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MiRNAs and long noncoding RNAs as biomarkers in human diseases

Expert Review of Molecular Diagnostics

March 2013
13(2):183-204

DOI:10.1586/erm.12.134

Source
PubMed

Authors:

Katrien Van Roosbroeck

University of Texas MD Anderson Cancer Center

Jeroen Pollet

Baylor College of Medicine

George A Calin

University of Texas MD Anderson Cancer Center

Noncoding RNAs (ncRNAs) are transcripts that have no apparent protein-coding capacity; however, many ncRNAs have been found to play a major biological role in human physiology. Their deregulation is implicated in many human diseases, but their exact roles are only beginning to be elucidated. Nevertheless, ncRNAs are extensively studied as a novel source of biomarkers, and the fact that they can be detected in body fluids makes them extremely suitable for this purpose. The authors mainly focus on ncRNAs as biomarkers in cancer, but also touch on other human diseases such as cardiovascular diseases, autoimmune diseases, neurological disorders and infectious diseases. The authors discuss the established methods and provide a selection of emerging new techniques that can be used to detect and quantify ncRNAs. Finally, the authors discuss ncRNAs as a new strategy for therapeutic interventions.

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10.1586 /ERM.12.134 183

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Loci harboring protein-coding genes are the

best-studied in the human genome, but com-

prise only less than 2% of it [1] . The fact that at

least 90% of the genome is actively transcribed

adds another layer of complexity to the human

transcriptome, suggesting that the majority of

the transcripts are noncoding RNAs (ncRNAs)

[2]. ncRNAs are transcripts that lack an open-

reading frame and, therefore, do not encode pro-

teins. They can be divided into small ncRNAs,

of which microRNAs (miRNAs) are the most

prominent, and long ncRNA, a very heterogene-

ous group of ncRNAs that are more than 200

nucleotides in length and that include, among

others, long intergenic ncRNAs (lincRNAs),

transcribed ultraconserved regions (T-UCRs),

pseudogenes and antisense RNAs (reviewed

by [3]). ncRNAs were originally thought to be

only ‘transcriptional noise’; however, increas-

ing evidence assigns a major biological role in

many physiological processes, and deregulation

of ncRNAs is implicated in many human dis-

eases. Although a lot of effort is being put into

discovering new ncRNAs and elucidating their

roles, both in normal physiology as well as in

disease, most of the research so far has focused

on miRNAs. Unraveling the roles and mecha-

nisms of action of most other ncRNAs is still in

its infancy and remains challenging. Especially,

long ncRNAs form a novel, poorly characterized

class of ncRNAs, and it is still a key challenge to

understand their precise roles in relation to phys-

iology and disease. Nevertheless, the function

of many ncRNAs has been elucidated in recent

years, pointing to a general role in a plethora of

human diseases.

In this article, the authors summarize some

emerging new roles for miRNAs and long ncR-

NAs, and describe mechanisms that cause abnor-

mal expression of ncRNAs. The authors review

established and new methods to detect RNAs,

and discuss the use of ncRNAs as biomarkers

in human diseases, as well as their targeting as

new strategies for therapeutic intervention. This

review mainly concentrates on, but is not only

restricted to, miRNAs and long ncRNAs in can-

cer; the authors also consider other human dis-

orders including cardiovascular, autoimmune,

neurological and infectious diseases. For reviews

concerning the use of miRNAs as biomarkers, the

reader is redirected to some excellent reviews [4–15 ] .

Emerging roles for ncRNAs

miRNAs

Since the discovery of miRNAs in 1993 [16 ,17]

and the demonstration of miRNA involvement

Katrien

Van Roosbroeck1,

Jeroen Pollet2 and

George A Calin*1

1Department of Experimental

Therapeutics, Unit 1950, The University

of Texas MD Anderson Cancer Center,

1881 East Road, Houston, TX 77054,

USA

2Department of Chemical and

Biomolecular Engineering, University of

Houston, Houston, TX 77204, USA

*Author for correspondence:

Tel.: +1 713 792 5461

Fax: +1 713 745 4526

gcalin@mdanderson.org

Noncoding RNAs (ncRNAs) are transcripts that have no apparent protein-coding capacity;

however, many ncRNAs have been found to play a major biological role in human physiology.

Their deregulation is implicated in many human diseases, but their exact roles are only beginning

to be elucidated. Nevertheless, ncRNAs are extensively studied as a novel source of biomarkers,

and the fact that they can be detected in body ﬂuids makes them extremely suitable for this

purpose. The authors mainly focus on ncRNAs as biomarkers in cancer, but also touch on other

human diseases such as cardiovascular diseases, autoimmune diseases, neurological disorders

and infectious diseases. The authors discuss the established methods and provide a selection of

emerging new techniques that can be used to detect and quantify ncRNAs. Finally, the authors

discuss ncRNAs as a new strategy for therapeutic interventions.

Keywor ds: autoimmune diseases • biomarkers • cancer • cardiovascular diseases • infectious diseases • miRNAs

• molecular diagnostics • neurological diseases • noncoding RNAs

miRNAs and long noncoding

RNAs as biomarkers in human

diseases

Expert Rev. Mol. Diagn. 13(2), 183–204 (2013)

Expert Review of Molecular Diagnostics

10.1586/ERM.12.134

1473-7159

1744-8352

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in human diseases, a lot of research has focused on elucidating

their roles in physiology as well as in human diseases. By now, it is

common knowledge that miRNAs negatively regulate expression

of protein-coding genes at the post-transcriptional and transla-

tional level through mRNA cleavage (Fig ure 1A) or translational

repression (Figu re 1B), and that miRNAs themselves are regulated

by transcription factors. However, recent studies suggest that

miRNAs work in a much more sophisticated way than initially

assumed and that complex interaction networks exist between

miRNAs, other ncRNAs and mRNA from protein-coding

genes [18]. For example, it has been shown that miRNAs and

their associated protein complexes, microribonucleoproteins or

microRNPs, can post-transcriptionally upregulate the expression

of target genes (Figure 1A) (reviewed in [1 9]).

Recent studies indicate that RNAs can inﬂuence each other’s lev-

els by competing for a limited pool of miRNAs. These competing

endogenous RNAs (ceRNAs) act as miRNA ‘sponges’ that divert

the miRNAs away from their mRNA targets, which results in

reduced downregulation of the target mRNA and translation into

protein (Figure 1C) [20] . ceRNA functions have been demonstrated for

pseudogenes including the pseudogenes of the PTEN tumor sup-

pressor gene (PTENP1) and the KRAS oncogene (KR AS1P) [21] for

mRNAs of protein-coding genes such as the ZEB2 mRNA, which

regulates PTEN protein levels through competition for at least four

miRNAs [2 2,2 3], and for long ncRNAs [24 ,25] . The muscle-speciﬁc

long ncRNA linc-MD1 has been shown to display decoy activity

for miR-133 and miR-135, and in this way is able to regulate the

expression of the MAML1 and MEF2C transcription factors, which

activate muscle-speciﬁc gene expression. In addition, linc-MD1

levels are strongly reduced in Duchenne muscular dystrophy

myeloblasts, pointing to a possible involvement of long non coding

ceRNAs in this hereditary disease [24] . Also, pseudogenes and

mRNA ceRNAs are suggested to be involved in the pathogenesis

of cancer. The PTENP1 locus is selectively lost in human cancer

and this loss is correlated with a decrease in PTEN [2 1] .

miRNAs can also act as an RNA decoy to modulate the func-

tion of regulatory proteins (Figur e 1D), as has been demonstrated

for miR-328 [26] . In addition to its gene-silencing activity [27] ,

miR-328, found to be downregulated in chronic myeloid leu-

kemia in blast crisis, has been shown to strongly compete with

CEBPA mRNA for binding to hnRNP E2. Consequently, CEBPA

mRNA is released from hnR NP E2-mediated translational repres-

sion and CEBPA mRNA translation is rescued [2 6]. Conversely, it

is well established that RNA-binding proteins can modulate the

function of miRNAs by competing for miRNA binding sites on

mRNAs [28 ,29].

Finally, miRNAs can also interact with and regulate other

ncR NAs such as T-UCRs [3 0] and noncoding antisense RNAs [31] .

Long ncRNAs

Our understanding of long ncRNAs and their functions is rap-

idly advancing nowadays [32,33] . However, a general mechanism

of action, as there is for miRNAs, has not been described due to

the heterogeneity of long ncRNAs. Long ncRNAs were originally

deﬁned as RNA molecules longer than 200 nucleotides that do

not encode a protein. This cutoff was arbitrarily chosen and was

not based on functionality, but on RNA puriﬁcation protocols

that exclude small RNAs [34] . Nevertheless, most long ncRNAs

described to date have been found to be related to transcriptional

regulation or mRNA processing, characteristics that they share

with miRNAs. However, unlike miRNAs, long ncRNAs show a

greater complexity of their functions and have a wider spectrum

of biological contexts. The emerging roles for long ncRNA are

nicely reviewed in [35] and will not be extensively discussed in this

review. However, a few functions will be highlighted. Many long

ncRNAs are involved in epigenetic regulation (Figur e 1e), typically

repression of target genes, which is achieved through coupling

with histone-modifying or chromatin-remodeling protein com-

plexes, most commonly PRC1 and PRC2 polycomb repressive

complexes [36] . The long ncRNAs ANRIL [37] , HOTAIR [38], H19

[39] , KCNQ1OT1 [4 0] and XIST [41] have all been linked to the

PRC2 complex. More rarely, long ncRNAs have been found to

be implicated in activation of epigenetic complexes [42] . In addi-

tion, long ncRNAs may regulate gene expression by inﬂuenc-

ing the activity of gene enhancers [43], and long ncRNAs with

enhancer-like functions have been revealed [4 4– 46] . Very recently,

enhancer-templated noncoding RNAs (eRNAs) have been discov-

ered, which are part of a highly specialized enhancer network that

regulates gene transcription at enhancer loci (Figu re 1F) [43] . Besides

transcriptional regulation, long ncRNAs are also involved in the

post-transcriptional regulation of genes and translational control.

For instance, the ncRNAs MALAT1 and NEAT1 are thought to

contribute to the regulation of alternative mRNA splicing, edit-

ing and export (Figur e 1g) [47, 48]. Also, antisense long ncRNAs may

regulate translation of their sense-coding transcript by stabiliz-

ing the coding mRNA through duplex formation (Fig ure 1H), as

was demonstrated for the Alzheimer’s disease-associated antisense

long ncRNA β-site amyloid precursor protein cleaving enzyme

(BACE1-AS) [49]. This increased stability of BACE1 mRNA leads

to elevated BACE1 protein levels.

A recent review [5 0] distilled the myriad functions of long ncR-

NAs into four archetypes of molecular mechanisms, which are

not mutually exclusive: signals; decoys, long ncRNAs that can

act as molecular sponges that pull away RNA-binding proteins,

which are themselves transcription factors, chromatin modiﬁers

or other regulatory proteins; guides, long ncRNAs that can recruit

chromatin-modifying enzymes to target genes, either in cis (near

the site of long ncRNA production) or in trans to distant target

genes and scaffolds. long ncRNAs may operate as ‘ropes’ that keep

together multiple proteins to form ribonucleoprotein complexes,

which may act on chromatin to affect histone modiﬁcations, or

that stabilize nuclear structures or signaling complexes.

Mechanisms causing abnormal expression of ncRNAs

Deregulation of ncRNAs is a general event in human diseases

including cancer, heart diseases, autoimmune diseases and neuro-

degenerative disorders [ 7,14,51– 53], and has been studied most inten-

sively for miRNAs in cancer [54] . miRNAs, and recently also

long ncRNAs, have been found to act as tumor-suppressor genes

and/or oncogenes, similar to the protein-coding genes. Several

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Post-transcriptional regulation Translational regulation

Competing endogenous RNA RNA decoy

Epigenetic regulation Gene enhancer regulation

Post-transcriptional regulation (e.g., RNA splicing) Translational regulation

miRNAsLong ncRNAs

miRNA

Cleavage

complex

Cleavage

complex

Cleavage

complex

mRNA target

mRNA cleavage

(repression)

mRNA stabilization

(upregulation)

mRNA target

miRNA

RNA binding

protein

RNA

binding

protein

miRNA

AAAA

Repression

Protein

Activation Protein

mRNA target

AAAA

mRNA

‘sponge’

Protein

Repression

Activation

Intron

retention

Exon

splicing

Activation

Stabilization

mRNA

AS RNA

5´

5´ Degradation

Enhancer

eRNA

Gene

H3K27me3

Repressor

complex

Activation

complex

Spliceosome

long ncRNA

H3K4me3

Protein

mRNA target

AAAA

mRNA target

AAAA

long ncRNA

A AB B

RNA binding protein

CProtein

AAAA 3´

mRNA AAAA 3´

Expert Rev. Mol. Diagn. © Future Science Group (2013)

Figure 1. General mechanisms of miRNA and long noncoding RNA function. (A) miRNAs can post-transcriptionally regulate gene

expression when binding to target mRNA sequences with perfect complementarity. This binding induces cleavage and degradation of the

targeted mRNAs (upper panel). Less frequently, miRNAs have been found to stabilize mRNAs, protecting them from degradation and

resulting in upregulation of gene expression (lower panel). (B) Imperfect binding of a miRNA to its target typically occurs in the 3′

untranslated region of the mRNA and leads to translational repression. In rare cases, this imperfect binding leads to the activation of

translation into proteins. (C) RNAs can inﬂuence each other’s levels by competing for a limited pool of miRNAs. These miRNA ‘sponges’ can

be pseudogenes, mRNAs of protein-coding genes or long ncRNAs. They divert the miRNA away from its target, which results in reduced

downregulation of the target mRNA and upregulation of protein translation. (D) miRNAs can also act as an RNA decoy to modulate the

function of regulatory proteins. They are able to bind to RNA-binding proteins, inhibiting the interaction of these proteins with their mRNA

targets (upper panel). Conversely, RNA-binding proteins can modulate the function of miRNAs by competing for miRNA binding sites on

mRNAs (lower panel). (E) Many long ncRNAs have been found to be involved in epigenetic regulation, typically repression, but infrequently

also activation, of target genes. This repression or activation is achieved predominantly through coupling with histone modiﬁcation protein

complexes, resulting in methylation of lysine residues in histones. Depending on which residue is modiﬁed, transcriptional repression (upper

panel) or activation (lower panel) may occur. (F) eRNAs are transcribed from enhancer loci and have been found to facilitate gene expression

of protein-coding genes. (G) Long ncRNAs also regulate gene expression at the post-transcriptional level, for example via RNA splicing. When

the long ncRNA masks the splice site from the spliceosome, this results in intron retention (upper panel). Long ncRNAs can also interact with

RNA-binding proteins that are involved in alternative splicing, facilitating mRNA exon splicing (lower panel). (H) Antisense long nRNAs are

able to bind to complementary mRNA sequences, which either protect the mRNA from degradation, resulting in protein expression

(upper panel), or induce cleavage and degradation of the target mRNA (lower panel).

AS RNA: Antisense RNA; ceRNA: Competing endogenous RNA; eRNA: Enhancer-templated noncoding RNA; ncRNA: Noncoding RNA.

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mechanisms have been described to be responsible for aberrant

expression of ncRNAs in human diseases. Extensive reviews on

this matter have already been published [32, 35,50,51, 54–57] , and the

authors would like to brieﬂy touch on the different causes of

ncRNA deregulation, which will make it easier for the reader to

understand the use of ncRNAs as biomarkers.

First, miRNAs and ncRNAs, such as T-UCRs, are frequently

located at fragile sites and disease-causing genomic loci includ-

ing regions of loss of heterozygosity (copy number loss), minimal

regions of ampliﬁcation (copy number gain) and common break-

points (rearrangements) [30 ,58– 60]. Second, alterations in the epi-

genetic regulation of ncRNAs are important in the pathogenesis

of human diseases [14 , 61] . This is not surprising as DNA is globally

hypomethylated in cancer [62] , and miRNAs and long ncRNAs

are inactivated by hypermethylation [63–65] or activated by hypo-

methylation [6 6]. Furthermore, in several human diseases, many

long ncRNAs interact with histone-modifying and chromatin-

remodeling complexes (discussed in the previous section), and

miRNAs can target genes important in the epigenetic machinery

leading to states of hyper- or hypo-methylation as well [67, 68].

ncRNAs can also be deregulated at the transcriptional level. Many

miRNAs and other ncRNAs are regulated by transcription factors

such as p53 [69,70] , MYC [71,7 2] or MeCP2 [73] . Although the occur-

rence of sequence variations in ncRNAs is still an emerging ﬁeld

of research, numerous recent reports point to a link with disease

susceptibility, an association that will be dealt with in the next

section [74–76]. Finally, miRNAs may be deregulated due to aber-

rations of enzymes involved in the miRNA biogenesis pathway,

for example TARBP2, DICER or XPO5 [32 ,77] .

ncRNAs as biomarkers

Cancer is hallmarked by a remarkable heterogeneity within groups

of patients diagnosed with the same tumor subtype, and even within

different cells of the same tumor mass. This heterogeneity, which

is a consequence of the fact that cancer is a genetic disease that is

caused by the accumulation of different genetic and epigenetic aber-

rations, is a huge obstacle in choosing the most effective treatment

for each individual patient. Biomarkers can aid in overcoming these

obstacles as they can be used to establish a diagnosis, classify tumors,

determine the stage of the disease and predict disease outcome and

evolution. Based on the levels of expression, speciﬁc biomarkers help

physicians to forecast the response to a speciﬁc therapy and to decide

on the optimal dose of a drug for an individual patient [78] . Besides

protein-coding genes and proteins, miRNAs and other ncRNAs

are extensively investigated for their potential use as biomarkers and

targets for therapeutic interventions in human diseases.

Characteristics of a good biomarker

Given that ‘a poor biomarker can be just as bad for the patient

as a bad drug’ [78], it is clinically very important to identify and

develop biomarkers that are as close to the ‘ideal’ biomarker as

possible. Biomarkers should have a unique expression proﬁle in

the disease compared with healthy individuals and should be

able to differentiate pathologies. They should, depending on how

the biomarker is to be used, show highly increased or decreased

expression levels in the diseased organ or tissue compared with

nondiseased organ or tissue, should give a reliable indication of the

disease before clinical symptoms appear, enabling early detection,

or allow detection of disease progression or therapeutic response.

Furthermore, ideal biomarkers are accessible through noninvasive

methods, have a long half-life in the sample and can be rapidly

detected by simple, accurate and inexpensive methods [8].

Although many current biomarkers are based on protein levels,

increasing interest has been shown in recent years for the use

of miRNAs and other ncRNAs as biological indicators for dis-

ease diagnosis, prognosis and prediction to therapeutic responses.

miRNAs are very stable, even in body ﬂuids such as plasma, serum,

urine and saliva [4]; their expression is speciﬁc to tissues, organs or

biological stages; and the expression level can be easily measured

by methods such as quantitative PCR and miRNA microarrays

[8]. Long ncRNAs are less stable than miRNAs, but still have half-

lives comparable to that of mRNAs of protein-coding genes [79 ,80] .

They show a greater tissue speciﬁcity compared with protein-cod-

ing mRNAs and miRNAs, which are frequently expressed from

multiple tissues, and show highly increased or decreased expression

levels in disease [81–8 3]. Similar to miRNAs, long ncRNAs are also

detectible in body ﬂuids [84– 86] . These features make them very

suitable as biomarkers, and many studies have been published on

this matter in recent years. Here, the authors highlight some of

the miRNAs and long ncRNAs that are used as biomarkers, both

in cancer and in other human diseases such as cardiovascular dis-

eases (CVDs), autoimmune diseases and neuro logical disorders.

A comprehensive list of long ncRNAs that have been or might be

linked to cancer can be found in [87] and [8 8].

ncRNAs in tissue samples & body ﬂuids

As already mentioned earlier in this article, one of the character-

istics of a good biomarker is the accessibility through noninvasive

methods. Thus far, clinical analysis of tumors is mainly based on

molecular analysis of the tumor tissue itself, and these diseased tis-

sues can usually only be obtained through invasive methods such

as surgery and biopsies. This makes biomarkers detected in tumor

tissue samples, although widely used, not the best options for diag-

nostic and prognostic purposes. Recently, circulating RNAs have

been discovered in different kinds of body ﬂuids including blood

(plasma and serum), urine and saliva, of which diagnostic samples

are easy to collect. Therefore, circulating RNAs, and miRNAs in

particular, might be extremely suitable as noninvasive biomarkers

for a plethora of human diseases. However, some level of caution

should be taken into consideration when assessing the usefulness

of circulating RNAs as biomarkers, as recent studies report on

the importance of the origin of biomarkers and their impact on

biomarker speciﬁcity. For example, a signiﬁcant proportion of

miRNAs derived from red and white blood cells have been found

to be present as contaminants in plasma preparation [89] , and differ-

ences in blood cell counts and hemolysis can alter plasma miRNA

biomarker levels by up to 50-fold [9 0]. In addition, inherent dif-

ferences in biological samples and the methods of collecting and

analyzing them can dramatically affect the detection and quanti-

ﬁcation of miRNAs and other ncRNAs [91, 92] . Therefore, in order

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to identify true disease-speciﬁc circulating RNAs, the approaches

used for quantiﬁcation of these RNAs should be optimized and

validated for accurate quantiﬁcation of circulating RNAs [91,92] .

The presence of cancer-speciﬁc miRNAs in plasma, serum, spu-

tum and urine of cancer patients is well documented [3, 4,93], despite

the fact that high amounts of ribonucleases circulate in the blood of

cancer patients [88]. These ﬁndings suggest that cell-free RNAs are

protected from RNase-mediated degradation, probably either due to

their packaging into microparticles, such as exosomes, microvesicles,

apoptotic bodies and apoptotic microparticles [94], or due to their

stabilization by binding to protein complexes [95–97]. Recently, high-

density lipoproteins were described to transport circulating miRNAs

in plasma [9 8]. The microvesicles and exosomes contain a variety of

proteins and nucleic acids including cytoplasmic and membrane

proteins, mRNAs of protein-coding genes, miRNAs, ncRNAs and

both genomic DNAs and cDNAs [99,10 0]. However, with the excep-

tion of prostate cancer 3 (PCA3) in exosomes isolated from urine

of prostate cancer patients [101] , long ncRNAs have not yet been

observed in microparticles extracted from body ﬂuids. Nevertheless,

long ncRNAs are expressed in different types of body ﬂuids includ-

ing blood [10 2] . For example, Panzitt et al. were able to detect the

highly upregulated in liver cancer ncRNA HULC in the periph-

eral blood cells of hepatocellular carcinoma (HCC) patients and

demonstrated that expression of this long ncRNA is upregulated in

these patients [8 5]. Since the extraction was done on peripheral blood

mononuclear cells and not on exosomes or microvesicles, protection

of the HULC long ncRNA from the RNases in the blood most

likely occurs through other mechanisms, such as binding to protein

complexes, as many long ncRNAs have protein-binding potential.

Disease-associated circulating cell-free RNAs are not restricted

to body ﬂuids of cancer patients, but can also be detected in other

human diseases, including but not limited to CVDs (reviewed by

[5, 6]), kidney disease [1 03 ] , autoimmune diseases such as inﬂam-

matory bowel disease [10 4 ] , Crohn’s disease [15] , systemic lupus

erythematosus (SLE) [105 ] and multiple sclerosis [1 0 6] , neurological

disorders including Alzheimer’s disease [11] , preeclampsia [107 ] ,

tuberculosis [10 8] , chronic hepatitis C and nonalcoholic fatty liver

disease [1 09 ] , and hepatitis B infection [110 ] .

Despite the increasing number of reports on the presence of

ncRNAs in body ﬂuids, the functions of these cell-free RNAs

remain poorly understood. One of the hypotheses ascribes a

hormone-like effect to circulating miRNAs, in which miRNAs

are released by a donor cell, spread to cells located in other parts

of the organism and affect acceptor cells at these distant sites [4] .

Methods of detecting ncRNAs

To be able to use ncRNAs as biomarkers for human disease, it

is essential to develop accurate determination methods. Several

established techniques are commonly used for the discovery, iden-

tiﬁcation and detection of ncRNAs (TABle 1). The ideal method

is sensitive enough to provide a quantitative expression analysis,

even with low amounts of starting material; can process multiple

samples in parallel; is speciﬁc enough to detect only the ncRNA

of interest without detecting closely related ncRNAs; is easy to

carry out; and does not need expensive reagents or equipment [111].

Northern blotting is one of the gold standards for the detection

of ncRNAs, but is not very suitable for clinical diagnostics as it is

time consuming and low throughput, necessitates relatively large

amounts of RNA and uses radioactivity for detection (TABle 1).

Custom ncRNA microarrays and quantitative reverse transcrip-

tion PCR (qRT-PCR) can be higher throughput, are more sensi-

tive, cheaper, easier to perform and already have some commercial

applications (TABle 1 & Box 1). These techniques, however, all use

ncRNAs extracted from the entire patient sample. It is generally

known that many tumors comprise a mix of normal and malig-

nant cells, and the amount of malignant cells can sometimes be

very low. For example, in Hodgkin’s lymphoma (HL), the number

of malignant Hodgkin’s and Reed Sternberg cells typically com-

prise less than 1% of the tumor mass [1 12 ] . RNA extraction of the

total tumor sample will thus result in a huge ‘contamination’ with

normal, nonmalignant cells and the biomarker that one wants to

measure will be below the detection limit. In situ hybridization

(ISH) overcomes this problem, as the ncRNA is detected within

the tissue, and if combined with immunostaining to identify the

cells of interest, ncRNA expression can be directly assessed in

the neoplastic cells (TABl e 1). In addition, ISH gives information

on the subcellular localization of the ncRNA. The most recently

established method to detect ncRNAs is RNA sequencing. This

technique is highly sensitive, highly speciﬁc, can be used for high-

throughput analysis and also discovers ‘de novo’ ncRNAs (TABle 1).

RNA sequencing generates a massive amount of complex data

that need to be analyzed by a well-trained bioinformatician. This,

together with the high costs of a single-RNA-seq run, makes this

method not yet appropriate for diagnostic purposes. However, the

rapidly reducing costs of deep sequencing and the announcement

of the ‘$1,000 genome’, the possibility to sequence a human-sized

human genome in 2 h at a cost of US$1000 [113 ] opens the door for

RNA-seq to be used for biomarker detection in patient samples.

There are many different platforms available to detect and quan-

tify ncRNAs (TABles 1 & 2); however, there are some technical chal-

lenges that make it difﬁcult to correlate measurements performed

on different platforms. An important factor is the different primer

design for the measurements by qRT-PCR and microarrays, the

two most commonly used detection methods [8]. In addition,

many research groups use different protocols for sample prepara-

tion, which makes comparisons between studies, even if the same

platform is used, difﬁcult. Some studies use an enriched small RNA

fraction, whereas others use total RNA; however, the inﬂuence of

the fractionation procedure has not yet been systematically inves-

tigated [114 ] . Furthermore, assessment of quantity and quality is

more challenging for miRNAs than for mRNAs of protein-coding

genes, for which sizes and relative abundance of ribosomal RNAs

can be used to check RNA integrity. Finally, technical and biologi-

cal variability has been assessed by a range of different normaliza-

tion processes, mostly based on the expression of reference genes.

Especially, for cell-free miRNA analysis, no known extracellular

reference RNA is currently suitable for a proper normalization [8].

As all established techniques have their own strengths and

weaknesses (TABle 1), there is a continuous search for novel detec-

tion methods that can be used to detect and quantify ncRNAs in

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patient samples. A selection of emerging new methods, developed

over the last 5–6 years, is given in TABl e 2. This table explains the

mechanism of action of the detection techniques, as well as the

advantages and limitations.

Clinical use of ncRNAs as biomarkers

An illustrative list of long ncRNAs that can be used as biomarkers,

and which will be discussed in the following sections, is shown

in TABle 3.

ncRNAs & disease susceptibility

It has been proposed that variations in the expression and/or

sequences of miRNAs and long ncRNAs, if inherited in the

germline, could be important in disease predisposition [10, 35,115] .

Genome-wide association studies often identify disease-associated

mutations (frequently SNPs) in noncoding, but transcribed, por-

tions of the human genome [116] . These sequence variations rarely

occur within the functional seed sequences of miRNAs [1 17] , but

are observed in primary miRNAs (pri-miRs) and precursor miR-

NAs (pre-miRs), the longer progenitors of the small miRNAs, in

a number of human cancers [118 –1 2 3] , as well as other human dis-

eases [124–127]. SNPs located within or near miRNA binding sites

(e.g., 3′ untranslated regions (UTRs) of protein-coding genes)

may also inﬂuence tumor susceptibility and serve as potential

biomarkers of disease risk (reviewed by [13, 128]). In addition, SNPs

within genes involved in miRNA biogenesis modify cancer risk

[12 9] and affect tumorigenesis and tumor progression [130 ] .

In this regard, two useful databases have recently been released.

The publicly available miRdSNP database combines PubMed

data on 630 disease-associated SNPs and their association with

miRNA binding sites on 3′ UTRs of human genes in 204 diseases

[131] . A catalog of miRNA seed region polymorphisms (miR-seed-

SNPs) consisting of 149 SNPs in six species was generated, and an

online tool for the detection of miRNA polymorphisms (miRNA

SNiPer) in vertebrates was developed [76].

Last, an increasing number of reports describe SNPs within or

near genomic sites where long ncRNAs are located. Two SNPs,

located within the boundaries of UCRs, were identiﬁed [132], which

were signiﬁcantly associated with familial breast cancer, whereas

polymorphisms in the antisense ncRNA ANRIL locus were found

Table 1. Established methods to detect and quantify noncoding RNAs.

Study (year) Method Advantages Limitations Ref.

Lau et al. (2001); Lee et al. (2008) Northern blot Gold standard for ncRNA

detection

Speciﬁc

Limited sensitivity (nM)

Low throughput

Time consuming

Limited for quantiﬁcation

Relatively large amounts of RNA

needed

Radioactivity generally used for

detection

[26 6,2 67]

Gupta and Mo (2011); Hanna

et al. (2012); Kloosterman et al.

(2006)

In situ hybridization Locates miRNA in tissue and

cell compartments

Low throughput

Invasive sample collection

Limited sensitivity

Very limited quantiﬁcation

[26 8–270 ]

Calin et al. (2004); Lodes et al.

(2009); Roderburg et al. (2012);

Schrauder et al. (2012); Zhao et al.

(2012); Tang et al. (2007)

Microarray Very high throughput In some cases, fair speciﬁcity

Medium sensitivity (pM)

Sensitivity and speciﬁcity can be

improved by LNA modiﬁcation of the

probes

Limited for quantiﬁcation

Results are often validated with

qRT-PCR

[156 , 271–275]

Benes and Castoldi (2010); Chugh

et al. (2010); Jensen et al. (2011);

Bianchi et al. (2012); Heneghan

et al. (2010); Heneghan et al.

(2010); Hu et al. (2012)

qRT-PCR Semi-high throughput

Good quantiﬁcation

Ampliﬁcation enables

superior sensitivity (fM)

Difﬁcult to distinguish single-

nucleotide differences

Not for ncRNA discovery

[276 –282]

Hu et al. (2012); Brase et al. (2011) RNA sequencing Most next-generation

platforms offer possibilities

for high throughput

High sensitivity (<fM)

High speciﬁcity

Can be used for discovery of

novel ncRNAs

Large amounts of complex data that

need to be analyzed

High costs

[282,2 83]

LNA: Locked nucleic acid; ncRNA: Noncoding RNA; qRT-PCR: Quantitative reverse transcription PCR.

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to be associated with CVDs such as coronary artery disease, diabe-

tes and multiple cancers, including Philadelphia chromosome posi-

tive acute lymphocytic leukemia [133–136]. In addition, expression of

a non-poly(A) circular ncRNA emanating from the ANRIL locus

correlates with enhanced risk of atherosclerosis [13 4] . Very recently,

a genetic variant in HULC has been described that contributes to

risk of hepatitis B virus-related HCC [13 7] . A ‘gene-desert’ region

on chromosome 8q24 upstream of the MYC oncogene harbors a

number of SNPs associated with prostate cancer [138 ,13 9]. In this

region, two long ncRNAs have been identiﬁed: PC AT-1 [140 ] and

PRNCR1 [141] . Although the relationship between both long ncR-

NAs and the 8q24 SNPs is not clear at present time, these ﬁndings

suggest that so-called ‘gene deserts’ may harbor long ncRNA genes,

and that SNPs in these regions may affect uncovered aspects of

biology [35]. Overexpression of PRINS has been found to correlate

with psoriasis susceptibility [1 4 2] , whereas a SNP in the promoter

of a B-cell speciﬁc antisense transcript (S AS- ZFAT ) determines

susceptibility to autoimmune thyroid disease [143].

ncRNA biomarkers in cancer

Cancer diagnosis

Many research groups sought diagnostic miRNA biomarkers that

distinguish cancer patients from cancer-free individuals and that

enable early detection. They typically established expression pro-

ﬁles to develop miRNA signatures of various cancer types. For

example, miRNAs signatures have been established that distinguish

between normal subjects and patients with solid tumors such as

lung cancer, breast cancer, gastric cancer, prostate cancer, colorectal

cancer, pancreatic cancer, HCC and glioblastoma, or hematological

malignancies such as chronic lymphocytic leukemia (CLL) and

acute lymphocytic leukemia, chronic myeloid leukemia and acute

myeloid leukemia, follicular lymphoma, diffuse large B-cell lym-

phoma and HL (reviewed by [9,10,115] ). The number of reports on

long ncRNAs in cancer diagnosis is much more restricted, but has

progressively grown over recent years ([1 02 ] ; reviewed by [88]). The

main causes for this rising interest are the increasing realization

that long ncRNAs may play important roles in physiological and

pathological processes in the cell [88] and the development of effec-

tive high-throughput expression analysis technologies (TABles 1 &

2). The most eminent long ncRNA used as a diagnostic biomarker

in cancer is undoubtedly PCA3, previously known as DD3, whose

expression was found to be highly speciﬁc in prostate tissue and

which is highly overexpressed in prostate cancer [14 4 ] . These fea-

tures, together with the detectability in urine and the expression

in early-stage tumors, resulted in the use of PCA3 as a diagnostic

biomarker already exploited in the clinic (Box 1). Two other long

ncRNAs, HULC and HOTAIR, are highly expressed in HCC [85]

and colorectal cancer that metastasizes to the liver [84 ], and in meta-

static breast cancer [81] , respectively, compared with noncancerous

tissues. In contrast, HOTAIR levels were found to be higher in

colorectal cancer tissues as compared with adjacent uninvolved

tissue [82] . Furthermore, T-UCR expression proﬁles can be used

to differentiate human cancers as genome-wide proﬁling revealed

that T-UCRs have distinct signatures in human leukemias and

carcinomas compared with the corresponding normal tissues [30] .

Fina lly, UCA1, also known as CUDR, was identiﬁed as a highly

speciﬁc (91.8%) and very sensitive (80.9%) biomarker detected in

the urine of bladder cancer patients [14 5] .

Tumor classiﬁcation

miRNA expression signatures can also be used to differentiate

between different tumor subtypes and to classify tumors of uncer-

tain cellular origin [1 46 ,147] . For instance, in breast cancer [148] ,

gastric cancer [149], acute myeloid leukemia [15 0] and HL [151] , it

has been described that miRNA signatures differentiate between

different molecular and histological subtypes. A four miRNA-

based molecular test that differentiates between speciﬁc cancer

(sub)types has been developed (Box 1). In addition, cancer subtypes

Box 1. Commercially available noncoding RNA-based diagnostic assays.

An increasing number of biotech companies started commercializing ncRNA-based diagnostic tests, primarily for certain neoplastic

diseases. For instance, Rosetta Genomics [402] offers four miRNA-based molecular tests and has a ﬁfth assay in the pipeline. The miRview®

mets² assay can differentiate between 42 different tumor types using a custom array platform containing 64 miRNAs and has an overall

sensitivity of 85% and a speciﬁcity of 99.3% [259] . The miRview lung test uses a qRT-PCR assay based on eight miRNAs (overall sensitivity

93.7%; overall speciﬁcity 97.9%) that differentiates primary lung cancers into four types: squamous cell carcinoma, nonsquamous NSCLC,

carcinoid and small-cell carcinoma [260], while miRview squamous is based on the expression level of hsa-miR-205 measured by qRT-PCR

and distinguishes squamous from nonsquamous NSCLC with an overall sensitivity and speciﬁcity of 100% [2 61,262]. The miRview meso

molecular assay is able to differentiate malignant pleural mesothelioma from carcinomas in the lung and pleura based on a three-miRNA

qRT-PCR assay that has an overall sensitivity of 100% and a speciﬁcity of 94% [263]. Finally, the miRview kidney diagnostic test, which

is still under development, is a two-step decision-tree classiﬁer that uses expression levels of six miRNAs: the ﬁrst step uses expression

levels of hsa-miR-210 and hsa-miR-221 to distinguish between the two pairs of subtypes; the second step uses either hsa-miR-200c with

hsa-miR-139-5p to identify oncocytoma from chromophobe or hsa-miR-31 with hsa-miR-126 to identify conventional from papillary

tumors [2 64] .

One US FDA-approved diagnostic assay for prostate cancer is based on the detection of the long ncRNA PCA3 in patient urine samples.

The Progensa™ PCA3 test from Gen-Probe Inc. [403] uses the transcription-mediated ampliﬁcation (TMA™) technology to determine a

PCA3 score from male urine. TMA is an isothermal nucleic acid-based method that can amplify RNA or DNA targets a billion-fold in less

than 1 h [2 65]. The PCA3 assay has a sensitivity of 60–70%, a speciﬁcity of 70–80% and a negative-predictive value of 90% [403]. PCA3 is

the ﬁrst long ncRNA to be used in clinical diagnostic assays, but with the recent developments in the ncRNA world, many more will most

likely follow soon.

ncRNA: Noncoding RNA; NSCLC: Non-small-cell lung cancer; qRT-PCR: Quantitative reverse transcription PCR.

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Table 2. Selection of emerging new methods to detect and quantify noncoding RNAs.

Study (year) Method ncRNA detection mechanism Advantages and limitations Ref.

Wang et al. (2011);

Wanunu et al. (2010 )

Nanopore-based

RNA detection

An applied voltage pulls ncRNAs either

through biological or solid state

nanopores. As the molecules

translocate, they partially block the ion

ﬂow through the pore, which is

detected as a drop in the measured

current. The detection of speciﬁc

ncRNAs can be carried out by adding a

complementary DNA probe to look for

a target-speciﬁc hybridization signature

signal

Single-molecule detection without the

need for labels or ampliﬁcation

Can quantify subpicomolar levels of

cancer-associated miRNAs and

distinguish single-nucleotide

differences between miRNA family

members

Can be made into a small instrument

Possibilities for high throughput

Extremely rapid

Requires sophisticated detection

instruments

Complex data analysis

[28 4,2 85]

Chan et al. (2010) TIRFM Complementary DNA probes are

hybridized to ncRNAs and labeled with

ﬂuorescent YOYO-1. The ﬂuorescent

hybrids are imaged by an electron-

multiplying charge-coupled device-

coupled TIRFM system and quantiﬁed

by single-molecule counting

Single-molecule sensitivity

Very accurate quantiﬁcation

Low throughput

Very advanced optical setup

[28 6]

Neely et al. (2006) Single-molecule

detection based

on dual

ﬂuorescent

labeling

Homogeneous assay using two probes,

each with a speciﬁc dye. If both probes

bind to the ncRNA, the detector will

see two ﬂuorescent peaks. Unbound

probes will appear as a single peak

Single-molecule sensitivity

Expensive labels

Very advanced optical setup

[28 7]

Gao and Yang (2006);

Gao and Yu (2007);

Peng and Gao (2011);

Cissell and Deo (2009)

Electrochemical

detection

ncRNAs are labeled with

electrocatalytic nanoparticles. The

labeled ncRNAs can hybridize with

complementary DNA immobilized on a

chip. The nanoparticles of the

hybridized ncRNA are able to catalyze

an oxidation reaction, which will

increase the current

Cost-effective sensitive sensors (high

fM)

Background signal has to be veriﬁed in

real samples

Need for special nanoparticle labels

[28 8–291]

Zhang et al. (2009) SiNWs Label-free and direct hybridization

assay for ultrasensitive detection of

miRNAs using silicon nanowires.

Peptide nucleic acids serve as a

complementary receptor. Resistance

change is directly corrected to the

concentrations of hybridized ncRNA

Label free, small device

Very sensitive (1 fM)

Very speciﬁc (single nucleotide)

Background signal has to be veriﬁed in

real samples

[292]

Sioss et al. (2012) Nanoresonator

chip

Detection of ncRNAs after

hybridization with complementary

DNA bound on metallic or silica

nanowires. Mass-amplifying gold

nanoparticles bind to the ncRNA–DNA

sandwich and will induce a large shift

in the resonance frequency of the

nanoresonator, which can be measured

optically

Promising technology for excellent

quantitative sensitivity and speciﬁcity

At this moment, still limited

reproducibility and sensitivity

Complex production process for

nanoresonators

[293]

Robertson and Vora

(2012)

Flow cytometry-

FISH

In situ hybridization of cells, induced

ﬂuorescence measured with ﬂow

cytometer

High throughput detection of in situ

hybridization

No quantiﬁcation

No location of the ncRNA

[294 ]

LOD: Limit of detection; ncRNA: Noncoding RNA; SERS: Surface-enhanced Raman spectroscopy; SiNW: Silicon nanowire; SNA: Spherical nucleic acid;

SPR: Surface plasmon resonance; TIRFM: Total internal reﬂection ﬂuorescence microscopy.

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can be further subcategorized by miRNA expression proﬁles

unique to speciﬁc genotypes [55] . For example in lung cancer, KRAS

mutation-positive tumors are associated with miR-495 upregula-

tion and EGFR mutation-positive tumors show upregulated miR-

21 and miR-25, whereas miR-155 is exclusively upregulated in lung

cancer tumors without KRAS or EGFR mutation [152 ,153 ] .

That also long ncRNAs can also be used to classify tumors was

illustrated for pediatric tumors [5 6] . The authors demonstrated

the presence and overexpression of a 250-kb stretch of noncod-

ing transcript, which they call a putative very long ncRNA, in

Ewing’s family of tumors, but not in the other cancer tumor

types analyzed (PAX-FKHR fusion-positive and -negative rhabdo-

myosarcoma, osteosarcoma, neuroblastoma and Wilms’ tumors).

However, further validation of these ﬁndings is necessary as this

study only considered a small number of patients for each subtype.

Furthermore, a novel ncRNA in rhabdomyosarcoma, NCRMS,

Table 2. Selection of emerging new methods to detect and quantify noncoding RNAs (cont.).

Study (year) Method ncRNA detection mechanism Advantages and limitations Ref.

Driskell et al. (2009) SERS Absorption of ncRNA to silver or gold

nanoparticles can be detected through

Raman scattering spectroscopy

High sensitivity (fM)

Provides a very speciﬁc molecular

ﬁngerprint

No labeling required

Sophisticated instrument needed

Low reproducibility

Difﬁculty of spectra interpretation,

especially in more complex matrices

[295]

Cissell et al. (2008) Bioluminescence Heterogeneous or homogeneous

competitive hybridization assays with

bioluminescent labeled DNA probes

Good sensitivity (LOD = 1 fM)

Expensive consumables

Difﬁcult to standardize

[296 ]

Fang et al. (2006);

Nasheri et al. (2011);

Sípová et al. (2010);

Wark et al. (2008)

SPR SPR is an optical detection method that

is sensitive for small changes of the

local refractive index that are, for

example, induced by the hybridization

of ncRNA to immobilized capture DNA.

The signal can be ampliﬁed by using

hefty labels. SPR imaging enables SPR

in a microarray format

Good sensitivity (10 fM)

Expensive read-out system

Risk for high background signal as

result of nonspeciﬁc binding

interactions

Limited throughput

[297–300]

Dong et al. (2012) Fluorescence

quenching on

graphene oxide

ssDNA strongly binds to graphene

oxide, which also quenches ﬂuorescent

dyes. Fluorescent probes hybridized

with ncRNA are protected from

adsorption to the graphene. The

sensitivity can be enhanced by

isothermal strand-displacement

ampliﬁcation

Ampliﬁcation process

High sensitivity (LOD = 2.1 fM)

Fluorescent labels are expensive

[3 01]

Alhasan et al. (2012) Scanometric

miRNA array

Isolated miRNAs are enzymatically

ligated to a universal linker followed by

hybridization onto miRNA microarray.

Universal SNA-functionalized gold

nanoparticle conjugates are

subsequently hybridized to detect

captured miRNA targets. The signal

intensity is ampliﬁed by depositing

gold with gold-enhancing solution and

before imaging

Sensitivity (1 fM)

Nonspeciﬁc adsorption of gold

nanoparticles may induce high

background signal

[30 2]

Jiang et al. (2012) Base stacking

hybridization

coupling with

time-resolved

ﬂuorescence

technology

The ncRNA and ﬂuorescent DNA tags

can hybridize together with an

immobilized DNA capture probe. If no

ncRNA is present, the short duplex

between the ﬂuorescent tag and

capture probe will be disrupted when

washing the surface, because the

binding is energetically unfavorable

Rapid, universal label

Sensitive (20 fM)

Needs ﬂuorescent tag

[303]

LOD: Limit of detection; ncRNA: Noncoding RNA; SERS: Surface-enhanced Raman spectroscopy; SiNW: Silicon nanowire; SNA: Spherical nucleic acid;

SPR: Surface plasmon resonance; TIRFM: Total internal reﬂection ﬂuorescence microscopy.

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Table 3. Illustrative list of long noncoding RNAs (potentially) used as biomarkers in human diseases.

Disease Involved ncRNA Name/description Application

Prostate cancer PCA3 or DD3 Prostate cancer 3 or differential

display code 3 long ncRNA

Diagnostic biomarker detected in exosomes

isolated from urine of prostate cancer patients

Liver cancer, CRC that

metastasizes to the liver

HULC Highly upregulated in liver long

ncRNA

Potential diagnostic biomarker highly

upregulated in blood of liver and hepatic

metastatic CRC patients

Generic variant contributes to risk of HBV-

related hepatocellular carcinoma

Coronary artery disease,

atherosclerosis, stroke, intracranial

aneurism, diabetes, ALL,

melanoma, glioma, lung cancer,

prostate cancer, NF type 1

ANRIL Antisense ncRNA in the INK4

locus

Polymorphisms in ANRIL as biomarker for

disease susceptibility

Prostate cancer PC AT-1 Prostate cancer-associated

ncRNA transcript 1

Polymorphisms in 8q24 region (region of

PC AT-1) associated with risk for prostate

cancer

Potential biomarker for prostate cancer

subtype stratiﬁcation

Prostate cancer PRNCR1 Prostate cancer ncRNA 1 Polymorphisms in 8q24 region (region of

PRNC1) associated with risk of prostate cancer

Psoriasis PRINS Psoriasis susceptibility-related

RNA gene induced by stress

Overexpression correlated with disease

susceptibility

Autoimmune thyroid disease SAS-ZFAT Small antisense transcript of

zinc-ﬁnger gene in AITD

susceptibility region

SNP in promoter of SAS-ZFAT determines

susceptibility to autoimmune thyroid disease

Metastatic breast cancer,

colorectal cancer, HCC, GIST,

pancreatic cancer

HOTA IR HOX antisense intergenic RNA Prognostic biomarker in several types of

cancer, as high expression is associated with

poor prognosis, metastasis and tumor

recurrence

Rhabdomyosarcoma NCRMS ncRNA in rhabdomyosarcoma Potential biomarker in tumor classiﬁcation due

to differential expression between

rhabdomyosarcoma subtypes

NSCLC, HCC, breast carcinoma,

pancreatic cancer, CRC, prostate

cancer, ESS of the uterus,

osteosarcoma, bladder cancer

MALAT1 Metastasis-associated lung

adenocarcinoma transcript 1

Prognostic marker in many human carcinomas,

as high expression is associated with poor

prognosis, poor response to therapy,

metastasis and tumor recurrence

HNSCC, bladder cancer, CRC,

cervical cancer

CUDR = UCA1 Cancer-upregulated drug-

resistant gene or urothelial

carcinoma associated 1

Overexpression associated with drug

resistance in HNSCC cell lines

Diagnostic biomarker detected in urine of

bladder cancer patients

HCC, ovarian cancer, bladder

cancer, HNSCC

H19 Imprinted (at IGF2 locus)

maternally expressed

untranslated mRNA

Potential diagnostic marker for HCC and

ovarian cancer

Potential prognostic marker in bladder cancer

and HNSCC, with higher expression associated

with higher grade disease and indicative for

tumor recurrence

May induce MDR1-associated drug resistance

in HCC cells

Myocardial infarction MIAT Myocardial infarction-

associated transcript

Polymorphisms in MIAT confer risk of

myocardial infarction

AITD: Autoimmune thyroid disease; ALL: Acute lymphocy tic leukemia; CRC: Colorectal cancer; ESS: Endometrial stromal sarcoma; GIST: Gastrointestinal stromal

tumor; HBV: Hepatitis B virus; HCC: Hepatocellular carcinoma; HNSCC: Head and neck squamous cell carcinoma; ncRNA: Noncoding RNA; NF: Neuroﬁbromatosis;

NSCLC: Non-small-cell lung cancer.

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shows differential expression between rhabdomyosarcoma

subtypes [15 4] .

Cancer prognosis

To date, the amount of prognostic ncRNAs outnumbers the

ncRNAs used for diagnostic purposes, mainly because diagnos-

tic biomarkers preferably enable early detection. One of the ﬁrst

miRNA signatures associated with prognosis was established in

CLL: a 13-miRNA expression signature has been identiﬁed that is

able to differentiate between CLL cases with low versus high levels

of ZAP-70, between unmutated and mutated IgV(H), and that

was associated with the presence or absence of disease progression

[155 ,156 ] . After these initial reports, many other prognostic miRNA

markers have been identiﬁed in a variety of cancers, including

non-small-cell lung cancer (NSCLC), breast cancer, gastric can-

cer, prostate cancer, HCC, glioblastoma, acute and chronic leuke-

mias, diffuse large B-cell lymphoma and HL (reviewed by [9,10]).

One well-known example of a prognostic long ncRNA is metas-

tasis-associated lung adenocarcinoma transcript 1 (MAL AT1),

which is highly expressed in many solid tumors [157,158] , predicts

poor prognosis in NSCLC [15 9,16 0] and is an independent prognos-

tic marker for predicting HCC recurrence after liver transplan-

tation [1 61] . Signatures for transcribed UCRs could differentiate

CLL patients with poor prognosis (high expression of ZAP-70)

from CLL patients with good prognosis (low ZAP-70 expression)

[30] , and were able to signiﬁcantly distinguish between short-term

and long-term neuroblastoma survivors [16 2] .

One major cause of cancer-related death is tumor metastasis,

a multistage process that includes dissemination of tumor cells

to and proliferation at distant sites [163]. Therefore, identiﬁcation

of biomarkers associated with disease progression is extremely

signiﬁcant as these markers may be used as a basis for the devel-

opment of new therapeutic strategies reducing the mortality

and morbidity, and improving the survival of metastatic cancer

patients [164 ] . miRNAs have been shown to act as either meta-

static activators or as metastatic repressors in a variety of human

cancers, including breast cancer, testicular germ cell tumors,

colon cancer, gliomas, pancreatic cancer, nasopharyngeal car-

cinoma and prostate cancer (reviewed by [9,16 3]). Several long

ncRNAs have also been implicated in metastasis. For instance,

HOTAIR has a unique association with patient prognosis and

correlates with metastasis in breast cancer patients [81] , colorectal

carcinoma [82], HCC [1 65] and gastrointestinal stromal tumors

[16 6] . MALAT1 was ﬁrst identiﬁed in metastatic NSCLC [159 ] ,

and its high expression was subsequently found to be related to

colorectal cancer metastasis [167] and to predict tumor recurrence

of HCC after liver transplantation [1 61] . In a recent report, the

relative abundance of a set of long ncRNAs was studied in pan-

creatic tissue samples and sets of intronic long ncRNAs whose

abundance is correlated with pancreatic ductal adenocarcinoma

or metastasis [168]. Finally, a unique 26-kb intergenic ncRNA

transcript, located on chromosome 2, was found to be highly

expressed in a primary Ewing’s family of tumors that did not

metastasize, whereas low expression was observed in primary

tumors that eventually metastasized [5 6 ] .

Recent reports indicate that sequence variations of ncRNAs can

also act as prognostic biomarkers. SNPs located in pre-miRNAs

[12 0, 169], in the miRNA binding site located in the 3′ UTR of

protein-coding genes [1 70 ,171] and even in the miRNA ﬂanking

region [1 72] have been found to have prognostic value in a variety

of human cancers.

These data show the potential of miRNAs and long ncRNAs

as biomarkers in cancer prognosis as well as to stratify metastatic

from nonmetastatic cancers. In addition, sequence variations of

ncRNAs may be potential prognostic biomarkers in human cancer.

Prediction of therapeutic responses

ncRNAs can be employed to predict response to therapy as their

expression is known to play a role in drug sensitivity and resist-

ance to therapy [1 0,173,174] . An 11 miRNA signature was estab-

lished in ovarian cancer cell lines, which differentiates between

cisplatin-resistant versus cisplatin-sensitive cells [17 5] . With respect

to single-miRNA biomarkers, low levels of miR-10b were found

to be associated with improved response to neoadjuvant therapy

and outcome in pancreatic ductal adenocarcinoma [176 ] , whereas

high expression in colorectal cancer confers resistance to chemo-

therapeutic agents [177 ] . Moreover, increased miR-21 expression

is correlated with chemoresistance in pancreatic cancer cells

[17 8– 181] , glioblastoma multiforme [18 2] , bladder cancer cells [1 83 ] ,

and head and neck squamous cell carcinoma cells [1 84 ] . In breast

cancer, miR-210 levels correlate with sensitivity to trastuzumab

[185 ] and miR-125b is predictive of chemoresistance [186 ,187] . In

addition, miR-125b is also associated with resistance to therapy

in ovarian cancer cells [188], glioblastoma stem cells [18 9] and pedi-

atric acute promyelocytic leukemia [19 0] . In NSCLC, miR-128b is

demonstrated to be a predictive factor of response to therapy [191].

To the best of our knowledge, only two long ncRNAs have been

associated with the prediction of chemotherapeutic response:

MALAT1, which is highly expressed in a poor responder group of

osteosarcoma [192] , and CUDR, whose overexpression is associated

with drug resistance in head and neck squamous cell carcinoma

cell lines [193] . In addition, it is suggested that the long ncRNA

H19 induces MDR1-associated drug resistance in HCC cells [19 4] .

Modulation of the ncRNAs involved in resistance to therapy may

sensitize cancer cells to chemotherapeutic agents [19 5,19 6] .

Sequence variations in ncRNAs may also be predictive of thera-

peutic responses. Among others, SNPs in miRNA binding sites

of protein-coding genes have been found to be correlated with

radiotherapy outcome in bladder cancer [19 7] , and with methotrex-

ate [198] and cisplatin resistance [19 9] . In addition, polymorphisms

in pri-miRNA and in components of the miRNA biogenesis

machinery have been found to be related to treatment outcome

in metastatic colon cancer patients treated with 5-ﬂuorouracil

and irinotecan [2 00] .

ncRNA biomarkers in CVD

According to the WHO, CVDs are the leading cause of death

worldwide [20 1] . In 2008, more than 17 million people died

from CVD, and although the mortality rates due to CVD have

reduced, the disease burden remains high [20 2]. Therefore, better

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diagnostic and therapeutic strategies are required to address these

high morbidity and mortality rates.

miRNAs play an essential role in cardiovascular biology and

are often deregulated in several heart diseases including myo-

cardial hypertrophy, heart failure and arrhythmia (reviewed by

[203] ). These deregulations can be caused by aberrant expression

of genes from the miRNA biogenesis machinery, as decreased

Dicer expression was observed in patients with end-stage dilated

cardiomyopathy and heart failure [204], or by aberrant expres-

sion of miRNAs themselves, for example deregulation of miR-1

and miR-133 in cardiac hypertrophy and arrhythmia [203] . Since

heart and vascular tissues are almost impossible to obtain from

patients with CVDs, most research has focused on the discovery

of circulating miRNA biomarkers for either diagnostic and/or

prognostic purposes ([205 –210] ; reviewed by [6 ,211] ).

A few long ncRNAs are reported to have a function in CVDs,

but their use as biomarkers still need to be validated. ANRIL was

found to be associated with atherosclerosis [21 2] and genetic vari-

ants at the 9p21 locus of ANRIL correlate with CVD susceptibil-

ity [13 3,13 4,213–215]. Similarly, altered expression of the long ncRNA

MIAT by SNPs confers risk of myocardial infarction [216 ].

ncRNA biomarkers in autoimmune diseases

Autoimmune diseases form a very heterogeneous group of more

than 100 distinctive diseases in which the patient’s immune system

in mistakenly activated to attack substances and tissues normally

present in the body. miRNAs are essential for normal immune

functions, immune cell development and prevention of autoim-

munity (reviewed by [217, 218]). Although most studies were per-

formed in cultured cells and animals, there is evidence that some

miRNAs play a critical role in the pathogenesis of auto immune

diseases such as SLE (e.g., miR-21, miR-125a, miR-146a and

miR-148a), rheumatoid arthritis (e.g., miR-124a, miR-146a and

miR-155) and multiple sclerosis (e.g., miR-17-5p, miR-20a, miR-

34a, miR-155 and miR-326), reviewed by [219] . Understanding the

involvement of long ncRNAs in autoimmunity is only in its infancy

and, to the best of our knowledge, no long ncRNA has been found

to be directly implicated in the development of the major types of

autoimmune diseases. Some preliminary data in a murine model

system point to a link between the long ncRNA GAS5 and disease

susceptibility to SLE [22 0]. In addition, the chromosomal locus of

GAS5, 1q25, has been associated with human SLE development

in genetic studies [221–223]. The identiﬁcation of abnormal ncRNA

expression in autoimmune diseases is still emerging, hence the

potential value of miRNAs and long ncRNAs as biomarkers is

still largely unexplored and warrants further investigation.

ncRNA biomarkers in neurological diseases

The nervous system has the broadest spectrum of miR NA expres-

sion of all human tissues, with approximately 70% of experi-

mentally detectable miRNAs being expressed in the brain [2 24] .

Thus, it is not surprising that miRNA deregulation has been

described in almost all neurological diseases studied [32], ranging

from neuro developmental, neurovascular and neurodegenerative

disorders to tumors of the central nervous system, nervous system

trauma and psychiatric disorders (reviewed by [22 5]). Although

differential miRNA expression proﬁles have been generated that

could potentially be used to diagnose, classify and predict the

prognosis of some neurological disorders, to date, there is no

diagnostic laboratory test available [111].

The majority of long ncRNAs are expressed in the nervous

system [2 26] and several are deregulated in neurological disorders

(reviewed by [51 ,22 7–230] ). Similar to CVDs, biological samples of

diseased tissues are very difﬁcult, if not impossible, to obtain.

For example, in Alzheimer’s disease, up until now, the diagnosis

relies principally on clinical criteria and a pathological conﬁrma-

tion can only be made by postmortem analysis of plaques and

tangles in the brain [231] . In the last few years, the search for useful

biomarkers in neurological disorders has begun to concentrate

on the detection in body ﬂuids, such as cerebrospinal ﬂuid and

plasma, although the main focus has been on proteins and pep-

tides [2 31,23 2]. Nevertheless, miRNAs begin to emerge as biological

body ﬂuid markers of neurological diseases [2 07,233–235]. Whether

long ncRNAs in body ﬂuids can also be useful as neurological

biomarkers remains to be elucidated.

ncRNA biomarkers in infectious diseases

To study the role of ncRNAs in viral infections, two aspects have

to be taken into account: the viral ncRNAs and host ncRNAs.

Some viruses encode their own ncRNAs, which target host

mRNAs or viral RNAs. These viral ncRNAs may be used as

diagnostic markers for viral infection or stage of infection [23 6].

On the other hand, parts of the viral life cycle, such as viral trans-

lation, may be regulated by host ncRNAs [237] . Finally, the virus

may alter the expression of host ncRNAs and, in this way, change

the regulation of host or viral ncRNA targets [2 37] .

The involvement of miRNAs in viral infection is reviewed else-

where [23 7,23 8]. An intron-less viral long noncoding poly adenylated

nuclear RNA was found to be highly expressed during the lytic

phase of infection of the Kaposi’s sarcoma-associated herpes

virus [239,240], the causative agent of various human cancers and

immuno proliferative disorders. Two recent studies used next-

generation whole transcriptome sequencing to identify ncRNAs

differentially expressed after virus expression. A ﬁrst study showed

differential expression of approximately 500 annotated, long

ncRNAs and 1000 nonannotated genomic regions after severe

acute respiratory syndrome corona virus infection in mice [ 24 1] .

In addition, the authors demonstrated that over 40% of ncR-

NAs were similarly regulated in vitro in response to inﬂuenza

virus infection, suggesting the existence of a signature proﬁle

associated with pathogenicity. In the second study, a software

tool ‘RandA’ (ncRNA Read-and-Analyze) that performs com-

prehensive ncRNA proﬁling and differential expression analysis

on data generated by deep sequencing while running on a local

computer was developed [2 42] . They used RandA to identify and

validate ncRNA shifts in samples infected with mycoplasma, HIV

and Epstein–Barr virus. Overall, the knowledge on the roles of

miRNAs and long ncRNAs in infectious diseases is limited and

further research is necessary in order to exploit them as useful

biomarkers.

Van Roosbroeck, Pollet & Calin

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ncRNAs as a new strategy for therapeutic

interventions

Their widespread role in physiological processes and deregula-

tion in human diseases makes ncRNAs very attractive targets for

novel therapeutics. Several strategies are currently being inves-

tigated that either silence overexpressed ncRNAs or reactivate

down regulated ncRNAs (reviewed by [32 ,203,243]). To date, the

main focus has been on miRNAs, because long ncRNA-based

research is only in premature stages. However, similar strategies

to restore deregulated miRNAs could potentially also be useful

for long ncRNAs.

Inactivation of overexpressed miRNAs can be achieved by

using antisense oligonucleotides, which are complementary to the

miRNA and inhibit miRNAs based on this base pair complemen-

tarity [3 2]. To increase the stability of antisense oligonucleotides,

different chemical modiﬁcations have been incorporated, leading

to the development of locked nucleic acid anti-miRs [24 4], anti-

sense miRNA oligonucleotides [245] and antagomirs [2 46] . Another

approach is the use of ‘miRNA sponges’, transcripts expressed from

strong promoters and containing multiple artiﬁcial miRNA bind-

ing sites that compete with natural miRNA targets for miRNA

binding [2 47] . The search for small-molecule inhibitors of miRNAs

is also ongoing [248] , although it is still under debate whether small

molecules are indeed able to inhibit miRNA function. On the

other hand, the ‘microRNA replacement strategy’ can be used

to re-activate downregulated miRNAs. These miRNA mimics

are synthetic dsRNAs that are designed to mirror the action of

mature endogenous miRNAs (reviewed by [249]). Although these

therapeutic strategies are very promising, some major hurdles

remain, including the problem of proper in vivo delivery and off-

target speciﬁcity [250 ]. Many biotech companies are trying to over-

come these obstacles to advance miRNA-based therapeutics to the

clinic. In fact, one locked nucleic acid-modiﬁed oligonucleotide,

SPC3649 or miravirsen, has gone forward from preclinical trials

in primates [ 251] to Phase I and IIa clinical trials in humans with

chronic hepatitis C virus infection [40 1] . SPC3649 was demon-

strated to block the liver-speciﬁc host miRNA miR-122, which

is critical to hepatitis C virus accumulation in the liver [ 251] . The

Phase IIa clinical trial recently ﬁnished and no ofﬁcial results have

been published yet. However, a late-breaking oral presentation

on the 2011 AASLD meeting reported that the drug was safe

and well tolerated, and resulted in a signiﬁcant dose-dependent

decrease in hepatitis C RNA levels in patients [252] . Miravirsen is

the ﬁrst miRNA-targeted drug to enter clinical trials, but most

likely, others will follow soon.

For more information on the use of ncRNAs as targets of

therapeutic interventions, the reader is redirected to some other

reviews [253–257].

Expert commentary

The discovery of informative biomarkers is not only important to

understand physiopathological processes of diseases, but is also

essential for improved diagnostics and therapeutics [8] . Variations

in expression and/or sequences of ncRNAs have been associated

with disease predisposition, which drives the development of

biomarkers for genetic disease susceptibility. Although in many

instances genetic screening might be very useful, it raises various

ethical questions that need to be answered [258] .

A critical issue, particularly in cancers, is early detection. Most

current diagnostic biomarkers are based on molecular analysis of

the tumor tissue itself, which is usually very small in early stages.

Consequently, many tumor types can only be detected in later

stages, when patients tend to be refractory to therapy, which leads

to high mortality rates. In this regard, the discovery of tumor-

speciﬁc ncRNAs in body ﬂuids such as serum, saliva and urine is of

key interest, as even in early stages, signiﬁcant changes in ncRNA

concentration levels could be detected [4] , and these samples can

be obtained through noninvasive methods. More research is, how-

ever, needed in order to establish circulating ncRNAs as diagnostic

and prognostic biomarkers, as the precise functions of these cell-

free RNAs remain poorly understood [4]. To effectively screen

for these ncRNA biomarkers, there is a need for accurate and

extremely sensitive determination methods. The ﬁeld of ncRNA

diagnostics is rapidly evolving. Several established techniques,

such as microarrays, ISH, real-time PCR and RNA sequencing,

are routinely used nowadays (TABle 1) . Meanwhile, novel methods

are being developed that are more sensitive and selective, and can

be used at high throughput and at low cost (TABle 2).

Even though the knowledge on ncRNAs has signiﬁcantly

advanced over recent years, much more research is needed to

elucidate the full extent to which ncRNAs exert their patho-

logical effect, before they can be widely used in diagnostics and

prognostics of human diseases.

Five-year view

In the last decade, the involvement of ncRNAs in human diseases

has become increasingly apparent, and many research groups have

started to explore the molecular use of ncRNAs in diagnostics and

therapeutics. In particular, miRNAs have been extensively inves-

tigated, although more and more research starts to focus on other

ncRNAs as well. As their importance in human diseases becomes

increasingly apparent, the molecular use of ncRNAs in diagnos-

tics and therapeutics is now beginning to be explored. The pres-

ence of ncRNAs in body ﬂuids makes them extremely suitable as

noninvasive biomarkers, and it is like they will become the main

source of biomarkers in the coming years. The ﬁrst diagnostic tests

employing miRNAs and long ncRNAs are commercially available

(Box 1) and many others are in the pipeline. ncRNAs can also be

used as ‘druggable’ targets, and although some hurdles associated

with in vivo delivery and off-target speciﬁcity still need to be over-

come before ncRNAs can be widely used as therapeutic targets, an

increasing number of biotech companies are trying to overcome

these obstacles, and their ﬁrst reports are promising for the near

future. In fact, the ﬁrst miRNA antagonist is in clinical trials for

patients with chronic hepatitis C infection [4 0 1] , and we expect many

more to follow soon.

miRNAs & long noncoding RNAs as biomarkers in human diseases

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Expert Rev. Mol. Diagn. 13(2 ), (2013)

196

Review

Financial & competing interests disclosure

K Van Roosbroeck is supported by a fellowship of the Henri Benedictus Fund/

Belgian American Education Foundation. J Pollet is supported by fellowships

of the Fulbright Association and the Belgian American Education Foundation.

GA Calin is an Alan M Gewirtz Leukemia & Lymphoma Society Scholar. He

is also supported as a Fellow at The University of Texas MD Anderson Research

Trust, as a University of Texas System Regents Research Scholar, and by the

CLL Global Research Foundation. Work in GA Calin’s laboratory is supported

in part by the NIH/NCI (C A135444), a Department of De fense Breast Cancer

Idea Award, Developmental Research Awards in Breast Cancer, Ovarian

Cancer, Brain Cancer, Prostate Cancer, Multiple Myeloma, Leukemia

(P50 CA100632) and Head and Neck (P50 CA097007) SPOREs, a SINF

MDACC_DKFZ grant in CLL, the Laura and John Arnold Foundation,

the RGK Foundation and the Estate of CG Johnson Jr. The authors have no

other relevant afﬁliations or ﬁnancial involvement with any organization or

entity with a ﬁnancial interest in or ﬁnancial conﬂict with the subject matter

or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Key issues

• miRNAs have more complex functions than initially assumed: they do not only negatively regulate expression of protein-coding genes,

but they can also upregulate expression of target genes, act as competing endogenous RNAs (ceRNAs) that divert miRNAs away from

their mRNA targets, function as RNA decoy to modulate the function of regulatory proteins, and interact with and regulate other

noncoding RNAs (ncRNAs).

• Most ncRNAs are good biomarkers as they are stable (even in body ﬂuids), their expression is speciﬁc to tissues, organs or biological

stages, they show increased or decreased expression levels in disease, and they can be easily measured by methods such as quantitative

PCR and microarrays.

• Variations in expression and/or sequences of miRNAs and long ncRNAs are involved in disease predisposition.

• ncRNAs are very useful cancer biomarkers as they can be used to establish a diagnosis, classify tumors, determine disease stage and

predict disease outcome and evolution.

• The use of ncRNAs as biomarkers is not only restricted to cancer, but extends to a plethora of other human diseases, including

cardiovascular diseases, autoimmune diseases, neurological disorders and infectious diseases.

• The clinical use of ncRNAs as biomarkers for cancer is translated into four commercially available diagnostic tests that distinguish

between different cancer subtypes.

• ncRNAs are very attractive targets for therapeutic interventions, and several strategies are currently being explored that either silence

overexpressed ncRNAs or re-activate downregulated ncRNAs.

• A ﬁrst miRNA antagonist, miravirsen, is in Phase IIa clinical trial for patients with chronic hepatitis C virus infection.

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Data Normalization of Urine miRNA Profiling from Head and Neck Cancer Patients Treated with Cisplatin

Article

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The microRNA (miRNA) expression profile by qRT-PCR depends directly on the most appropriate normalization strategy adopted; however, currently there is no universally adequate reference gene. Therefore, this study aimed to determine, considering RNA-Seq results, the most adequate endogenous normalizer for use in the relative quantification of urine miRNAs from head and neck cancer patients, treated with cisplatin chemoradiotherapy. The massive sequencing was performed to identify the miRNAs differentially expressed between the group with cisplatin nephrotoxicity (n = 6) and the one without (n = 6). The candidate endogen normalizer was chosen according to four criteria: (1) the miRNA must be expressed in most samples; (2) the miRNA must have a fold change value between 0.99 and 1.01; (3) the miRNA must have a p-value ≥ 0.98; and (4) the miRNA must not be commented on by the final GeneGlobe (Qiagen, Hilden, Germany) analysis. Four miRNAs met all the criteria (hsa-miR-363-5p, hsa-miR-875-5p, hsa-miR-4302, and hsa-miR-6749-5p) and were selected for validation by qRT-PCR in a cohort of 49 patients (including the 12 sequencing participants). Only hsa-miR-875-5p was shown to be an adequate normalizer for the experimental condition under investigation, as it exhibited invariant expression between the two groups.

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The dynamic spectrum of microRNA (miRNA) has grown significantly over the years with its identification and exploration in cancer therapeutics and is currently identified as an important resource for innovative strategies due to its functional behavior for gene regulation and modulation of complex biological networks. The progression of cancer is the consequence of uncontrolled, nonsynchronous procedural faults in the biological system. Diversified and variable cellular response of cancerous cells has always raised challenges in effective cancer therapy. miRNAs, a class of non-coding RNAs (ncRNAs), are the natural genetic gift, responsible to preserve the homeostasis of cell to nurture. The unprecedented significance of endogenous miRNAs has exhibited promising therapeutic potential in cancer therapeutics. Currently, miRNA mimic miR-34, and an antimiR aimed against miR-122 has entered the clinical trials for cancer treatments. This review, highlights the recent breakthroughs, challenges, clinical trials, and advanced delivery vehicles in the administration of miRNA therapies for precision oncology.

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Aug 2022
PLOS ONE

Background The role of the long non-coding RNAs (lncRNAs) in the pathogenesis of systemic lupus erythematosus (SLE) is mostly unknown, despite increasing evidence that lncRNAs extensively participate in physiological and pathological conditions. Aim To detect the level of lncRNA-Cox2, HOTAIR, IL-6, and MMP-9 in the serum of SLE patients and to correlate these levels with disease activity and patients’ clinical and laboratory data to evaluate the value of these biomarkers for SLE diagnosis and assessment of disease activity. Methods Blood samples from 58 SLE patients, and 60 healthy controls (HCs) were used for detection of lncRNAs-Cox2 and HOTAIR expression levels by real-time polymerase chain reaction. Both IL-6 and MMP-9 serum levels were assayed by enzyme-linked immunosorbent assay. Lupus activity was assessed with the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Results The serum expression levels of lncRNA-Cox2 and HOTAIR were significantly up-regulated in SLE patients vs HCs (fold change [median (IQR) was 1.29(0.81–1.71, P <0.0001) and 2.68(0.95–3.67), P = 0.038) for lncRNA-Cox2 and HOTAIR, respectively. Serum levels of both IL-6 and MMP-9 were significantly high in SLE patients compared with HCs ( P≤ 0.001 for each). The up-regulated lncRNA-Cox2 was positively associated with the presence of neurological manifestations in SLE patients ( P = 0.007). Furthermore, HOTAIR expression level had significantly positive correlation with IL-6 ( r = 0.578, P <0.0001), MMP-9 level (r = 0.762, P <0.0001), nephritis grades ( r = 0.296, P = 0.024) and proteinuria ( r = 0.287, P = 0.035). LncRNA-Cox2 showed sensitivity and specificity 72.4%, and 100.0% respectively. HOTAIR sensitivity was 60.3%, and specificity was 100.0%. By multiple logistic regression analysis, lncRNA-Cox2 and HOTAIR were found as SLE independent predictors. Conclusion LncRNA-COX2 and HOTAIR can be used as new non-invasive biomarkers for the diagnosis of SLE.

Does the diverse source of miRNAs affect human health? An approach towards diagnosis and therapeutic management

Article

Jul 2022

MicroRNAs are small non-protein coding RNAs of 19–24 nucleotides that operate as regulators of gene expression in the eukaryotic system at both the transcriptional and post-transcriptional levels. Promising evidences suggest that most of the miRNAs and the related components of the RNA interference pathway are decisively significant in the regulation of nearly all cellular processes of both animals and plants. MicroRNA has crucial role in different metabolic processes like cell division, growth, proliferation, differentiation as well as apoptosis of the cell. Scientists are given more insight into this novel idea of cross kingdom regulation system by miRNA after discovery of exogenous plant MIR168a influencing the transport system of cholesterol in mice by down regulating the receptor adopter protein 1 expression of law density lipoprotein. These types of influential exploration about cross kingdom regulation of miRNAs have taken more intention towards its role in animal as well as microbial system. In human health, the animal sources, plant, microbes derived miRNAs and diet based miRNA plays significant role. Dysregulation of miRNAs is correlated with development of various pathological events such as neurological disorders, cardiovascular diseases, cancer, AMR, which are the most threatened field for human society. Here we detailed about the role of miRNA in human health with diverse source of miRNAs and its role in therapeutic and diagnosis of disease like cancer in human.

MiR‐379‐5p inhibits the proliferation, migration, and invasion of breast cancer by targeting KIF4A

Article

Full-text available

May 2022

Background: Many studies have shown that microRNAs (miRNAs) play an essential role in gene regulation and tumor development. This study aimed to explore the expression of miR-379-5p and its mechanisms of affecting proliferation, migration, and invasion in breast cancer (BC). Methods: MiRNAs and mRNAs expression data of BC and normal breast tissue samples were downloaded from the TCGA and GEO databases. qRT-PCR was used to detect the expression of miR-379-5p in human normal breast epithelial cell lines and human BC cell lines. The proliferation ability of transfected cells was detected by colony formation and EdU assays. The mobility and invasion ability of transfected cells was measured by wound healing and transwell assays. The relative protein expression of transfected cells was detected by western blot. Dual luciferase reporter assay was performed to identify the targeted binding of miR-379-5p and KIF4A. Results: MiR-379-5p was lowly expressed in BC tissue samples and BC cell lines. The target genes of miR-379-5p were involved in many cancer-related signaling pathways. PPI analysis and the cytoHubba algorithm of Cytoscape identified 10 genes as the hub genes. Survival analysis showed that only KIF4A expression in 10 hub genes was significantly associated with the prognosis of BC patients and was significantly upregulated in BC. Overexpression of miR-379-5p inhibited proliferation, migration, and invasion in the BC cell line MDA-MB-231, which could be reversed by KIF4A. Conclusions: MiR-379-5p inhibits proliferation, migration, and invasion of BC by targeting KIF4A.

High Expression of a tRNAPro Derivative Associates with Poor Survival and Independently Predicts Colorectal Cancer Recurrence

Article

Full-text available

May 2022

Colorectal cancer (CRC) is the second most lethal cause of cancer-related deaths in Europe. Fragments of tRNAPro are conserved among vertebrates, characterized by pleiotropic regulatory functions and have been found to discriminate colorectal tumors from normal colorectal mucosa. In the current study, we investigated the prognostic utility of 5′-tiRNA-ProTGG levels in CRC. For this purpose, total RNA was extracted from 155 malignant colorectal tumors and 74 adjacent non-cancerous tissue specimens, polyadenylated and reverse-transcribed using an oligo-dT adapter as primer. Real-time quantitative PCR (qPCR) was used to assess the levels of 5′-tiRNA-ProTGG. Kaplan-Meier survival analysis demonstrated that high 5′-tiRNA-ProTGG levels predict both poor disease-free survival (DFS) and overall survival (OS) of CRC patients. Of note, high 5′-tiRNA-ProTGG levels retain their unfavorable prognostic value in patients with rectal cancer and/or moderately differentiated CRC (grade II). More importantly, multivariate cox regression analysis highlighted that the overexpression of 5′-tiRNA-ProTGG constitutes an adverse prognostic factor predicting short-term relapse of CRC patients independently of the established prognosticators in CRC. Finally, bioinformatics analysis unveiled a potentially critical role of 5′-tiRNA-ProTGG regarding the maintenance of cellular homeostasis, signaling, cell communication, and cellular motility.

Biomarkers for disease identification

Chapter

Full-text available

Jun 2024

Integrative analysis of circular RNA regulatory network in papillary thyroid carcinoma

Article

Sep 2023

The Diagnostic and Prognostic Value of miR-155 in Cancers: An Updated Meta-analysis

Article

Mar 2023
MOL DIAGN THER

Background: MicroRNA-155 has been discussed as a biomarker in cancer diagnosis and prognosis. Although relevant studies have been published, the role of microRNA-155 remains uncertain because of insufficient data. Methods: We conducted a literature search in PubMed, Embase, and Web of Science databases to obtain relevant articles and extract data to evaluate the role of microRNA-155 in cancer diagnosis and prognosis. Results: The pooled results showed that microRNA-155 presented a remarkable diagnostic value in cancers (area under the curve = 0.90, 95% confidence interval (CI 0.87-0.92; sensitivity = 0.83, 95% CI 0.79-0.87; specificity = 0.83, 95% CI 0.80-0.86), which was maintained in the subgroups stratified by ethnicity (Asian and Caucasian), cancer types (breast cancer, lung cancer, hepatocellular carcinoma, leukemia, and pancreatic ductal adenocarcinoma), sample types (plasma, serum, tissue), and sample size (n >100 and n <100). In prognosis, a combined hazard ratio (HR) showed that microRNA-155 was significantly associated with poor overall survival (HR = 1.38, 95% CI 1.25-1.54) and recurrence-free survival (HR = 2.13, 95% CI 1.65-2.76), and was boundary significant with poor progression-free survival (HR = 1.20, 95% CI 1.00-1.44), but not significant with disease-free survival (HR = 1.14, 95% CI 0.70-1.85). Subgroup analyses in overall survival showed that microRNA-155 was associated with poor overall survival in the subgroups stratified by ethnicity and sample size. However, the significant association was maintained in cancer types subgroups of leukemia, lung cancer, and oral squamous cell carcinoma, but not in colorectal cancer, hepatocellular carcinoma, and breast cancer, and was maintained in sample types subgroups of bone marrow and tissue, but not in plasma and serum. Conclusions: Results from this meta-analysis demonstrated that microRNA-155 was a valuable biomarker in cancer diagnosis and prognosis.

Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells

Article

Full-text available

Jan 2007

Exosomes are vesicles of endocytic origin released by many cells. These vesicles can mediate communication between cells, facilitating processes such as antigen presentation. Here, we show that exosomes from a mouse and a human mast cell line (MC/9 and HMC-1, respectively), as well as primary bone marrow-derived mouse mast cells, contain RNA. Microarray assessments revealed the presence of mRNA from approximately 1300 genes, many of which are not present in the cytoplasm of the donor cell. In vitro translation proved that the exosome mRNAs were functional. Quality control RNA analysis of total RNA derived from exosomes also revealed presence of small RNAs, including microRNAs. The RNA from mast cell exosomes is transferable to other mouse and human mast cells. After transfer of mouse exosomal RNA to human mast cells, new mouse proteins were found in the recipient cells, indicating that transferred exosomal mRNA can be translated after entering another cell. In summary, we show that exosomes contain both mRNA and microRNA, which can be delivered to another cell, and can be functional in this new location. We propose that this RNA is called " exosomal shuttle RNA " (esRNA). Exosomes are small (50–90 nm) membrane vesicles of endocytic origin that are released into the extracellular environment on fusion of multivesicular bodies (MVB) with the plasma membrane 1. Many cells have the capacity to release exosomes, including reticulo-cytes 2 , dendritic cells 3 , B cells 4 , T cells 5 , mast cells 6 , epithelial cells 7 and tumour cells 8. The functions of exosomes are not completely understood, although it has been shown that exosomes can participate in the signalling events contributing to antigen presentation to T cells 4 and the development of tolerance 9. Several mechanisms have been hypothesized describing the interactions of exosomes and recipient cells. Exosomes can bind to cells through recep-tor–ligand interactions, similar to cell–cell communication mediating , for example, antigen presentation 4. Alternatively, exosomes could putatively attach or fuse with the target-cell membrane, delivering exosomal surface proteins and perhaps cytoplasm to the recipient cell 10,11. Finally, exosomes may also be internalized by the recipient cells by mechanisms such as endocytosis 12. Exosomes were isolated from a mast-cell line (MC/9), primary bone marrow-derived mast cells (BMMC) and a human mast-cell line (HMC-1) through a series of microfiltration and ultracentrifugation steps modified from what has been previously described 4. To confirm that the structures studied indeed are exosomes, they were examined by electron microscopy (Fig. 1a), flow cytometric analysis (FACS; Fig. 1b), and proteomic analysis (see Supplementary Information, Table S1). The electron micrographs of the exosomes revealed rounded structures with a size of approximately 50–80 nm, similar to previously described exo-somes 4,13–15. The identity of the studied vesicles was further confirmed as exosomes by FACS analysis (Fig. 1b), which show the presence of the surface protein CD63 — a commonly used marker of exosomes. Finally, extensive protein analysis of the MC/9 exosomes was performed on multiple samples using LC-MS/MS technology. A total of 271 proteins were identified (see Supplementary Information, Table S1) from three preparations of the isolated vesicles, of which 47 proteins were present in all three samples. More importantly, a large number of the proteins found in the preparations were the same as proteins previously identified in exosomes produced by other cells (that is, exosomes derived from intestinal epithelial cells, urine, dendritic cells, microglia, melanoma, T-cells and B-cells). In particular, 60% of the 47 proteins found in all samples of mast-cell exosomes have been previously found in other types of exosomes. Moreover, 39% of the 271 total proteins found in the analysed exosome samples have also been previously found in other types of exosomes. Thus, the electron microscopy, the FACS, and the detailed protein analyses each provided significant evidence in favour of the identification of the isolated vesicles as exosomes. The presence of nucleic acids was examined in exosomes derived from MC/9, BMMC and HMC-1 cells to define a potential mechanism by which exosomes may mediate cell–cell communication. These assessments showed that isolated exosomes contain no DNA (see Supplementary Information, Fig. S1). However, substantial amounts of RNA were detected by agarose gel electrophoresis, spectrophotometry

A miR-200b/200c/429-binding site polymorphism in the 3' untranslated region of the AP-2α gene is associated with cisplatin resistance

Article

Full-text available

Jan 2011
PLOS ONE

Identification of miRNA Changes in Alzheimer's Disease Brain and CSF Yields Putative Biomarkers and Insights into Disease Pathways

Article

May 2008

MicroRNAs have essential functional roles in brain development and neuronal specification but their roles in neurode- generative diseases such as Alzheimer's disease (AD) is unknown. Using a sensitive qRT-PCR platform we identified regional and stage-specific deregulation of miRNA expression in AD patient brains. We used experimental validation in addition to literature to reveal how the deregulated brain microRNAs are biomarkers for known and novel pathways in AD pathogenesis related to amyloid processing, neurogenesis, insulin resistance, and innate immunity. We additionally recovered miRNAs from cerebrospinal fluid and discovered AD-specific miRNA changes consistent with their role as potential biomarkers of disease.

New microRNA-based diagnostic test for lung cancer classification.

Article

May 2011

A second-generation microRNA-based assay for diagnosing tumor tissue origin.

Article

May 2011

10575 Background: Cancer of unknown primary (CUP) constitutes 3%-5% of all newly diagnosed cancer cases. It presents a major diagnostic challenge as knowing the tumor tissue of origin (ToO) of the cancer is crucial for choosing the optimal treatment. MicroRNAs are a family of non-coding, regulatory RNA genes involved in development and differentiation that were shown to be involved in carcinogenesis. MicroRNAs, highly stable in clinical samples and tissue-specific, serve as ideal biomarkers for cancer diagnosis. Our first generation assay identifies the ToO using a set of 48 microRNAs measured on a qRT-PCR platform and differentiates 25 different tumor types. We present here the development and validation of a second generation clinical assay that can identify 42 different tumor types using a custom array platform. Methods: Over 1300 primary and metastatic tumor formalin-fixed and paraffin-embedded (FFPE) tissue samples were used for the training and development of the assay. High-purity RNA was extracted...

Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors

Article