INTRODUCTION

In 1993, the first microRNA (miRNA), a new class ofnoncoding RNA molecules, was discovered while study-ing the temporal regulation of Caenorhabditis elegansdevelopment. These RNA products were shown to havean antisense complementarity to multiple sites in the 3’untranslated region (UTR) of mRNA (lin-14) leading tothe inhibition of their translation and to a decrease in theamount of their protein product (LIN-14) [1,2]. Sevenyears later, miRNA let-7 was discovered in C. elegans[3] with its gene conserved throughout several speciesand most importantly in humans [4]; thus triggering aninterest in the field of microRNA research.

miRNA, a large family of small RNA moleculesapproximately 18-25 nucleotides in length, has beennewly established as important post-transcriptional generegulators [5]. It is estimated that miRNAs regulatearound sixty percent of all protein coding mammaliangenes [5]. Currently, 1872 precursors and 2578 mature

miRNAs [mirbase.org] are identified in humans and varyin their expression in a tissue-specific or developmental-stage-specific manner [5]. Changes in miRNA expres-sion have been implicated in many pathologies includingcancer [6]. Remarkably, specific miRNAs can be dys-regulated in certain cancers and not in others, or vary inthe pattern of their dysregulation between cancers (i.e.up-regulation or down-regulation) [7]. Interestingly,these changes can be detected not only at the tissue levelbut also in body fluids and accordingly the role ofmiRNAs as potential diagnostic and prognostic bio-markers in many cancer types has recently emerged.

miRNA BIOGENESIS AND FUNCTION

The precursors of miRNAs (pre-miRNAs) can be gener-ated in two distinct pathways (Fig.1). The first pathway,known as the canonical pathway, involves the transcrip-tion of long primary RNA sequences called pri-miRNAsfrom independent genes via RNA polymerase-II [5]. pri-miRNAs fold into a hairpin and are spliced by the com-plex Drosha (RNase-III family member) into a ~70nucleotide hairpin known as pre-miRNA. The secondpathway for pre-miRNA biogenesis is known as themirtron pathway that involves the direct generation ofpre-miRNA from introns of protein coding genes by-passing Drosha via Spliceosomes [5]. The pre-miRNAs

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MMIISSEE AAUU PPOOIINNTT// IINN--DDEEPPTTHH RREEVVIIEEWWCIRCULATING microRNAs: POTENTIAL BIOMARKERS IN CANCER DETECTION, DIAGNOSIS AND PROGNOSIShttp://www.lebanesemedicaljournal.org/articles/64-2/review2.pdf

Nader MAKAREM1*, Ahmad MOURAD1*, Farah J. NASSAR2, Rihab NASR1

Makarem N, Mourad A, Nassar FJ, Nasr R. Circulating micro-RNAs: Potential biomarkers in cancer detection, diagnosis andprognosis. J Med Liban 2016 ; 64 (2) : 116-122.

Makarem N, Mourad A, Nassar FJ, Nasr R. Les microARN cir-culants : biomarqueurs potentiels pour la détection, le diagnosticet le pronostic du cancer. J Med Liban 2016 ; 64 (2) : 116-122.

1Department of Anatomy, Cell Biology & Physiology, Facultyof Medicine, American University of Beirut, (AUB), Beirut,Lebanon.

*Co-first authors.2Department of Biology, Faculty of Arts and Sciences, AUB.

Correspondence to: Rihab Nasr, PhD.e-mail: rn03@aub.edu.lb

ABSTRACT • A large family of small 18-25 nucleotide longnon coding RNA molecules now known as microRNA (miRNA)was described two decades ago, and has been recently es-tablished as post-transcriptional gene regulators. miRNAswere shown to be involved in the regulation of diverse phys-iological and developmental processes. Moreover, dysregula-tion of specific miRNAs has been implicated later in severalpathologies including cancer. Owing to their presence andstability in body fluids, miRNAs have been investigated asnovel circulating non-invasive biomarkers. Accordingly, theirrole as potential diagnostic, prognostic or predictive biomark-ers for many cancer types has recently emerged. This reviewtackles the use of circulating miRNAs in cancer detection,diagnosis and prognosis, giving examples using commonsolid tumors and discussing the advantages of their use, thechallenges facing this novel circulating biomarker and recom-mendations to overcome them.

Keywords: microRNA, cancer, biomarker, prognosis, diagnosis

RÉSUMÉ •Les microARN (miARNs), découverts il y a deuxdécennies, forment une grande famille de petites moléculesd’ARNs non codants de 18 à 25 nucléotides de longueur.Récemment, plusieurs recherches ont prouvé que lesmiARNs sont des régulateurs post-transcriptionnels degènes impliqués dans la régulation du développement et dedivers processus physiologiques. De plus, la dérégulationde certains miARNs semble être à l’origine de plusieurspathologies, dont le cancer. En raison de leur présence etde leur stabilité dans des fluides corporels, les miARNs ontété étudiés en tant que biomarqueurs diagnostiques, pro-nostiques ou prédictifs non invasifs pour de nombreux typesde cancer. Cet article aborde l’utilisation des miARNs cir-culants dans la détection, le diagnostic et le pronostic decertaines tumeurs solides et examine les avantages de leurutilisation, les défis auxquels ces nouveaux biomarqueurscirculants feront face et certaines recommandations pourles surmonter.

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are then exported into the cytoplasm via Exportin 5 to befurther spliced by the complex Dicer (RNase-III familymember) into a ~20bp duplex, miRNA/miRNA*. Thethermodynamic stability of the strands determines whichone will be loaded into a final large functional proteincomplex called the miRNA-induced silencing complex(miRISC). The remaining passenger strand, miRNA*, isdegraded or released by the cell (in some cases it isloaded into another miRISC complex). Effective mRNAtargeting requires its continuous base pairing with theseed region (from nucleotides 2 to 8) of the miRNA inthe miRISC complex. Binding of miRISC to mRNAleads to either degradation of mRNA or prevention of itstranslation.

These post-transcriptional regulators were shown tobe involved in the regulation of not only diverse physio-logical and developmental processes but also of a vari-ety of diseases such as cancer.

miRNAs IN CANCER

A significant alteration exists in the miRNome (total cel-lular miRNA expression level) in malignant tissues ascompared to normal ones [8]. miRNAs can be classifiedas tumor suppressors or oncogenes depending on theirdownstream targets. Hence, the decrease in tumor sup-pressor miRNA would lead to the overexpression of itsoncogenic mRNA targets while an increase in oncogenicmiRNA would cause an inhibition of its tumor suppres-sive mRNA targets. This dysregulation in miRNA ex-pression could be due to the localization of its genes infragile chromosomal loci that can be amplified, deletedor mutated upon tumor development. Interestingly, thesepatterns of amplifications or deletions are tissue specificand can be traced back to their origin even after metasta-sis [6]. Moreover, overexpression or underexpression oftranscription factors and even defects in the biogenesis

FIGURE 1. Biogenesis of miRNATwo pathways exist for the biogenesis of miRNA. The first pathway, known as the canonical pathway involves specific miRNA codinggenes that are transcribed from DNA via RNA polymerase-II. After transcription, the primitive miRNA known as pri-miRNA is cleaved byDrosha into precursor miRNA, pre-miRNA. The second pathway, known as the non-canonical pathway or mirtron pathway, involvesgeneration of pre-miRNA from introns via the spliceosome acting on mRNA transcribed from independent genes not directly related tothe coding of miRNAs. Pre-miRNA is then exported into the cytoplasm and cleaved by Dicer into miRNA/miRNA* duplex formed of thestrand that will be loaded into the miRNA-induced silencing complex (miRISC) and a passenger strand. The passenger strand will bedegraded or may be itself loaded into another miRISC. The miRISC then proceeds to cleave mRNA resulting in repression of their trans-lation based on complementarity between the miRNA sequence in the complex and the mRNA in the cytoplasm.

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machinery described previously can result in possiblemiRNA dysregulation. Finally, epigenetic changes, name-ly DNA methylation and histone modifications, can alsocause dysregulation of miRNA expression in cancer [9].

As stated previously, not only can miRNAs differen-tiate between normal and malignant tissues but they canalso specify the tumor origin as well as indicate its sub-type. Rosenfeld et al. have shown that specific miRNAsignatures can classify the tissue of origin in cancer withunknown primary origin [10]. miRNAs have also differ-ent expression profiles in basal breast cancer as com-pared to luminal breast cancer [11]. In fact, miRNA pro-filing was found to be superior to mRNA profiling forthe classification of breast cancer molecular subtypes[12]. Remarkably, miRNA expression can be altered inthe different stages of cancer from initiation, progression,migration, invasion, metastasis, angiogenesis and drugresistance, highlighting their potential clinical utility asdiagnostic, prognostic as well as predictive biomarkers.

miRNAsBIOMARKERS IN BODY FLUIDS

The World Health Organization (WHO) defines a bio-marker as “any substance, structure, or process that canbe measured in the body or its products and influence orpredict the incidence of outcome or disease” [13]. Anideal biomarker must fulfill several criteria which broad-ly include being specific, sensitive, predictive, robust,translatable and non-invasive [14]. Recently, owing totheir characteristics, miRNAs have been investigated asnovel potential circulating non-invasive biomarkers.

First, miRNAs have been reported in several biologi-cal fluids including blood plasma, serum, saliva, urine,breast milk, seminal plasma, tears, amniotic fluid, cere-brospinal fluid, peritoneal fluid, pleural fluid, bronchiallavage and colostrum [15]. Since most cancer diagnosisand potential treatment decisions rely on invasive histo-logical techniques, circulating miRNAs offer a uniqueadvantage of being acquired peripherally in a non-inva-sive manner such as a simple blood sample withdrawalor urine sample collection. Recently, Haj-Ahmad and hiscolleagues have detected two urinary miRNAs, miR-484and miR-1825 that can be used for prostate cancer diag-nosis among the benign prostatic hyperplasia patients [16].

Second, circulating miRNAs appear to be stable, sug-gesting that they must be protected against ribonucleasesthat degrade extracellular free RNAs. Intensive studieshave provided evidence that miRNAs can be secretedeither in microvesicles (exosomes and shedding vesicles),apoptotic bodies, high density lipoprotein (HDL) particlesor associated with Argonaute family of proteins [17] thatshield them from degradation [18]. Indeed, miRNAs inplasma/serum are resistant to degradation under harshconditions such as storage at room temperature for 24hours, repetitive freeze-thawing cycles or low/high pH[19]. Blood samples can even be stored for relatively longperiods of time, with little change in the total amount of

miRNAs in the sample, owing to their superior stabilitycompared to other RNAs such as mRNA [20]. Anotherkey advantage of miRNAs is their ease of detection.Circulating miRNAs can be quantified using microarray,deep sequencing or reverse transcription real time PCR(RT-qPCR). RT-qPCR is considered the most suitable userfriendly technique with high sensitivity and specificityeven though microarray and deep sequencing can make aglobal miRNA profile.

Circulating miRNAs could also potentially serve asdiagnostic and prognostic biomarkers in cancer. Fordiagnosis, circulating miRNAs can be used to identifyearly stage tumor and even monitor asymptomatic high-risk patients (Table I). For example, plasma miR-29a andmiR-92a were found to be dysregulated in both colorec-tal carcinoma (CRC) and advanced adenoma as com-pared to healthy controls, so that they could be used as adiagnostic marker for early CRC [21]. Moreover, thesignature of 34 miRNAs in serum can detect early stagenon-small cell lung carcinoma (NSCLC) with 80% over-all accuracy among asymptomatic high-risk patients [22].

miRNAs as prognostic markers can predict the overallsurvival of patients as well as the outcome, progressionand recurrence of the disease (Table I). For instance, arecent study in NSCLC has identified plasma miRNAs(miR-155-5p, miR-223-3p, miR-126-3p, miR-20a-5p,miR-152-3p, and miR-199a-5p) that can predict the re-currence of the disease as well as the survival of thepatients [23]. Plasma miR-141 in patients with colorectalcancer has been described as an indicator of distantmetastasis and poor prognosis [24]. Other miRNAs couldalso be used to detect cancer metastasis such as the po-tential use of circulating miR-373 and miR-10b for iden-tifying lymph node metastasis of breast cancer [25].miRNAs could also act as a predictive marker to help intreatment decisions and to monitor therapy resistance andsensitivity. For example, the upregulation of miR-125b inthe serum has been correlated with chemoresistance ofbreast cancer patients having neoadjuvant chemotherapyof 5-fluorouracil, epirubicin and cyclophosphamide [26]as well as of NSCLC patients taking pre-operative cis-platin-based therapy [27]. All these properties of miRNAmake them a better biomarker as compared to otherssuch as circulating tumor cells whose isolation anddetection methods are still not well optimized, especial-ly that they are rare (one in a billion normal cells) andheterogeneous and they might not be released in all can-cer patients [28].

CIRCULATING miRNAs CHALLENGES AND RECOMMENDATIONS

Several challenges face the use of miRNAs as circulatingnon-invasive biomarkers. Circulating miRNAs have aheterogeneous origin. Indeed, miRNAs could originatefrom blood cells, tumor cell secretions or lysis, inflamma-tory cells in the vicinity of tumor cells or organs involvedby tumor invasion or inflammation [29].

TABLE I CIRCULATING miRNA AS DIAGNOSTIC, PROGNOSTIC OR PREDICTIVE OF COMMON SOLID TUMORS

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A recent study has shown that out of 109 serummiRNAs dysregulated in renal cell carcinoma, only 36of those were upregulated in tumor tissue. This suggeststhat some but not all of miRNA biomarkers of canceractually originate from the tumor cells themselves [30].Thus, more studies should be conducted in order to showthe origin of dysregulated miRNAs in cancer.

Challenges are also faced in the process of miRNApreparation. This is mainly due to the absence of a stan-dardized protocol for detecting circulating miRNAs, fromspecimen collection to data analysis [31]. Sample extrac-tion and processing are very delicate steps that mightaffect the miRNA level. For instance, isolating the plasmafrom blood requires the addition of anticoagulants, eitherethylenediaminetetraacetic acid (EDTA) or citrate, andnot heparin which may interfere with the downstreamanalysis such as RT-qPCR [32]. Blood cells can contributemajorly to the miRNA level – up to 50-fold – so that thetime taken between sampling and processing as well asthe speed of centrifugation steps and aspirating plasmawithout touching the blood cell pellet must be optimized.An option is to choose miRNA biomarkers that are nothighly expressed by blood cells so that the results wouldreflect the cancer disease rather than the presence of bloodcells [33]. The fraction of blood in which miRNA levelswill be detected should be carefully selected. When com-paring serum and plasma, plasma was more recommend-ed due to the possible release of RNA in the serum duringthe stressful process of coagulation [34]. However, in arecent study, it was suggested that the choice of serum orplasma depends on the miRNA of interest and the pro-posed protocol [35].

Another challenge for miRNAs is the normalization ofthe samples when using RT-qPCR. There is no unifiedconsensus regarding what endogenous control should beused for normalization. Most studies use small nucleolarmiRNAs with a stable expression level such as RNU6B,RNU44, RNU48, and RNU47 or utilize other invariantmiRNAs like miR-16 and miR-223 [32].

However, there are instances where the internal con-trol used in specific tissues cannot be used in circulation.For example, RNU6B, which has been extensively usedfor the normalization of miRNAs in tissues, is not rec-ommended for data normalization in circulation since itcan be dysregulated in a disease specific manner such asin liver fibrosis [36]. For normalization, spiked-in syn-thetic miRNAs from C. elegans can also be added afterthe denaturing step of plasma and serum and used as aninternal control that can accommodate for alterations inefficiency of RNA recovery between samples [32].

Furthermore, miRNAs are differentially expressedbetween ethnic groups making an absolute baseline fordiagnosis and prognosis difficult to quantify. Hence, ele-vated or downregulated levels of a certain miRNA maynot have the same implications between different ethnicor population groups in terms of diagnosis, prognosis orpredictive value. For example, the prognostic value ofmiR-21, miR-181b, and miR-203 differed when assessing

paired normal and tumor colorectal archival tissues col-lected from 106 African American and 239 non-HispanicCaucasian colorectal cancer patients [37]. Similarly in theplasma of patients having NSCLC, African Americanshad a significant reduction in eight diagnostic miRNAs(let-7a, miR-17-5p, miR-27a, miR-29c, miR-146b, miR-106-a, miR-155, and miR-221) as compared to EuropeanAmericans [38]. We have recently reported that somemiRNAs, which were shown by many studies in the Westto be dysregulated in breast cancer, were not similarlyexpressed in Lebanese breast cancer patients [39].

Therefore, further studies need to address the issue ofcreating one unified database or list of aberrantly ex-pressed miRNAs which must be standardized to specificpopulations and ethnic groups as well.

CONCLUSIONS

Growing evidence suggests that circulating miRNAs pro-vide a specific, non-invasive and stable detection modelfor different types of cancer that can be a promising diag-nostic and prognostic tool. Even though their role as bio-markers is still being investigated extensively, miRNAshave not yet been clinically utilized. A standardized pro-tocol of the sample extraction to data analysis must beperformed in order to get universal reproducible resultsthat can be validated in large cohorts. Importantly, onemiRNA is not sufficient to be used as diagnostic or prog-nostic biomarker but rather a panel of dysregulated tumor-associated miRNA signatures must be chosen for thispurpose. Larger prospective trials should be conducted inorder to confirm their clinical role. A database that canaccumulate all the information about circulating miRNAsregarding their pattern of expression, cancer types, samplesources, method of detection and potential use as diag-nostic, prognostic and predictive marker can be formu-lated to be later used as reference for validation in a clini-cal setting. If well optimized, miRNAs in body fluids holdgreat potential in becoming a breakthrough tool for earlycancer detection and prognosis.

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