise. Though many aspects of miRNA biogenesis and functioning have already been unraveled, more investigations will need to be done to be able to fully grasp the regulation and the genesis of the correct expression pattern of the full miRNA repertoire in the cell. We also anticipate that, especially for the investigation of genetic variants in disease, we will need to undertake comprehensive and integrative approaches to be able to fully appreciate how certain genetic variants can undermine this regulatory system and lead to disease, while others remain completely harmless.

# Author Contributions

All authors contributed to the conception of the work. SC wrote the manuscript. MS, PD, and JD provided input and revised the manuscript. All authors approved the final manuscript.

# Acknowledgments

This research was funded by Research Foundation – Flanders (G027410N to JD) and by the University Research Fund (UA-KP BOF/MS-2013 6264 to MS).

inherited hearing loss in an Italian family by altering pre-miRNA processing. *Hum. Mol. Genet.* 21, 577–585. doi: 10.1093/hmg/ddr493

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2015 Cammaerts, Strazisar, De Rijk and Del Favero. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Noncoding RNAs in human saliva as potential disease biomarkers

Xianzhi Lin1 †, Hsien-Chun Lo1 †, David T. W. Wong2, 3, 4 and Xinshu Xiao1, 2, 4 \*

*1 Department of Integrative Biology and Physiology, University of California, Los Angeles, Los Angeles, CA, USA, 2 Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA, 3 School of Dentistry, University of California, Los Angeles, Los Angeles, CA, USA, 4 Jonnson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, CA, USA*

#### Keywords: RNA-Seq, miRNA, piRNA, circular RNA, extracellular RNA, biomarker, saliva

#### Edited by:

*Mohammadreza Hajjari, Shahid Chamran University of Ahvaz, Iran*

#### Reviewed by:

*Edward K. L. Chan, University of Florida, USA Daniel Wai Hung Ho, The University of Hong Kong, China Jun-An Chen, Academia Sinica, Taiwan Helen K. W. Law, The Hong Kong Polytechnic University, China*

#### \*Correspondence:

*Xinshu Xiao<EMAIL_ADDRESS>† *These authors have contributed equally to this work.*

#### Specialty section:

*This article was submitted to RNA, a section of the journal Frontiers in Genetics*

#### Received: *03 March 2015* Paper pending published: *27 March 2015* Accepted: *22 April 2015* Published: *07 May 2015*

#### Citation:

*Lin X, Lo H-C, Wong DTW and Xiao X (2015) Noncoding RNAs in human saliva as potential disease biomarkers. Front. Genet. 6:175. doi: 10.3389/fgene.2015.00175* Human saliva emerged as a research material in as early as the 17th century when investigators sought to understand the basis for salivary secretion (Garrett, 1975). Over the centuries, the focus of salivary research has evolved greatly and a wide range of topics has been examined (Garrett, 1975; Schipper et al., 2007). It is now known that the functions of saliva include at least lubrication, digestion of food, remineralization, prevention of demineralization, protection against microbial and viral infection, speech facilitation, and maintenance of oral and general health (Schipper et al., 2007; Malathi et al., 2014).

One active research area related to human saliva is the discovery of biomarker molecules for a variety of diseases. Compared to other body fluids, saliva is easily accessible in a noninvasive manner. However, it is also immediately exposed to the outside environment, thus may be confounded by a wide variety of environmental factors. Nevertheless, previous studies have established that at least some human molecules in saliva are highly stable and potential biomarkers have been examined for a number of oral and systemic diseases (Bonne and Wong, 2012; Schafer et al., 2014).

# Key Components of Human Saliva

Saliva consists of both cellular and fluid contents. Epithelial cells, leukocytes, and erythrocytes are the three major human cell types, which co-exist with bacterial cells in human whole saliva (Aps et al., 2002). The fluid content of saliva is primarily generated by the salivary glands, but with additional contributions from blood, oral tissue, bacteria, viruses, and food remnants (Schipper et al., 2007). It mainly consists of water, macromolecules (such as glycoproteins, enzymes), small organic molecules, inorganic components (e.g., electrolytes), and metabolites from oral bacteria (Almstahl and Wikstrom, 2003; Aps and Martens, 2005; Schipper et al., 2007).

Many biomarker studies focused on profiling and quantification of proteins or RNA molecules in saliva. Since the diseases investigated for salivary biomarkers are often systemic, it is of great interest to identify circulating protein or RNA molecules that may have originated from diseaserelevant cells (such as tumor cells). Such molecules reside outside of the cells in saliva and are often captured in cell-free saliva (CFS), the fraction of saliva with cellular contents removed (often by centrifugation). Most of the salivary RNAs appear to be highly degraded compared to full-length mRNAs in cellular compartments, possibly due to presence of RNA degradation enzymes in saliva and other body fluids (for circulating mRNAs) (Park et al., 2007). Notably, certain miRNA and mRNA molecules were shown to be highly stable, possible owing to protection by exosomes or protein complexes (Park et al., 2006, 2009; Palanisamy and Wong, 2010; Palanisamy et al., 2010).

# Technologies for Salivary RNA Profiling

About a decade ago, microarrays were applied to characterize the global profile of mRNAs in saliva (Li et al., 2004; Park et al., 2007). These studies revealed that there were over one thousand distinct mRNA molecules in human CFS (Li et al., 2004). In addition to mRNAs derived from coding genes, many noncoding RNAs (ncRNAs) were also detected. Data from these studies demonstrate that there are hundreds of microRNAs (miRNAs) in human saliva, and most of them likely exist in exosomes (Michael et al., 2010; Gallo et al., 2012).

However, microarray techniques have inherent limitations, such as the dependence on gene annotation and crosshybridization noise. In recent years, more powerful techniques based on next generation sequencing (NGS) revealed additional coding and ncRNA species in human saliva (Spielmann et al., 2012; Bahn et al., 2015). In contrast to the hybridization-based microarrays, RNA sequencing (RNA-Seq) offers single nucleotide information, high sensitivity and accuracy in transcript detection, and the capability to detect novel RNA species and transcript isoforms (Lee et al., 2011, 2013; Li et al., 2012). An increasing number of bioinformatic tools are emerging for analysis of RNA-Seq data, ranging from rapid short read aligners to detailed examination of RNA expression patterns (Oshlack et al., 2010). Owing to these improvements, the catalog of human genes, especially ncRNA genes, has been greatly expanded (refer to Sai Lakshmi and Agrawal, 2008; Kozomara and Griffiths-Jones, 2014; Xie et al., 2014 for ncRNA databases).

# ncRNA Molecules in Saliva

In 2012, the Wong group reported the first global characterization of the human salivary transcriptome using high-throughput RNA-Seq (Spielmann et al., 2012). This study demonstrated that saliva harbors a wide variety of RNA species. More than 4000 distinct RNA molecules derived from coding or noncoding human RNAs were identified, including a small number of miRNAs. This study established that the RNA content in saliva is very diverse, which should be fully explored in future biomarker studies.

Recently, another in-depth analysis of human salivary extracellular ncRNA revealed novel insights regarding its RNA content and provided a comparative view of salivary ncRNAs relative to those of other body fluids (Bahn et al., 2015). Using human CFS, this study confirmed previous findings that miRNAs are stably and abundantly present in saliva (Patel et al., 2011), often harbored within exosomes (Gallo et al., 2012). miRNA expression profiles of healthy individuals were quantified and compared. Highly concordant miRNA expression was observed across individuals. Furthermore, considerable similarity was observed between miRNA expression levels of saliva and other body fluids (blood, cerebral spinal fluid (CSF)). Thus, these data suggest that salivary miRNAs could serve as candidate biomarkers, at least with equivalent promise as those derived from more invasive fluids.

 The number of abundant piRNAs is less than that of miRNAs in saliva, despite the large number of annotated piRNAs in various databases (Sai Lakshmi and Agrawal, 2008; Bahn et al., 2015). Nevertheless, piRNA expression levels were highly concordant between healthy individuals, similarly as miRNA levels. However, in contrast to the consistent expression profile of miRNAs across body fluids, piRNAs were highly exclusive to saliva with very low abundance in blood or CSF. These observations indicate that salivary piRNAs may have originated from cells in the oral mucosa or salivary glands, rather than circulating from systemic organs via blood. Nevertheless, salivary piRNAs may impose systemic functional impact, which needs to be further investigated.

Another novel finding in this study was the discovery of circular RNAs (circRNAs) in CFS, which is the first report of the presence of circRNAs in an extracellular fluid (Bahn et al., 2015). CircRNAs were originally identified in RNA viruses (Sanger et al., 1976; Kos et al., 1986). Later, intracellular circRNAs generated from specific exons of coding genes were reported (Nigro et al., 1991; Cocquerelle et al., 1992; Capel et al., 1993). Recent studies demonstrated that circRNAs exist in many different cell types and species (Salzman et al., 2012, 2013; Jeck et al., 2013; Memczak et al., 2013). Some circRNAs are likely noncoding (Capel et al., 1993; Memczak et al., 2013; Guo et al., 2014), but others may code for proteins (Wang and Wang, 2015). The function of most circRNAs remains unknown. Two circRNAs were shown to function as miRNA sponges (Hansen et al., 2013; Memczak et al., 2013). However, this function may not apply to the majority of other circRNAs as they lack bioinformatic evidence of significant miRNA complementarity (Guo et al., 2014). The discovery of circRNAs in CFS indicates that this type of molecule may have extracellular function and should be considered as a type of candidate biomarker (Li et al., 2015).

Although mRNAs are highly degraded in saliva and other body fluids, small ncRNAs are often stable with reproducible expression across individuals.  The similarity between miRNA profiles of saliva and other body fluids (Weber et al., 2010; Bahn et al., 2015) strongly supports the potential of using miRNAs (and possibly other ncRNAs) from human CFS as biomarkers for various human diseases.

# Salivary ncRNAs as Potential Biomarkers for Diseases

Although at an early stage, salivary ncRNA studies have revealed potential disease biomarkers. Thus, far, most studies focused on miRNA expression in saliva. **Table 1** summarizes a number of

*aValidated by qRT-PCR.*

*bArea under Receiver Operating Characteristic (ROC) curve.*

*cChange in disease relative to control. The number in the parentheses represents the fold change.*

*dA combination of four miRNA exhibits discriminating power.*

studies where miRNAs were assessed as putative biomarkers for oral squamous cell carcinoma (OSCC) (Park et al., 2009), parotid gland tumors (Matse et al., 2013), and esophageal cancer (Xie et al., 2013). In addition to oral and esophageal diseases, salivary ncRNAs were also examined as potential biomarkers for systemic diseases. In a clinical study focusing on Sjögren's Syndrome, a chronic autoimmune disease, the authors observed different miRNA expression patterns in minor salivary glands of Sjögren's Syndrome patient compared to healthy individuals (Alevizos et al., 2011). The disease group can be clearly distinguished from the normal group using the miRNA expression profile by principal components and hierarchical clustering analyses. A very recent study focused on pancreatic cancer, using samples of patients with pancreatic cancer, benign pancreatic tumor or healthy controls (Xie et al., 2015). The authors observed significant down-regulation of miR-3679-5p and up-regulation of miR-940 in the cancer group compared to the other groups (**Table 1**), suggesting salivary miRNA may potentially be used for early detection of pancreatic cancer.

In addition to human ncRNAs, exogenous ncRNAs in saliva may also serve as potential disease biomarkers. The humanassociated microbial communities have profound impact on the individual's physiological outcome (Human Microbiome Project Consortium, 2012). In human saliva, over 1500 bacteria have been identified and completely sequenced [Human Oral Microbiome Database; http://www.homd.org/]. Some studies have shown that saliva can be used to detect microbial infection (Schafer et al., 2014). In addition, both DNA and RNA viruses were detected in human saliva from viral infected hosts (Liou et al., 1992; Chen et al., 1997; Vieira et al., 1997; Shugars et al., 2001; Hermida et al., 2002; Mackiewicz et al., 2004; Goncalves et al., 2005; Balamane et al., 2010; Pride et al., 2012), thus could serve as biomarkers of viral infection. Thus, far, little is known regarding the landscape and function of exogenous ncRNAs in saliva.

# Future Challenges and Perspectives

A comprehensive ncRNA expression profile is emerging for human saliva including the presence of miRNAs, piRNAs, and circular RNAs (Ogawa et al., 2013; Bahn et al., 2015). More RNA species may be discovered in the future given the rapid evolution of new technologies and powerful bioinformatic methods. The value of saliva as a body fluid for biomarker discovery is just becoming widely recognized. However, there are a number of challenges in this field, most of which are general to usage of any body fluid in biomarker discoveries. One challenge lies in the unbiased isolation of short and long RNA molecules from saliva samples. Although this topic is under intensive investigation, improved methods that can retain most RNA species unbiasedly in an operator-independent manner are highly desired.

Another challenge is accurate quantification of ncRNA abundance, which is key to biomarker assessment. RNA yield from different samples may vary greatly, which calls for effective experimental and bioinformatic methods for normalization of RNA expression. Most RNA-Seq studies discussed above calculated RNA expression levels by normalizing the number of reads of a particular RNA molecule against the total number of mapped reads (i.e., the RPKM measure Mortazavi et al., 2008). However, to estimate the absolute concentration of an RNA molecule in a sample, synthetic spike-in RNAs with known concentration should be added to the RNA sample before library generation. This approach necessitates accurate measurement of RNA concentration of the sample and synthesis of a large number of spike-in RNAs with varying sequence contents and concentrations (see Williams et al., 2013 for a demonstration of this approach). This challenging approach, though highly desirable and necessary for clinical usage of a biomarker, has not been widely adopted.

A third major challenge is a better understanding of the biogenesis pathways of human ncRNAs in saliva, which constitutes the basis to assess whether and to what extent

# References

ncRNA expression can reflect a person's health condition. Salivary RNAs could be derived from systemic organs or local cells of the oral cavity. Cellular origins of candidate biomarkers for various diseases should be further examined to substantiate our understanding of the validity of the biomarkers. Indeed, the presence, origin, and functional roles of disease biomarkers are all essential questions general to studies of different types of biomarkers and diseases. A valid disease biomarker should be directly involved in disease mechanisms or indirectly associated/correlated with key pathways driving the pathogenesis of disease. The ultimate question is how knowledge gained in biomarker studies could be utilized to develop effective strategies for disease prevention and treatment, which closely relies on a clear understanding of disease mechanisms.

# Acknowledgments

This work was supported by NIH grants R01HG006264 (to XX) and UH2 TR000923 (DTWW).

**Conflict of Interest Statement:** David T. W. Wong is co-founder of RNAmeTRIX Inc., a molecular diagnostic company. He holds equity in RNAmeTRIX, and serves as a company Director and Scientific Advisor. The University of California also holds equity in RNAmeTRIX. Intellectual property that David Wong invented and which was patented by the University of California has been licensed to RNAmeTRIX. Additionally, he is a consultant to PeriRx. The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2015 Lin, Lo, Wong and Xiao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Phage display and targeting peptides: surface functionalization of nanocarriers for delivery of small non-coding RNAs

Babak Bakhshinejad\*

Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran

Keywords: phage display, peptide library, targeting, delivery, nanocarrier, small non-coding RNA

# Introduction

#### Edited by:

Mohammadreza Hajjari, Shahid Chamran University of Ahvaz, Iran

#### Reviewed by:

Tohru Yoshihisa, University of Hyogo, Japan Venugopal Thayanithy, University of Minnesota, USA João Conde, Massachusetts Institute of Technology, USA

#### \*Correspondence:

Babak Bakhshinejad<EMAIL_ADDRESS><EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to RNA, a section of the journal Frontiers in Genetics

Received: 25 February 2015 Accepted: 26 April 2015 Published: 12 May 2015

#### Citation:

Bakhshinejad B (2015) Phage display and targeting peptides: surface functionalization of nanocarriers for delivery of small non-coding RNAs. Front. Genet. 6:178. doi: 10.3389/fgene.2015.00178 Small non-coding RNAs are known as a clinically relevant category of non-coding RNAs (ncRNAs) that have gained growing attention for their therapeutic values. miRNA and siRNA are of the most important and well-documented types of small ncRNAs. These regulatory RNA molecules are short–approximately 22 nucleotides long–with a similar mechanism of generation in which they are excised from longer double-stranded RNA precursor molecules. Both miRNA and siRNA target protein-coding genes via an antisense-based strategy and follow the same processing manner to reach gene-silencing effect (Chen et al., 2015). Functionally, small ncRNAs play a determining part in the modulation of cellular gene expression. The impressive role of these molecules as the controlling machinery of cells has proposed promising capacities for their potential translation into the clinic. In this context, tuning the regulatory components (such as ncRNAs) rather than the regulated components (protein-coding gens) has been suggested to be a more convenient and more effective approach to correct many cellular dysfunctions (Taft et al., 2010). This emerging concept symbolizes the dramatic potential of small ncRNAs, as a significant part of the regulatory apparatus of cells, to treat human diseases.

# Small ncRNA Delivery: Active Targeting and the Dream of Magic Bullet

Within the recent years, nanotechnology-based approaches have emerged as potentially powerful and efficient systems for cellular delivery of ncRNAs. The exploitation of nanocarriers shows promise to bypass a variety of biological barriers for systematically administered RNA therapeutics (Miele et al., 2012; Segovia et al., 2015). Nanoparticles provide protection from serum nucleases for RNA-based therapeutic molecules, decrease their sequestration by phagocytes of the reticuloendothelial system (RES) and are able to carry both hydrophilic and hydrophobic substances (Aagaard and Rossi, 2007; Peer et al., 2007). The excellent characteristics of nanocarrier platforms aid in enhancing overall bioavailability, extending in vivo stability, and improving the cellular delivery of RNA therapeutics such as small ncRNAs.

For the successful delivery of small ncRNA, one of the most important problems that needs to be solved is targeting of these drugs into the desired sites. The delivery of small ncRNAs to the diseased cells, tissues or organs not only raises their silencing potency, but also leads to a considerable reduction of side effects by avoiding normal cells (Daka and Peer, 2012). Active targeting plays a highly critical part in achieving efficient in vivo biodistribution and optimized systemic delivery of ncRNA-based therapeutics. Active targeting becomes possible when the surface of nanocarriers that are embedded with small ncRNAs is modified through the covalent attachment of a cell/tissue-specific ligand.  In this manner, the delivery of therapeutic small ncRNA molecules will be poised to materialize the dream of constructing magic bullet; the concept that was theoretically fathered by Paul Ehrlich to describe targeted transportation of therapeutic agents to the desired sites of the body. Peptides are known as one of the most interesting categories of targeting ligands that have recently received huge attention. Due to some unique properties, peptides have become favorable targeting ligands for formulating powerful and sophisticated drug delivery platforms.

# Phage Display Libraries: A Treasure of Cell-targeting Peptides

Phage display that is defined as the ability to genetically modify bacteriophages for introducing specific amino acids onto their surface is a worthwhile tool in biomedicine. The birth of phage display, as a kind of virus-based molecular chimera, goes back to three decades ago when Smith for the first time reported the expression of a foreign polypeptide on the surface of phage particles (Smith, 1985). The power of phage display for gaining momentum as a revolutionary strategy for gene/drug delivery studies comes from two distinctive features: (i) establishing a physical connection between the phenotype (the displayed ligand) and the genotype (the DNA sequence encoding the displayed ligand) within the same viral particle and (ii) producing very large libraries of ligands displayed on the surface of the phage particles. The most widely-used phage display libraries are random peptide libraries (RPL). In simple terms, a random peptide library is a collection of phages carrying on their surface extremely diverse types of peptides.

Due to the phenotype-genotype link, phage peptide libraries can be screened against any target of interest for selecting targetspecific binders. This screening strategy displays similarity to the approach of natural selection but with distinctive difference of being performed in the test tube instead of nature (Bakhshinejad and Sadeghizadeh, 2014). Affinity selection is a hallmark of phage display screening through which binding peptides specific to any target including organic and inorganic structures can be obtained. The procedure of affinity-based library screening is called "panning." Whole cells in the culture are one of the most important targets used in panning studies. Cell panning involves incubating the phage library with target cells. Some phages interact with and bind to the target with higher affinity. The unbound or weakly-bound phages are then washed away and the bound phages are recovered. Subsequently, the recovered phages are amplified through infecting host bacterial cells. This process is repeated for several rounds to ultimately isolate the most strongly binding phages whose surface displayed peptides are identified by DNA sequencing (Bakhshinejad et al., 2014).

# Phage Library Selected Peptides and Targeted Delivery of miRNA/siRNA-based Nanocarriers