C H A P T E R

12
Circulating Noncoding RNAs as
Clinical Biomarkers
Francesco Russo1,2,a, Flavia Scoyni3,a, Alessandro Fatica3, Marco
Pellegrini1, Alfredo Ferro4, Alfredo Pulvirenti4,a, Rosalba Giugno4,a
1Laboratoryof Integrative Systems Medicine (LISM), Institute of Informatics and Telematics (IIT)
and Institute of Clinical Physiology (IFC), National Research Council (CNR), Pisa, Italy;
2Department of Computer Science, University of Pisa, Pisa, Italy; 3Department of Biology and

Biotechnology Charles Darwin, Sapienza University of Rome, Rome, Italy; 4Department of Clinical
and Experimental Medicine, University of Catania, Catania, Italy

O U T L I N E

1. Introduction 240 5.2 ExoCarta: The Exosome Database 251

5.3 Vesiclepedia: The Extracellular Vesicles
2. MicroRNAs 242
Database251
2.1 miRNA Biogenesis and Function 242
5.4 HMDD: The Human miRNA Disease
2.2 miRNAs as Diagnostic Biomarkers 243
Database252
2.3 Effects of Drugs and Diet on Circulating
miRNAs244 6. Conclusion and Future Perspective 252
3. Long Noncoding RNAs 245 List of Abbreviations 253
4. Circular RNAs 249 Acknowledgments253
5. Resources for Circulating RNAs 250 References253
5.1 miRandola: Extracellular Circulating
miRNAs Database 250

a These authors contributed equally to this work.

Epigenetic Biomarkers and Diagnostics

http://dx.doi.org/10.1016/B978-0-12-801899-6.00012-7 239 Copyright © 2016 Elsevier Inc. All rights reserved.
240 12. CIRCULATING ncRNAs
1. INTRODUCTION
For decades, it was thought that the major
players in gene expression regulation were only
proteins. RNA was considered to play a minor
role in gene expression by converting genetic
information from DNA into functional proteins
or to serve as structural molecules for translation
or RNA maturation. Many noncoding RNAs
(ncRNAs) can act as biologically crucial regula-
tory molecules [1,2]. These RNA molecules are
generally referred as noncoding because they
are not translated into a protein product but
nevertheless they have emerged as key factors
in maintaining normal cellular function and
therefore play several roles in human diseases,
including cancer.
The human genome consists of a vast collec-
tion of ncRNAs, which can be grouped according
to their sizes and functions. MicroRNAs (miR-
NAs) are the best-characterized class of small
(around 22 nucleotides) ncRNAs, with 2588
mature human transcripts listed in miRBase v21
(http://www.mirbase.org/) [3], the database of
published miRNA sequences and annotation.
miRNAs function as posttranscriptional regula-
tors of gene expression [4] by targeting specific
mRNAs, leading to translational repression or
promoting degradation [5] of mRNA. miRNAs
have been proposed as tissue biomarkers and
more recently as promising noninvasive bio-
markers because they circulate in the blood-
stream in highly stable extracellular forms [6].
The human genome also contains long
ncRNAs (lncRNAs, molecules longer than 200
nucleotides), which are structurally similar to
protein-coding genes with proximal promoter
sequences and consist of exons and introns
but possess no open reading frames (ORFs).
lncRNAs constitute the majority of the tran-
scriptome but little is known about their biologi-
cal functions and their potential role as novel
noninvasive biomarkers.
In order to establish specific extracellular RNA
molecules as clinically relevant biomarkers, both
reproducible recovery from biological samples
and reliable measurements of isolated RNA are
key instrumental variables.
The main challenge associated with circulat-
ing miRNA diagnostics is a lower efficacy of
miRNA isolation from body fluids compared
to miRNA isolation from cells and tissues. This
limits the sensitivity of this approach for clinical
purposes [7]. Furthermore, extracellular circu-
lating miRNA exists in both free- and microves-
icle-associated states (such as exosomes) [6,8].
Therefore, the method chosen for blood plasma
processing can significantly affect the results
of extracellular miRNA analysis [9] and many
methods and commercial kits for RNA extrac-
tion from body fluids exist (see Table 1). Some
RNA isolation methods could be superior to oth-
ers for the recovery of RNA from biological flu-
ids. Furthermore, addition of a carrier molecule,
such as glycogen, could be beneficial for some
isolation methods.
Exosome isolation protocols vary depend-
ing on the biological fluid of origin, but gener-
ally involve serial centrifugation at low speed,
followed by ultracentrifugation at 100,000  g
[10,11]. Alternatively, exosomes can be isolated
by immunocapture or size-exclusion methods
[10,12]. Recently, a method for exosome isolation
called ExoQuick (System Biosciences, Mountain
View, California, USA) has been made commer-
cially available [13].
Real-time polymerase chain reaction (PCR)
is the most commonly used approach for
miRNA quantification. The sensitivity and
specificity of real-time PCR and its flexibility,
makes it a rather attractive approach to mea-
suring miRNA abundance. The data stored in
databases such as miRandola (the database
of circulating miRNAs) [14,15], clearly show
that next-generation sequencing (NGS) is not
widely used yet, perhaps because a larger
amount of RNA is needed [16]. Nevertheless,
some groups have started to use this technol-
ogy for profiling circulating miRNAs [17,18].
Yang and colleagues [17] for the first time
 1. Introduction 241
TABLE 1 Some Methods and Commercial Kits for RNA Extraction from Body Fluids
Kit Company Description

MaxRecovery BiooPure BiooScientific A single-phase reagent for extraction of total RNA or enriched
RNA Isolation Reagent small RNA (including miRNA) from solid tissues, cultured cells,
and cell-free fluids such as serum and plasma.

mirVana miRNA Ambion Uses a rapid procedure to isolate small RNAs using an efficient
Isolation Kit glass fiber filter (GFF)-based method. The method isolates total
RNA ranging in size from kilobases down to 10-mers.

TRI Reagent RT Molecular Research Center TRI Reagent RT is used for RNA isolation from tissues, pelleted
cells, and cells grown in monolayer.

TRI Reagent RT-Blood Molecular Research Center TRI Reagent RT-Blood is specifically designed to isolate RNA
from whole blood, plasma, or serum samples.

TRI Reagent RT-Liquid Molecular Research Center TRI Reagent RT-Liquid Samples is used for cell suspensions and
Samples other liquid samples.

RNAzol RT Molecular Research Center RNAzol RT is used to isolate RNA from tissues, cells, liquid
samples, or blood. One milliliter is sufficient to process up to
100 mg tissue yielding 50–700 μg of large RNA (>200 bases) and
8–120 μg of small RNA (200–10 bases).

MiRNeasy Serum/ Qiagen The miRNeasy Serum/Plasma Kit is designed for purification
Plasma Kit of cell-free total RNA—primarily miRNA and other small
RNA—from small volumes of serum and plasma.

PureLink microRNA Ambion The Ambion PureLink miRNA Isolation Kit provides a
Isolation Kit column-based method for isolating high-quality total miRNA from
animal and plant cells, as well as from bacteria and yeast samples.

mirPremier Sigma Provides a method for purifying and enriching miRNA along
with other small RNAs, allowing researchers to obtain miRNA
directly from cells or tissues.

miRCURY RNA Exiqon The miRCURY Isolation Kits for exosome isolation are optimized
Isolation Kits—Exosome for isolation of exosomes from various biofluids and for
Isolation integration with the miRCURY RNA Isolation Kits. Exosomes can
be isolated from various biofluids such as cell-conditioned media,
urine, serum, or plasma.

QIAamp Circulating Qiagen For isolation of free-circulating DNA and RNA from human
Nucleic Acid Kit plasma or serum.

analyzed the expression profiles of circulating
miRNAs in the serum of four pregnant women
with preeclampsia (PE, MIM #189800) and
one healthy pregnant woman as normal con-
trol, using the SOLiD sequencer. Their results
showed that miRNAs in serum of pregnant
women could be detected more comprehen-
sively by NGS.
In another recent study, small RNA fraction
from cerebrospinal fluid (CSF) was profiled
through NGS (HiSeq 2000) for the first time by
Burgos and colleagues [18]. The aim of their
study was to maximize RNA isolation from
RNA-limited samples and apply these methods
to profile miRNA in human CSF by small RNA
deep sequencing. More recently, one study [19],
242 12. CIRCULATING ncRNAs

TABLE 2 Classes of Circulating ncRNAs

ncRNA Length (nt) Function Extracellular form

MicroRNA (miRNA) 21–23 Posttranscriptional regulation Ago2, vesicles, HDL

Piwi-interacting RNA (piRNA) 24–30 Genome stabilization Vesicles

Long noncoding RNA (lncRNA) >200 Transcription, splicing, transport regulation Vesicles

Ribosomal RNA (rRNA) 120–4700 Translation Vesicles

Transfer RNA (tRNA) 70–100 Translation Vesicles

Small nuclear RNA (snRNA) 70–350 Splicing, mRNA processing Vesicles

Small nucleolar RNA (snoRNA) 70–300 RNA modification, rRNA processing Vesicles

aimed at the discovery and characterization of
plasma-derived exosomal RNAs through NGS
techniques, demonstrated that a wide variety
of RNA species are embedded in circulating
vesicles. Authors detected significant fractions
of miRNAs (the most abundant) and other
RNA species including ribosomal RNA (9.16%
of all mappable counts), lncRNA (3.36%), Piwi-
interacting RNA (1.31%), transfer RNA (1.24%),
small nuclear RNA (0.18%), and small nucleolar
RNA (0.01%) (see Table 2). Fragments of cod-
ing sequence (1.36%), 5′ untranslated region
(0.21%), and 3′ untranslated region (0.54%) were
also present.
Circulating nucleic acids in plasma and
serum also include DNA [20]. In 1948, Mandel
and Metais reported the existence of circulat-
ing extracellular nucleic acids in human blood
[21]. Circulating DNAs are small fragments of
genomic DNA present in the plasma or serum
[22]. Many researchers proposed the application
of circulating cell-free DNA as a noninvasive
biomarker for early detection and prognosis of
cancer. For example, the detection of point muta-
tions, microsatellite alterations, DNA hyper-
methylations, and losses of heterozygosity in
circulating cell-free DNA have been character-
ized in esophageal cancer (MIM #133239) [23].
In this chapter we present a brief descrip-
tion of miRNAs, lncRNAs, and the recently
discovered class of circular RNAs (circRNAs).
Then, we discuss the application of these
ncRNAs as noninvasive biomarkers and, finally,
we present the major online resources for bio-
marker annotation and identification. These
tools are fundamental resources for extracellular
nucleic acids giving researchers a comprehen-
sive overview about noninvasive biomarkers.
2. MicroRNAs
2.1 miRNA Biogenesis and Function
miRNA genes are widely distributed in ani-
mals, plants, and viruses and constitute one of
the most abundant gene families [24]. In ani-
mals, miRNAs represent the dominating class of
small RNAs in most somatic tissues.
In humans, the majority of miRNAs reside in
introns of their host genes. They share their reg-
ulatory elements and primary transcript, and
have a similar expression profile. Other miRNA
genes are transcribed from their own promot-
ers, but few primary transcripts have been
fully identified. miRNAs can be also encoded
by exonic regions. Often, several miRNA loci
constitute a polycistronic transcription unit
[25]. miRNAs in the same cluster are generally
co-transcribed, but the individual miRNAs can
be additionally regulated at the posttranscrip-
tional level.
 2. MicroRNAs
miRNA transcription is carried out by
RNA Pol II and is controlled by RNA Pol II-
associated transcription factors and epigenetic
regulators [4,26–29]. After the transcription
phase, the primary miRNA (pri-miRNA)
undergoes several steps of maturation [25].
The pri-miRNA contains a stem-loop struc-
ture, in which mature miRNA sequences are
embedded. The nuclear RNase III Drosha ini-
tiates the maturation process by cropping the
stem loop to release a small hairpin-shaped
RNA [30]. Together with its essential cofactor
DiGeorge syndrome chromosomal (or critical)
region 8 (DGCR8), Drosha forms a complex
called microprocessor [31–34]. Drosha cleaves
the hairpin [35,36], and the resulting pre-
miRNA is exported into the cytoplasm, where
maturation can be completed. Upon export
to the cytoplasm, pre-miRNA is cleaved near
the terminal loop by the RNAse III enzyme
Dicer. The resulting small RNA duplex [37–41]
is subsequently loaded onto an Argonaute
(AGO) protein to form an effector complex
called RNA-induced silencing complex (RISC)
[42–44]. Only one of the two strands of the
original pre-miRNA stem remains bound to
the RISC (the guide strand) as mature miRNA,
whereas the other strand (passenger strand)
may be eliminated.
In RNA silencing, miRNA functions as a guide
by base pairing with its target mRNAs, whereas
AGO proteins function as effectors by recruit-
ing factors that induce translational repression,
mRNA deadenylation, and mRNA decay.
miRNA-binding sites are usually located in
the 3′ untranslated region of mRNAs [45]. The
sequence of mature miRNAs at the 5′ end that
spans from nucleotide position 2 to 7 is crucial
for target recognition and has been termed the
“miRNA seed.”
miRNAs are released from cells in mem-
brane-bound vesicles (e.g., exosomes and
microvesicles), which protect them from blood
RNase activity. Exosomes are 50- to 90-nm ves-
icles arising from multivesicular bodies and
243
released by exocytosis [46]. They consist of a
limiting lipid bilayer, transmembrane proteins,
and a hydrophilic core containing proteins,
mRNAs, and miRNAs. Exosomes are present
in a wide range of biological fluids, including
blood and urine. They are recognized by their
size and the fact that they are formed intra-
cellularly within multivesicular endosomes,
while microvesicles (100–1000 nm in diameter)
are shed from the plasma membrane surface
directly [47]. Key mechanisms by which exo-
somes may exert their biological functions on
cells include (1) direct contact between surface
molecules of vesicles and cells, (2) endocyto-
sis of vesicles, and (3) vesicle–cell membrane
fusion [48]. Exosomes may horizontally trans-
fer RNAs, including miRNAs that have been
shown to be functional after exosome-mediated
delivery [49]. Moreover, recent evidence indi-
cates that viruses can export and deliver func-
tional miRNAs through exosomes [50,51].
A significant portion of circulating miRNAs
in human plasma and serum is associated with
the Argonaute2 (Ago2) protein [6,8]. Ago2 is
the effector component of the miRNA-induced
silencing complex that directly binds miRNAs
and mediates mRNA repression in cells [52],
[53]. It has been hypothesized that most extra-
cellular miRNAs might be by-products of dead
cells that remain in extracellular space due to
the high stability of the Ago2 protein and Ago2-
miRNA complex [6].
2.2 miRNAs as Diagnostic Biomarkers
miRNAs have been used to identify cancer
tissue of unknown primary origin [54], demon-
strating the effectiveness of miRNAs as biomark-
ers. Moreover, it has been reported that miRNA
expression accurately separated carcinomas
from benign samples such as benign prostatic
hyperplasia (BPH, MIM #600082) and also fur-
ther classified the carcinoma tumors accord-
ing to their androgen dependence, indicating
the potential of miRNAs as a novel diagnostic
244 12. CIRCULATING ncRNAs
and prognostic tool for prostate cancer (MIM
#176807) [55].
Although there are only about 2000 different
miRNAs found in human cells (see miRBase,
http://www.mirbase.org/), current estimates
indicate that more than one-third of the cellular
transcriptome is regulated by miRNAs [45].
miRNAs have been considered as a promis-
ing next generation of diagnostic biomarkers
because of the strong correlation between their
expression patterns and disease progression.
Recently, miRNAs have been found in extra-
cellular human body fluids including plasma,
serum, urine, and saliva [56–59]. Some circulat-
ing miRNAs in the blood have been success-
fully proven as biomarkers for several diseases,
including cardiovascular diseases [59] and can-
cer [56,60]. It remains unclear, however, if this
approach can be extended to all reported cir-
culating miRNAs, since variations in isolation,
detection, and quantification protocols make it
hard to compare the significance of the results.
Recently, it has been shown that breast cancer
cells secrete exosomes with specific capacity for
cell-independent miRNA biogenesis, while nor-
mal cell-derived exosomes lack this ability [61].
In the same study, the authors found that exo-
somes derived from cancer cells and serum from
patients with breast cancer (MIM #114480) con-
tain the RISC-loading complex proteins (Dicer,
TRBP, and AGO2). Moreover, cancer exosomes
alter the transcriptome of target cells in a Dicer-
dependent manner, which stimulates nontumori-
genic epithelial cells to form tumors. The authors
concluded that the presence of Dicer in exosomes
may serve as biomarker for detection of cancer.
One of the most frequent circulating miRNAs is
miR-21. It has been successfully characterized as a
promising biomarker for several diseases includ-
ing hepatocellular carcinoma [60], non-small-cell
lung cancer (NSCLC, MIM #211980) [62] and other
solid cancers [63], as well as cardiovascular dis-
eases, such as myocardial fibrosis [64].
Recently, it has been shown that serum miR-21
is a potential biomarker of epigenetic therapy in
myelodysplastic syndrome (MDS, MIM #614286)
patients [65]. Furthermore, Song et al. [66] explore
regulation of miR-21 expression by epigenetic
change and its impact on chemoresistance and
malignant properties of pancreatic cancer showing
that the miR-21 upregulation induced by histone
acetylation in the promoter zone is associated with
chemoresistance to gemcitabine and enhanced
malignant potential in pancreatic cancer cells.
Another promising circulating miRNA is
miR-141. Serum levels of miR-141 (an miRNA
expressed in prostate cancer) can distinguish
patients with prostate cancer from healthy con-
trols [56]. Moreover, plasma miR-141 may repre-
sent a novel biomarker in detecting colon cancer
(MIM #114500) with distant metastasis and high
levels of miR-141 in plasma are associated with
poor prognosis [67].
So far, most approaches for the identification
of circulating diagnostic RNAs have focused on
quantifying circulating RNAs that are overex-
pressed or depleted in the cancer cells they may
have originated from [56,68],69]. This approach
has only revealed some of the highly abundant
cellular miRNAs and other ncRNAs, suggesting
that only a subset of cellular RNAs is released
into the extracellular environment [70]. This
release does not appear to be a mere mechani-
cal consequence of cellular decay, and in a recent
study, Pigati and colleagues [71] have found that
nearly 30% of the released miRNAs in vitro and
in vivo do not reflect cellular profile, suggesting
that some miRNAs are retained or released selec-
tively. Some selectively released miRNAs were
enriched in body fluids conditioned by mam-
mary cells, including mammary fluids, blood,
and milk. This subset of miRNAs may have value
in breast cancer diagnosis and biology.
2.3 Effects of Drugs and Diet on
Circulating miRNAs
Circulating miRNAs appear to be affected by
various parameters, including drugs and diet.
For instance, De Boer and colleagues [72] have
 3.  LONG NONCODING RNAs
shown that aspirin intake should be accounted
when considering circulating miR-126 as diag-
nostic biomarker for cardiovascular diseases, or,
more generally, when studying the possible role
of miRNAs as mediators of cardiovascular dis-
ease. Table 3 summarizes some recent reports on
the effects of drugs on circulating miRNAs.
Likewise, diet-derived exogenous miRNAs
(or “xenomiRs”) should also be taken into
account because they affect total miRNA profiles
as part of a circulating miRNA homeostasis that
is altered in many diseases. Because miRNAs
are found in both animals and plants, almost
all fresh foods contain small RNAs that could
contribute to the circulating miRNA population.
Even processed food (e.g., cooked rice, potatoes,
cabbage [73], and baby milk) [74] contain miR-
NAs, albeit at reduced concentrations.
The fact that these miRNAs could enter the
bloodstream is supported by oral delivery of
pharmacological preparations of small inter-
fering RNA (siRNA) [75–77]. When protected
by lipids, proteins, or polysaccharides, from
the acidic and enzymatic environment of the
digestive tract, ingested small RNA molecules
may enter into circulation through the gut.
Protection is achieved by artificial shells for
therapeutic siRNAs, but multiple protective
means are available for food-acquired miR-
NAs, including natural lipid vesicles [78] and
protein complexes [73].
245
Recently, Vickers and colleagues [79] reported
evidence that the high-density lipoprotein (HDL,
a delivery vehicle for the return of excess cellu-
lar cholesterol to the liver for excretion) trans-
ports endogenous miRNAs and delivers them to
recipient cells with functional targeting capabili-
ties. Many of the genes significantly altered in
response to atherosclerotic HDL-miRNAs deliv-
ery have a role in lipid metabolism, inflamma-
tion, and atherosclerosis (NDST1 [80], NR1D2
[81,82], BMPR2 [82], VEGFA, and FLT1 [83]). The
exact process of how HDL is loaded with miR-
NAs and which proteins, if any, facilitate this
association is not known. However, the impor-
tance of this study is that HDL-miRNAs could
potentially serve as novel diagnostic markers in
much the same way exosomal miRNAs have.
Circulating miRNA resources can be used to
estimate the potential of reported miRNAs and
thus help prioritizing their systematic clinical
evaluation. Table 4 shows some data retrieved
from the miRandola database [14,15] (see the
following sections) about potential biomarkers
based on extracellular miRNAs.
3.  LONG NONCODING RNAs
Over the last decade, development of new
technologies for genome-wide analyses of the
eukaryotic trascriptome has revealed that around

TABLE 3 The Effects of Drugs on Circulating miRNAs

miRBase ID Expr. Drug Sample Disease

Hsa-miR-134 Down Lithium Plasma Bipolar disorder

Hsa-miR-21-5p Up Platinum Plasma Non-small-cell lung cancer

Hsa-miR-210 Up Trastuzumab Plasma Breast cancer

Hsa-miR-126-3p Down Aspirin Plasma Type 2 diabetes

Hsa-miR-152 Up Docetaxel Malignant effusions Lung cancer

Hsa-miR-122-5p Up Acetaminophen Serum Acetaminophen-induced acute liver injury

Hsa-miR-192-5p Up Acetaminophen Serum Acetaminophen-induced acute liver injury

246 12. CIRCULATING ncRNAs

TABLE 4 Some Data Retrieved from miRandola and PubMed about Potential Biomarkers Based on Extracellular
miRNAs and Long Noncoding RNAs
ID RNA type Sample Disease or cell line Expr.

Hsa-miR-21-5p miRNA Plasma, serum Esophageal cancer, aortic stenosis, breast cancer, Up
colorectal cancer, gastric cancer, glioblastoma,
hepatocellular carcinoma, lung cancer,
non-small-cell lung cancer, pediatric Crohn
disease, solitary pulmonary nodules

Hsa-miR-210 miRNA Plasma, serum Breast cancer, conventional renal cell cancer, Up
preeclampsia, solitary pulmonary nodules

Hsa-miR-29a-3p miRNA Plasma, serum Advanced colorectal neoplasia (carcinomas and Up

advanced adenomas), colorectal cancer, colorectal
liver metastasis , preeclampsia

Hsa-miR-499a-5p miRNA Plasma Acute heart failure, acute myocardial infarction, acute Up
viral myocarditis, ST elevation myocardial infarction,
troponin-positive acute coronary syndrome

Hsa-miR-208b miRNA Plasma Acute myocardial infarction, acute viral myocarditis, Up

ST elevation myocardial infarction

Hsa-miR-208a miRNA Plasma Acute myocardial infarction, troponin-positive acute Up

coronary syndrome

Hsa-miR-200b-3p miRNA Plasma, serum Biliary atresia, metastatic breast cancer, serous Up
epithelial ovarian cancer

Hsa-miR-1 miRNA Plasma Acute myocardial infarction, ST elevation myocardial Up

infarction

Hsa-miR-122-5p miRNA Serum Acetaminophen-induced acute liver injury, chronic Up

hepatitis C infection, endometriosis

Hsa-miR-126-3p miRNA Plasma,urine Endurance exercise, urothelial bladder cancer Up

Hsa-miR-133a miRNA Plasma, serum ST elevation myocardial infarction, troponin-positive Up

acute coronary syndrome

Hsa-miR-141-3p miRNA Plasma, serum Colorectal cancer, pregnancy, prostate cancer Up

Hsa-miR-20a-5p miRNA Plasma, serum Chronic hepatitis c infection, multiple myeloma, Up

pediatric Crohn disease

Hsa-miR-92a-3p miRNA Plasma, serum Advanced colorectal neoplasia (carcinomas and Up

advanced adenomas), chronic hepatitis c infection

Hsa-miR-122-5p miRNA Serum Liver injury Down

Hsa-miR-126-3p miRNA Plasma Acute myocardial infarction, type 2 diabetes Down

Hsa-miR-133a miRNA Serum Malignant astrocytomas Down

Hsa-miR-141-3p miRNA Plasma Pregnancy Down

Hsa-miR-20a-5p miRNA Plasma Endometriosis Down

Hsa-miR-92a-3p miRNA Serum Breast cancer Down

 3.  LONG NONCODING RNAs 247
TABLE 4 Some Data Retrieved from miRandola and PubMed about Potential Biomarkers Based on Extracellular
miRNAs and Long Noncoding RNAs—cont’d
ID RNA type Sample Disease or cell line Expr.

POU3F3 lncRNA Plasma Esophageal squamous cell carcinoma Up

TapSAKI lncRNA Plasma Acute kidney injury Up

LIPCAR lncRNA Plasma Cardiac remodeling after myocardial infarction Up

LIPCAR lncRNA Plasma Future cardiovascular death after cardiac failure Down

H19 lncRNA Plasma Gastric cancer Up

PCA3 lncRNA Urine Prostate cancer Up

HULC lncRNA Blood Hepatocellular carcinoma Up

TUC399 lncRNA Cell medium Hep3B, HepG2, PLC/PRF/5 Up

linc-RoR lncRNA Cell medium HepG2 and PLC-PRF5 Up

two-thirds of the human genome is pervasively
transcribed. This extensive transcription gener-
ates a huge array of RNA molecules of which less
than 2% are protein-coding RNAs [84–87].
A large proportion of the human transcrip-
tome produces the heterogeneous class of
lncRNAs, which are generally referred to as
molecules longer than 200 nt, often polyadeni-
lated and lacking of evident ORFs [88–90].
lncRNAs are produced from RNA polymerase
II and can be antisense, interleaved, or overlap-
ping with protein-coding genes or produced
from region devoid of coding genes (termed lin-
cRNAs, long intergenic or intervening ncRNAs)
[84–87]. In addition, some of them are produced
from enhancer regions (termed eRNAs) or tran-
scription start site. Defining lncRNAs simply
on the basis of size and lack of protein-coding
capability is intellectually far from satisfying for
these noncoding molecules, which are charac-
terized by heterogeneous functions. lncRNAs
for their intrinsic nature are able to mediate base
pairing with other nucleic acids in a sequence-
specific manner and at the same time are able
to form complex structures that allow interac-
tion with proteins. In particular, their modular
and flexible characteristic leads them to act as
scaffold for partners that normally would not
interact with a protein–protein interaction and,
in addition, their nucleic acids nature make
them able to localize the scaffold-based com-
plex in a sequence-specific manner [91,88,90].
Thus, in many cases, the secondary structure is
more important than nucleotide sequence. This
is reflected in the lack of sequence conservation
for the majority of lncRNAs between different
species [92–95]. The production of such flex-
ible and multifunctional RNA molecules led
on to an important evolutionary advantage for
the selection of regulatory molecules [88,90,91].
Indeed, the increase in the noncoding content of
genome with organismal complexity supports
the idea that the noncoding regions were funda-
mental to the genetic programming of complex
eukaryotes [96].
The function of lncRNAs is often related to
their localization inside the cell. Most nuclear
lncRNAs function by recruiting chromatin-
modifying machineries to specific genomic loci
[88,90,91,97]. In particular, they can act as a
guide for DNA methyltransferase 3 and histone
modifier as polycomb repressive complex PRC2
[98,99], and histone H3 lysine 9 (H3K9) meth-
yltransferases [100,101]. These modifications
248 12. CIRCULATING ncRNAs
correlate with the leading of repressive het-
erochromatin and the resultant transcriptional
repression. lncRNAs have also been shown to
lead transcriptional activation by recruiting
chromatin-modifying complexes as the his-
tone H3K4 methyltransferase MLL1 complex
[102,103]. In addition, the lncRNA transcrip-
tion itself can negatively affect gene expres-
sion [104,105]. Based on the target sites, it is
possible to distinguish between cis- and trans-
acting lncRNAs. Cis-acting lncRNAs control
the expression of genes located near their tran-
scription sites, instead trans-acting can regulate
gene expression at independent loci [88,90,91].
lncRNAs-mediated mechanisms of gene regula-
tion have been also identified in the cytoplasm
[96]. The regulation promoted by cytoplasmic
lncRNAs is often based on sequence comple-
mentary with protein-coding transcripts and
upon recognition of the target by base paring,
they can modulate gene expression at the post-
transcriptional level by regulating RNA transla-
tion and/or stability. Such examples include the
positive regulation by the ubiquitin carboxyl-
terminal hydrolase L1 antisense RNA 1 (Uchl1-
as1) [106] and the negative regulation by the
tumor protein p53 pathway corepressor 1 (Trp-
53cor1; also known as lincRNA-p21) [107].
In the cytoplasm, lncRNAs can also act as
competing endogenous RNAs (ceRNAs) [108].
This process is based on binding to and seques-
tering of specific miRNAs, acting as “miRNAs
sponges” to lead to the derepression of the target
mRNA. This phenomenon represents a new reg-
ulatory loop in which different types of RNAs
(both coding and noncoding) can compete with
each other for shared miRNAs binding [109].
Due to the great potential of lncRNA to
modulate gene expression, there is an increas-
ing interest in the likely involvement of these
RNAs in disease etiology. Indeed, different stud-
ies have already shown that changes in lncRNA
expression may contribute to the development
of human diseases. Many studies demonstrate
a correlation between lncRNAs misregulation,
and so gene expression alteration, and occur-
rence of pathology, but now what is emerging
is a potential role in gene regulation outside the
cell and between different cells.
Recent publications have shown that in
human fluids it is possible to detect the pres-
ence of lncRNAs (see Table 4), and this dis-
covery is important in the development of
new diagnostic and prognostic tools, thanks
to the possible association between circulating
lncRNA concentration and pathology devel-
opment and evolution. A systematic analysis
of human plasma-derived exosomal RNAs by
deep-sequencing technique detected a signifi-
cant fraction of other RNA species in addition
to well known and abundant miRNAs among
which lncRNAs represent around 3% of the
mapped counts [19].
Cell-free RNA stability analysis demon-
strated that the majority of circulating RNA
is fragmented and unstable in plasma [110].
Nevertheless, at present, with the well-known
instability of RNA species, detection of circu-
lating RNA was perhaps rather surprising and
probably associated to an RNA-packaging pro-
cess to avoid nucleases degradation as apop-
totic bodies. At present, few lncRNAs have
been identified and characterized as potential
biomarkers in human fluids. In particular, by
examining the expression of lncRNAs in periph-
eral blood of major depressive disorder (MDD,
MIM #608516) patients, it has been shown that
there is a substantial difference in the expres-
sion of lncRNAs and mRNAs compared to the
control group [111]. Among these differentially
expressed mRNAs, 17 genes were documented
as depression-related genes in previous stud-
ies and it has been speculated that the differ-
entially expressed lncRNAs might contribute
to MDD by regulating these coding genes. The
detection of circulating lncRNAs in peripheral
blood not only represents a new layer of com-
plexity in the molecular architecture of MDD,
but it also reveals the potential to use them as
diagnostic markers and therapeutics.
 4.  CIRCULAR RNAs
Another example is provided by the mea-
surement of lncRNA PCA3 (prostate cancer
antigen 3) in patient urine sample, which it
has been shown to allow more sensitive and
specific diagnosis of prostate cancer than the
widely used prostate-specific antigen (PSA)
serum level [112–114]. Comparing the perfor-
mance characteristic of PCA3 assay in a cohort
of over 300 patients showed an increased spec-
ificity in cancer detection with respect to PSA
assay and minimized unnecessary biopsies
[115]. The lncRNA HULC (highly upregulated
in liver cancer) is highly expressed in hepato-
carcinoma (HCC, MIM #114550) patients and
can be detected in the HCC patient blood in
tissue samples [116]. This study highlights the
potential role of HULC as novel biomarker for
HCC.
Another study identified a previous
unrecognized role of lincRNAs regulator of
reprogramming (linc-ROR) as a mediator of
cell-to-cell communication through the trans-
fer of extracellular vesicle. This mechanism is
involved in stress cell-to-cell signaling in order
to activate stress responses such as activa-
tion of survival pathways. Cellular cross-talk
pathway may then result in chemoresistance
within the tissue and may contribute to loss
of therapeutic effect of therapeutic drugs, such
as sorafenib [117].
These studies demonstrate that circulating
lncRNAs are present in human fluids and may
play an important regulative role in cell-to-cell
signaling and modulation of gene expression.
Even if the mechanisms by which this could
happen are not understood at all, cell-to-cell
exchange of lncRNAs could lead drastic changes
due to the wide landscape of function that these
RNAs are able to dispatch both at physiological
and pathological levels.
For the time being, circulating lncRNA stud-
ies represent a very promising field that in the
next future will provide great advantages in the
development of prognostic and diagnostic non-
invasive tool for human diseases.
249
4.  CIRCULAR RNAs
circRNAs are recent and new-growing
components of the lncRNAs class. Although
biologists identified the existence of circular
transcripts more than 20 years ago [118], these
circular molecules were long considered arti-
facts of aberrant splicing reactions [119] or pre-
rogative of few viral pathogens [120,121].
However, recent works, by using specific
computational pipeline for the identification
of circular molecule, have revealed that in
many cells the production of circRNAs is not
as rare as once thought [122–124]. Evidence
from expression data in Archaea and Mamma-
lia indicated that circRNAs are abundant, con-
served, and stably accumulated within the cell
[125,118,119,126–128,122].
CircRNAs can be generated from exons
(exonic circRNA) or introns (intronic circRNAs)
through independent ways: direct ligation of
5′ and 3′ ends of linear RNAs, as intermediates
in RNA processing reaction, or by “backsplic-
ing,” wherein a downstream 5′ splice site (splice
donor) is joined to an upstream 3′ splice site
(splice acceptor) [129].
Circular molecules present many advantages
compared to linear molecule that could explain
the evolutionary conservation from Archaea to
Mammalia [125,124]. RNA circles can be much
more stable than linear RNAs due to the inacces-
sibility to degradative enzymes [130–132], and in
addition circRNAs can create constrained folded
molecule, which may be useful for interacting
with specific protein or other RNA molecules.
The functions of circRNAs remain to be fully
understood. However, one recent study demon-
strates that some intronic circRNAs enhance the
transcription of the genes from which they are
produced. Antisense oligonucleotides against
a circRNA intron reduce expression of the resi-
dent gene, while oligonucleotides against other
introns, or the region between the branchpoint
and the 3′ splice site of the circRNA intron,
which would be lacking in the stable circRNA
250 12. CIRCULATING ncRNAs
intron, do not have any effect [133]. Moreover,
intronic circRNAs can associate with RNA poly-
merase II [133]. These observations suggest that
circRNAs might modulate RNA polymerase II
in cis and thereby alter the expression of their
hosting gene. Consistent with this idea, some
intronic circRNAs accumulate in the nucleus
and, where examined, can be localized to their
sites of transcription [134,135,133]. One of the
first circRNA identified in mammals is the one-
antisense to the mRNA transcribed from the
sex-determining region Y locus, which is highly
expressed in testes of adult mouse and is essen-
tial for sex determination [136]. More interest-
ingly, recent studies identified a new example
of ceRNA [108] belonging to the circRNA class
[128,137,138]. Whereas the linear ceRNAs have
a short half-life that allows a rapid control of
sponge activity, circRNAs have improved sta-
bility, and their turnover can be controlled by
the presence of a perfectly matched miRNA
[128,137,138]. In particular, in human neuronal
cells a circRNA running antisense to the cerebel-
lar degeneration-related protein 1 (CDR1) locus
[128] has been identified, referred to as CDR1as
(antisense) or CiRS-7 (circular RNA sponge for
miR-7), which show about 70 conserved matches
to the miR-7 seed [138], [137]. The striking pres-
ence of such abundant number of sites for miR-7
suggested a possible function as miRNA sponge,
inhibiting an endogenous miRNA and leading
to miR-targets derepression. So circRNAs may
serve as transcription regulators [133] or as
sponges for small RNA regulators [137,138]. Cir-
cRNAs could also act as RNA-binding proteins
“sponge” or recruiter and bind RNAs besides
miRNAs to form RNA–protein complexes. In
addition, some circRNAs could be easily trans-
lated producing protein since internal ribosome
entry site (IRES)-mediated translation can in
principle occur on circRNAs [139]. Evidence
that circular intronic RNAs can get passed on to
offspring in Xenopus oocytes hints at their role
in RNA-mediated inheritance and epigenetics
[140]. It has also been demonstrated that they
are differentially expressed in different cell types
and conditions [124].
Due to their emerging role as gene expression
regulators, circRNAs are very likely to become
important players in cancer development [127]
and pathologies as other type of ncRNAs (see
Sections 2 and 3). Studies about emerging circu-
lar structured RNA molecules have just started
and are a completely new field of study that
will contribute to better understand the articu-
lated regulatory networks occurring in complex
organisms. Furthermore, the striking stability of
these molecules suggests that soon or late cir-
cRNAs could be used as diagnostic marker and
in addition, they could be easily identified in
human fluids.
5.  RESOURCES FOR CIRCULATING
RNAs
Here, we present four online resources
recently developed, collecting noninvasive bio-
marker data based on literature mining. miRan-
dola and Human miRNA Disease Database
(HMDD) are specific for miRNAs, while Exo-
Carta and Vesiclepedia are databases of mem-
brane-bound vesicles. These resources provide a
variety of information including diseases, RNA
isolation methods, and the experimental proto-
cols, essential data for researchers. Nowadays,
these resources are mainly focused on miRNAs.
5.1 miRandola: Extracellular Circulating
miRNAs Database
miRandola (http://atlas.dmi.unict.it/miran­­
­ ola/) is the first comprehensive database of extra-
d
cellular circulating miRNAs [14,15]. It is manually
curated and miRNAs are classified into different
categories, based on their main extracellular forms:
solely complexed with Ago2 protein [6,8] encapsu-
lated within exosomes [49] or bound to HDL [79].
The last database update is based on the compi-
lation of 139 papers, resulting in 2366 entries,
 5.  RESOURCES FOR CIRCULATING RNAs
599 unique mature miRNAs, and 23 samples. The
database provides users a variety of information
including diseases, samples description, potential
biomarker role of miRNAs, the isolation method,
and the experimental protocol.
By using the search section of miRandola
(http://atlas.dmi.unict.it/mirandola/browse.
php), users can mine information choosing
between miRNA-Ago2, miRNA-exosome,
miRNA-HDL, and miRNA-circulating. The lat-
ter, which constitutes the largest group, is used
when authors of the corresponding paper do
not distinguish between Ago2, exosome, or
HDL. The miRNA-exosome type has 862 entries
retrieved from papers mined from PubMed
(http://www.ncbi.nlm.nih.gov/pubmed/) and
ExoCarta [141], a database of exosomal proteins,
RNA, and lipids (more details in the next sections).
In miRandola there are 173 miRNA-Ago2-
type entries [6,8]. Extracellular miRNA in
human blood plasma can be also immunopre-
cipitated with anti-Ago1 antibody; furthermore
Ago1- and Ago2-associated miRNA profiles are
significantly different [142]. This indicates that
many other tissues and organs are probably
contributing to the extracellular miRNA con-
tent of biological fluids (provided that the rela-
tive expression of AGO1 and AGO2 is cell-type
specific).
The database also contains 20 entries retrieved
from a work of Vickers and colleagues [79]. Alto-
gether, they make a total of 16 unique mature
miRNAs derived from HDL isolated from
plasma. One of the most abundant miRNAs that
was found in Vickers’ work is miR-223, a highly
conserved miRNA shown to be highly corre-
lated with atherosclerosis [143]. Interestingly,
miR-223 is one of the most frequent miRNAs in
the database (together with miR-21 and miR-16).
The existence of HDL-associated miRNAs
in the blood circulation have been recently
confirmed by an independent research group,
however the analyzed HDL-miRNAs (including
miR-223) constituted only a minor proportion of
the total circulating miRNAs [144].
251
The miRNA-circulating type constitutes the
largest group. The classification of this biotype
is used whenever authors report an extracel-
lular miRNA without further characterization.
miRandola contains 1311 entries of this kind.
The vast number of entries for the miRNA-
circulating type, probably depends on the fact
that the extracellular miRNA research topic is
very new, yet it is growing very fast.
5.2 ExoCarta: The Exosome Database
ExoCarta (http://www.exocarta.org) is a
manually curated database of exosomal pro-
teins, RNA, and lipids [141]. It collects infor-
mation from both published and unpublished
exosomal studies, the mode of exosomal purifi-
cation, and characterization and the biophysical
and molecular properties. Currently, ExoCarta
contains 13,333 protein entries (4563 proteins),
2375 mRNA entries (1639 mRNAs), 764 miR-
NAs, and 194 lipid entries retrieved from 146
studies.
The database has two modules. The Query
module (http://exocarta.org/query.html) allows
users to search for gene symbols and protein
names while for more options to search data by
tissue/cell-type or organism users can select the
Browse module (http://exocarta.org/browse).
ExoCarta collects data from five species
(Homo sapiens, Rattus norvegicus, Mus musculus,
Ovis aries, and Cavia porcellus) and 52 samples.
All the data can be downloaded from the
download web page of the database.
5.3 Vesiclepedia: The Extracellular
Vesicles Database
Vesiclepedia is a manually curated com-
pendium of molecular data (lipid, RNA, and
protein) identified in different classes of extra-
cellular vesicles (EVs), membraneous vesicles
released by a variety of cells into the extracellu-
lar microenvironment [145]. Based on the mode
of biogenesis, EVs can be classified into three
252 12. CIRCULATING ncRNAs
classes: (1) ectosomes or shedding microvesicles
(large EVs ranging between 50 and 1000 nm in
diameter) (2) exosomes, and (3) apoptotic bod-
ies (they are released from fragmented apoptotic
cells and are 50–5000 nm in diameter).
Currently, Vesiclepedia comprises 92,897 pro-
tein entries, 27,642 mRNA entries, 4934 miRNA
entries, 584 lipid entries, and 33 species collected
from 538 studies.
The importance of this database is that the
authors have initiated a continuous community
annotation project with the active involvement
of EV researchers setting a gold standard in data
sharing for the field.
5.4 HMDD: The Human miRNA
Disease Database
The HMDD (http://cmbi.bjmu.edu.cn/
hmdd) is a collection of experimentally sup-
ported human miRNA and disease associa-
tions [146]. Currently, HMDD collected 10,368
entries that include 572 miRNA genes, 378 dis-
eases from 3511 papers.
Users can browse the HMDD by miRNA
names or disease names by clicking one miRNA
or disease in the Browse page. Moreover, the
authors provided a fuzzy search function for the
entries by the full or partial names of miRNAs
or diseases in the Search page. All data in the
database can be freely downloaded and users
can also submit novel data into the database.
HMDD collects specific classes of entries,
for example, entries whose experimental evi-
dence is from genetics, epigenetics, circulating
miRNAs, and miRNA–target interactions. The
information provided for the circulating miRNA
data concerns the miRNA name, the associated
disease, the PubMed ID, and a short description.
Interestingly, by analyzing the publication
time regarding the miRNA–disease associa-
tions in the past decade included in HMDD, the
authors show that this simple analysis could
predict the tendency of miRNA–disease rela-
tionship investigations. Especially, entries from
circulating miRNAs increase most dramatically
in recent years, and it is expected that more data
will be generated in the coming years suggest-
ing that the identification of circulating miR-
NAs as biomarkers is one of the hottest topics in
miRNA research.
6.  CONCLUSION AND FUTURE
PERSPECTIVE
An ideal biomarker should be accessible by
applying noninvasive protocols, being inexpen-
sive to quantify, specific to the disease of inter-
est, translatable from model systems to humans,
and a reliable early indication of disease before
clinical symptoms appear [147]. Extracellular
RNAs are promising candidate biomarkers, but
optimal and standardized conditions for pro-
cessing blood specimens for RNA measurement
remain to be established.
The establishment of a standardized acqui-
sition, processing, storage procedures, as
well as the development of assays that are
accurate, precise, specific, and robust, with
regard to quantitation of miRNAs, are very
much needed. Plasma and serum processing
[148,149], choice of anticoagulant [150], and
hemolysis [148,151] have been reported to
affect miRNA measurement.
Given the increasing amount of data available
about extracellular nucleic acids, databases are
useful reference tools for anyone who wishes to
investigate the role and function of ncRNAs as
noninvasive biomarkers. The online resources
presented in this chapter are mainly manually
curated and the updation of data needs many
biocurators and time. For this reason, in the
spirit of modern collaborative research projects,
scientists can collaborate through direct data
submissions, in order to expand the knowledge
base and give their contribution to the research
topic. This will facilitate a better understanding
of the role of circulating ncRNAs, while stimu-
lating the discussion on the current knowledge
and the controversial topics mentioned in this
chapter.
 References
LIST OF ABBREVIATIONS
BPH  Benign prostatic hyperplasia
ceRNA  Competing endogenous RNA
circRNAs  Circular RNAs
CNA  Circulating nucleic acids
HDL  High-density lipoprotein
lncRNA  Long noncoding RNA
MDD  Major depressive disorder
MDS  Myelodysplastic syndromes
miRNA  MicroRNA
miRISC  miRNA-induced silencing complex
ncRNAs  Noncoding RNAs
NGS  Next-generation sequencing
NSCLC  Nonsmall cell lung cancer
PSA  Prostate-specific antigen
Acknowledgments
Francesco Russo has been supported by a fellowship sponsored
by Progetto Istituto Toscano Tumori-Grant 2012 Prot. A00GRT.
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