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Vet Pathol. 2014 July ; 51(4): 759–774. doi:10.1177/0300985813502820.

MicroRNAs: History, Biogenesis, and Their Evolving Role in

Animal Development and Disease
M. Bhaskaran1 and M. Mohan2
1Infectious Disease Aerobiology, Division of Microbiology, Tulane National Primate Research
Center, Covington, LA, USA
2Division of Comparative Pathology, Tulane National Primate Research Center, Covington, LA,
USA

Abstract
The discovery of microRNAs (miRNAs) in 1993 followed by developments and discoveries in
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small RNA biology have redefined the biological landscape by significantly altering the
longstanding dogmas that defined gene regulation. These small RNAs play a significant role in
modulation of an array of physiological and pathological processes ranging from embryonic
development to neoplastic progression. Unique miRNA signatures of various inherited, metabolic,
infectious, and neoplastic diseases have added a new dimension to the studies that look at their
pathogenesis and highlight their potential to be reliable biomarkers. Also, altering miRNA
functionality and the development of novel in vivo delivery systems to achieve targeted
modulation of specific miRNA function are being actively pursued as novel approaches for
therapeutic intervention in many diseases. Here we review the current body of knowledge on the
role of miRNAs in development and disease and discuss future implications.

Keywords
animal microRNA; miR; oncomiR; apoptomiR; circulating miRNA; biomarkers
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MicroRNAs: Introduction and a Brief History

MicroRNAs (miRNAs) are a class of small (~19–24 nucleotides in length), endogenous,
evolutionarily conserved RNAs that function as posttranscriptional regulators of gene
expression. They primarily function by binding to complementary target sequences in
messenger RNA (mRNA) and interfering with the translational machinery, thereby
preventing or altering the production of the protein product. Follow-up studies also revealed
that in addition to repressing translation, miRNA binding to its target mRNA also triggered
the recruitment and association of mRNA decay factors, leading to mRNA destabilization,

© The Author(s) 2013

Corresponding Author: M. Bhaskaran, Infectious Disease Aerobiology, Division of Microbiology, Tulane National Primate
Research Center, 18703 Three Rivers Rd, Covington, LA 70433-8915, USA. [email protected].
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
 Bhaskaran and Mohan Page 2

degradation, and resultant decrease in expression levels. miRNAs were discovered in 1993
by Lee and colleagues93 in the nematode Caenorhabditis elegans. In these organisms, the
downregulation of LIN-14 protein was found to be essential for the progression from the
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first larval stage (L1) to L2. Furthermore, the downregulation of LIN-14 was found to be
dependent on the transcription of a second gene called lin-4. Interestingly, the transcribed
lin-4 was not translated into a biologically active protein. Instead, it gave rise to 2 small
RNAs approximately 21 and 61 nucleotides in length. The longer sequence formed a stem-
loop structure and served as a precursor for the shorter RNA. Later this group,93 along with
Wightman et al,183 found that the smaller RNA had antisense complementarity to multiple
sites in the 3′ UTR of lin-14 mRNA. The binding between these complementary regions
decreased LIN-14 protein expression without causing any significant change in its mRNA
levels. These 2 studies together brought forth a model wherein base pairing occurred
between multiple lin-4 small RNAs to the complementary sites in the 3′ UTR of lin-14
mRNA, thereby causing translational repression of lin-14 and subsequent progression from
L1 to L2 during C. elegans development.

This novel mode of regulating gene expression was first thought to be a phenomenon
exclusive to C. elegans. In 2000, 2 separate groups discovered that a small RNA, let-7, was
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essential for the development of a later larval stage to adult in C. elegans.141,159 More
importantly, homologues of this gene were subsequently discovered in many other
organisms, including humans.133 The period that followed was marked by a deluge of
information wherein multiple laboratories cloned numerous small RNAs from humans, flies,
and worms. These RNAs were noncoding, around 19 to 24 nucleotides in length, and
derived from a longer precursor with a stem-loop or fold-back structure.9 Many were found
to be evolutionarily conserved across species and exhibited cell-type specificity. The
recognition and confirmation of the existence of these small RNAs, now termed microRNAs
(mi-RNAs), led to intense research aimed at identifying new members of this family. This
resulted in the discovery of multiple miRNAs across different species of plants and animals.
An miRNA registry, named miRBase, set up in 2002 serves as the primary online repository
for all potential miRNA sequences, annotation, nomenclature, and target prediction
information.53,55 The current release (miRBase 20) contains 24 521 entries representing
hairpin precursor miRNAs that express 30 424 mature miRNA products in 206 species. The
biological significance of a vast majority of annotated miRNAs, however, remains unknown
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and requires functional validation.

Biogenesis of miRNAs
miRNA genes can vary widely in their location in the genome. Earlier studies had revealed 2
distinct classes of miRNAs: those that originated from overlapping introns of protein coding
transcripts and others that are encoded in exons, underscoring the complexities associated
with miRNA maturation.143 Clusters of miRNA genes that coex-press polycistronically with
the potential to be transcribed as a single unit were also discovered.86,90 It is estimated that
approximately 50% of miRNAs are expressed from non– protein coding transcripts.147 The
rest are mostly located in the introns of coding genes and are generally cotranscribed with
their host genes and processed separately.147 Since this is a rapidly evolving field, there is
potential for future developments to significantly overhaul the current understanding of

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miRNA genesis. Based on current knowledge, it can be stated that genomic regions capable
of generating mature functional miRNAs can be present on diverse locations within the
genome.
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A general overview of the steps involved in miRNA biogenesis is illustrated in Fig. 1.

miRNA coding transcripts are initially transcribed by RNA polymerase II as long primary
miRNAs (several hundred nucleotides long) with a 5′ guano-sine cap and a 3′
polyadenylated tail. These can be either non-coding or coding (present within the intron of a
coding gene). The primary miRNA is then processed into ~70- to 120-nucleotide-long
precursor RNA (pre-miRNA) by a multiprotein complex called Microprocessor (Fig. 1A).
This complex contains a ~160-kDa nuclear RNase III enzyme called Drosha.94 This enzyme
is highly conserved in animals but not in plants.185 Drosha dimerizes with another double-
stranded RNA (dsRNA) binding protein, called DiGeorge syndrome critical region gene 8
(DGCR8) or Pasha, to form the functional Microprocessor complex (Fig. 1A).34,52,59,88 The
newly transcribed pre-miRNA with a typical 5′ phosphate and ~2-nucleotide 3′ overhang is
then exported into the cytoplasm by exportin 5 (Exp-5), a Ran-dependent nuclear transport
receptor protein (Fig. 1B).110,195 In the cytoplasm, the pre-miRNAs are finally processed
into mature ~18- to 23-nucleotide-long duplexes by another RNase III enzyme, Dicer-1,
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with help from dsRNA-binding proteins, protein kinase RNA activator and transactivation
response RNA binding protein (Fig. 1C,D).28,95,96,195 The 2 miRNA strands are then
separated, depending on various factors such as thermodynamic asymmetry of the duplex
and stability of base pairing at the 5′ end. One strand, termed the guide strand, along with
the aforementioned and other RNA binding proteins that include trinucleotide repeat-
containing gene 6A (TNRC6A), associates with catalytic Argonaute (AGO) proteins,
forming a microribonuclear protein complex (miRNP) called RNA-induced silencing
complex (RISC) (Fig. 1E).154 The miRNA strand with the most unstable base pairing at the
5′ end usually acts as the guide strand, while the strand with stable base pairing at the 5′ end
(known as the passenger or miR* strand) is usually degraded or, in rare cases, even
associates with AGO proteins, enabling both strands to serve as functional miRNAs.33,129
The guide strand directs the complex to the target mRNA through sequence
complementarity and causes its translational repression (Fig. 1F). Ago2 proteins have been
localized to cytoplasmic bodies called GW/P-bodies (processing bodies), where miRNAs
bound to their mRNA targets are believed to be stored for degradation or translational
repression (Fig. 1G).23 However, recent evidence also demonstrates thatmiRNA
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biogenesiscan be Microprocessor independent. Examples include pre–miRNA-like hairpins

named “Mirtrons” formed from spliced and disbranched short hairpin introns, some small
nucleolar RNAs (snoRNAs), and endogenous short hairpin RNAs (shRNAs).7,41,128,149

Various mechanisms have been proposed on the modus operandi of miRNAs that results in
posttranscriptional repression of target mRNA. The repression can be the result of either
reduced translational efficiency or due to an actual decrease in the mRNA levels. Degree of
complementarity between the miRNA and the target regions in the mRNA is thought to have
a role wherein sufficient complementarity is believed to result in mRNA degradation while
less complementarity leads to translational inhibition.67 Nevertheless, there are exceptions to
this rule where even near-perfect complementarity leads to translational suppression but not

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cleavage.6 An alternative mechanism of action has also been proposed where binding of
miRNAs leads to faster deadenylation of mRNAs, thereby decreasing mRNA stability and
accelerating their degradation.186 Most recent studies show that mRNA destabilization with
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a resultant decrease in the mRNA levels is predominantly responsible for the reduced
protein output than just inhibition of the translational machinery.57 An independent study
also demonstrated that miRNAs can function as “decoys” and interfere with the functioning
of regulatory proteins by binding to mRNA without necessitating a seed region.37 The
notion that the 3′ UTR is the sole target of miRNA interactions is also changing as some
recent studies have demonstrated that miRNA binding sites can be present both in 5′UTR
and within the coding sequences.111,168

At least 1 study also identified miRNAs to function as activators of target mRNA

transcription that play a role in maintaining quiescence during cell cycle, alluding to the
possibility that, in rare instances, they can perform dual roles as activators and repressors.174
Bioinformatic predictions indicate that mammalian miRNAs may regulate up to 30% of all
protein coding genes.101 Emerging evidence also suggests that miRNAs not only play a
major role in regulating gene expression but also represent a very critical cellular factor with
enormous capability to fine-tune biological processes. In this context, it is remarkable to
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note that these tiny RNA molecules with such important and ubiquitous roles in regulating
cellular processes managed to evade the scientific community's radar for most of the 20th
century.

Nomenclature of miRNAs
A uniform system of annotation and nomenclature has been adopted to ensure uniformity
and ease of cataloging miRNAs.2,53–55 miRNAs are numbered sequentially in the order they
are discovered. Those that have been experimentally confirmed are assigned a number that
is attached to the prefix “miR” followed by a dash (eg, miR-21). In the identifier hsa-
miR-21, the first 3 letters indicate the organism (eg, hsa for Homo sapiens, mml for Macaca
mulatta). The mature miRNA is denoted as miR-21 (with a capitalized R), while the
uncapitalized mir-21 refers to both the miRNA gene and the predicted stem-loop component
of the primary transcript, also known as the precursor miRNA. Identical mature miRNA
sequences that originate from discrete precursor sequences and genomic loci are given
identifiers that contain a numeric suffix such as hsa-miR-219-1 and hsa-miR-219-2. On the
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other hand, closely related mature sequences that differ by 1 or 2 nucleotides are named with
a lettered suffix. This would mean that hsa-miR-130a and has-miR-130b are derived from
precursors hsa-mir-130a and hsa-mir-130b, respectively. Recent deep sequencing studies
have revealed that individual pre-miRNAs can give rise to multiple mature miRNA species/
sequences that can vary in length or sequence after being subjected to a variety of
modifications that include trimming at the 5′/3′ ends, substitutions, insertions, deletions, or
additions at the 3′ end.119 Termed isomirs, their biological significance is a subject of
intense investigation. Also, efforts are being made to modify the nomenclature system so as
to incorporate these variants. Some miRNA precursors give rise to two ~21- to 23-
nucleotide-long mature miRNAs. When the predominantly expressed miRNA species can be
definitively established, it is named as mentioned above (eg, miR-136) while the one
originating from the opposite strand of the precursor is designated the same name but with

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an asterisk next to the number (miR-136*). However, when determination of the

predominantly expressed species is not possible, identifiers such as miR-502-5p (from the 5′
strand) and miR-502-3p (from the 3′ strand) are assigned. miRNAs can also be found as
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clusters and present in close proximity within the genome. For example, the miR-17 cluster
contains 6 precursor miRNAs located within a 1-kilobase region of chromosome 13 that can
give rise to 7 mature miRNAs—namely, miR-17, miR-91, miR-18, miR-19, miR-19b,
miR-20, and miR-92.96 These clusters have been denoted in the literature as either an
miR-17 cluster (designated with only the smallest numbered miRNA) or an miR-17-92
cluster (contains both the lowest and highest numbered miRNA).

MiRNAs in Development and Organogenesis

Animals that do not express miRNAs fail to survive or reproduce normally.12,76,81,182 The
universal impairment of the miRNA pathway by knocking down Dicer in fruit flies and mice
caused embryonic lethality with abnormal morphology in almost all organs and resulted in
significant lack of stem cells.3,12,61,62,81,193 Embryonic stem cell differentiation, a cardinal
event in the development of organs and organ systems, is significantly modulated by
miRNAs.114 Studies show that miRNAs can either promote or inhibit stem cell renewal
depending on the cell type or culture environment. In mice, even though Dicer deficiency
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did not stop the formation of stem cell colonies, it severely impaired the differentiation
capability of embryonic stem cells and caused considerable defects in cellular
morphology.75 A subgroup of miRNAs from the miR-290 cluster (cluster implies that these
miRNAs are coexpressed from a single transcript) was shown to regulate the embryonic
stem cell (ESC) cycle and is now termed the ESCC family of miRNAs (ESC cell cycle–
promoting miRNAs).114,178 ESCC miRNAs directly target the cell cycle inhibitors p21 and
LATS2, thus facilitating G1-S phase transition.178 Moreover, transcription factors such as
Oct3/4, Nanog, and Sox2, which are critical for maintaining pluripotency, have been shown
to bind to the promoter of the miR-290 cluster and sustain its expression, thereby promoting
self-renewal and maintenance of the pluripotent state.103,112 ESC miRNA knockout
(through deletion of Dicer or Dgcr8) in mouse models resulted in an altered cell cycle
profile with an extended G1 phase.75,179 As ESCs transition from a self-renewing to a
differentiated state, several ESCC miRNAs show a gradual decrease in expression levels. In
contrast, miRNA let-7 acts as a suppressor of pluripotency and is known to antagonize the
effects of the miR-290 cluster. Unlike the miR-290 cluster, upregulation of let-7 was
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detected in the differentiated state, suggesting that its antagonistic effect may help stabilize
the differentiated state.114,115

Similarly, miRNAs are also important in regulating the proliferation and differentiation of
hematopoietic stem cells. miR-125b performs a specialized role not only in regulating
hematopoiesis at the stem cell level but also in modulating inflammation and innate
immunity by specifically promoting the differentiation and activation of macrophages.25,155
This proinflammatory effect of miR-125b was shown to be mediated predominantly via
regulation of the nuclear factor (NF)–κB pathway. Interestingly, the dysregulation of
miR-125b has been reported in multiple human cancers, including leukemia, and causes
acute myeloid and lymphoid leukemias in mouse models.42,126

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miRNAs have been described to play a major role in orchestrating the coordinated
development of various organ systems. Although ubiquitously expressed, temporal and
spatial expression of distinct sets of tissue-specific miRNAs is important in modeling tissue
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development and differentiation. miR-273 is required for establishing left-right asymmetry

during neuronal development.64 In mouse heart, the fact that even deletion of 1 of the 2
genes coding for miR-1 (miR-1-2) caused severe and irreparable defects in cardiac
morphology suggests critical roles for this miRNA in regulating cardiogenesis.198 The
highly conserved miR-1 is the most abundant miRNA in adult heart, and its known functions
include controlling cardiac morphogenesis, electrical conduction, and the cell cycle. miR-1
has been proposed to regulate cardiogenesis by fine-tuning the expression of Hand2, a
transcription factor essential for cardiac development.199 Other validated targets of miR-1
include insulin-like growth factor 1, calmodulin, and myocyte enhancer factor 2A, all of
which have been well documented to cumulatively contribute to the development of cardiac
hypertrophy.1,39,68 In mice, deletion of miR-208 markedly impaired the ability of the heart
to respond to stress stimuli.173 Double gene knockout of yet another muscle-enriched
miRNA, miR-133a, in mice resulted in increased proliferation and apoptosis of myocytes,
ventricular septal defects, and embryonic lethality. Those that survived developed severe
cardiac dilatation and failure.106 In skeletal muscle, upregulation of miR-27 and subsequent
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downregulation of its target protein, Pax3, were found to be important in reducing myocyte
proliferation and facilitating myogenic differentiation.32 Besides cardiac and skeletal
muscle, miRNAs also exert specific functions in skin development. miR-203 is induced
during differentiation and stratification of mouse skin, which in turn controls the basal to
suprabasal transition by repressing p63, a member of the p53 family of transcription
factors.99,194 On the contrary, p63 represses miR-34a/c expression to maintain cell cycle
progression and thus antagonize the effects of miR-203.5,153 Along similar lines, expression
of miR-124 was found to be essential for proper development of the nervous system.36,163

Systematic expression of miR-127 was found to be essential for proper branching of lung in
rat fetal lung cultures. Its untimely overexpression caused defective branching and
malformation of lung buds.13 Transgenic overexpression of the miR-17-92 cluster induced
proliferation and inhibited differentiation in lung epithelial progenitor cells.109 Another
important functional role for miRNAs was documented in insulin secretion, where miR-375
expression in pancreatic islets directly altered exocytosis of insulin from pancreatic beta
cells.139 In mice, miR-143 regulates adipocyte differentiation and miR-122 regulates lipid
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metabolism by altering the expression of multiple target genes.43,44 In the proximal

convoluted tubules of mouse kidney, miR-192 suppresses Na+/K+-ATPase, the major
transporter of solutes and fluids in renal epithelial cells, and contributes to renal handling of
fluid balance during increased sodium/water intake.118 There studies, together with the
recent developments in high-throughput miRNA profiling that have investigated the spatial
and temporal expression patterns in specific organ systems, including whole organisms,
have unequivocally established the role and relevance of miRNAs in animal development
and organogenesis.

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miRNAs in Diseases
miRNAs in cancer—Apoptosis plays a significant role in both animal development and
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disease, and the dysregulation of this process has been invariably linked to the progression
of various neoplastic processes. miRNAs that regulate apoptosis, termed apoptomiRs, can be
either pro- or antiapoptotic. The first miRNA described as a regulator of apoptosis was the
Drosophila gene bantam, which directly suppressed the proapoptotic factor hid, thus
facilitating proliferation.18,152 Understandably, many miRNAs that play a role in modulating
apoptosis have also been linked to the initiation and progression of various neoplastic
processes. Approximately 50% of miRNAs are located at genomic sites that are disrupted or
amplified in various cancers.21 The first evidence of miRNAs playing a role in cancer
(termed oncomiRs) development came to light in 2002 in a study20 that attempted to find
tumor suppressor genes at chromosome 13q14, which is frequently deleted in chronic
lymphocytic leukemia (CLL).69 CLL is characterized by the presence of substantially
increased numbers of predominantly nondividing malignant B cells overexpressing the
antiapoptotic B-cell lymphoma 2 (Bcl2) protein. In patients with CLL, the tumor suppressor
locus on chromosome 13q14 was found to be frequently altered. However, instead of coding
for a tumor suppressor protein, this region contained 2 miRNA genes, miR-15a and
miR-16-1, which, when overexpressed, were found to negatively regulate antiapoptotic Bcl2
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gene at the posttranscriptional level. Later, many miRNAs that have tumor suppressor roles
were identified. The miR-34 family, for example, has been shown to exert significant tumor
suppressor capabilities. Upregulation of p53 (a potent tumor suppressor/cell cycle regulator)
caused increased miR-34 expression that resulted in G1 arrest in a complementary and
parallel fashion to mRNAs that are directly activated by p53.17,152 Also, miR-34 was shown
to inhibit the silent mating information regulator 1 (SIRT1) gene that resulted in the
upregulation of p53, p21, and PUMA (p53-upregulated modulator of apoptosis), thus
regulating cell cycle and apoptosis and functioning as a tumor suppressor by modulating the
SIRT1-p53 pathway.190 Furthermore, miR-34 has mediated growth arrest via direct
regulation of cell cycle regulatory factors, such as cyclin E2 (CCNE2), cyclin-dependent
kinase 4 (CDK4), E2F3, and the hepatocyte growth factor receptor (c-Met), ultimately
leading to increased caspase-dependent cell death.152,181 In a separate study, miR-34
inhibited the proliferation/growth of human pancreatic tumor–initiating cells, and its
overexpression in p53-deficient human pancreatic cancer cells partially restored the tumor-
suppressing function of p53.70 MCL-1, a member of the BCL-2 family, was also
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demonstrated to be posttranscription-ally regulated by miR-29a, b, and c.121,152 Forced

expression of miR-29b to induce tumor cell apoptosis by reducing MCL-1 expression may
represent a novel intervention for cancer therapy. Along similar lines, let-7a exerts tumor
suppressor functions by directly targeting the expression of RAS and HMGA2, 2 widely
recognized oncogenes.71,97 Other examples of tumor suppressor miRNAs include miR-7,
miR-124, miR-137, miR-146b, miR-15b, miR-128, and miR-326.151 Furthermore, global
knockdown of mature miRNAs by selectively targeting Dicer1, RNASEN, and its cofactor
DGCR8 increased the oncogenic potential of transformed cell lines, resulting in accelerated
tumor formation in mouse models of K-RAS–driven lung cancer and Rb-driven
retinoblastoma.47,84,85,87

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Apart from functioning as tumor suppressors, miRNAs can also promote tumor development
(oncogenes) depending on the functions of the target protein(s) they regulate. These
oncogenic miRNAs include miR-155 and the miR-17-92 cluster that accelerated tumor
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development in B-cell lymphomas.38,63 Ectopic expression of miR-155 in transgenic mice

resulted in pre–B-cell expansion, splenomegaly, and lymphopenia that preceded the
development of lymphoblastic leukemia and lymphoma.31 mir-155 is now known to play a
critical role in the development of lymphomas, although the components of its upstream
regulatory pathways and downstream targets remain unclear. It is interesting to note that
even before the discovery of miRNAs in mammalian cells, Tam et al166 had reported that
the “bic” locus, the common retroviral integration site for the avian leukosis virus, generated
a noncoding RNA.151 Later, after the discovery of miRNAs, it was found that this transcript
harbored the mature miR-155 coding sequence, thus offering a potential explanation for the
function of bic.151 Members of the mir-17-92 cluster are potent activators of cell
proliferation and are frequently overexpressed in several neoplasms, including lymphoma,
multiple myeloma, medulloblastoma, and cancers of the lung, colon, breast, and prostate.47
miR-21 is another commonly upregulated miRNA in cancers that include glioblastoma,
lymphomas, and cancers of the breast, ovary, colon, rectum, pancreas, lung, liver,
gallbladder, prostate, stomach, thyroid, and cervix.151 Increased expression of miR-21 was
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found in glioblastoma tumors and cell lines, and its inhibition resulted in increased cell
death, suggesting that miR-21 could play the role of an oncogene that inhibited cell death in
these tumors.24 Furthermore, in glioblastoma cells, knockdown of miR-21 induced the
activation of caspase-3, transforming growth factor–β, p53, and mitochondrial apoptotic
pathways mainly through upregulation of its validated targets, heterogeneous nuclear
ribonucleoprotein K, p53-related TAp63, and PDCD4, acting in synergy with the
aforementioned proteins.27,132,151

An individual miRNA can also potentially perform dual functions as an oncogene and tumor
suppressor if its targets include antiproliferative genes and growth-promoting genes,
respectively. miR-26 functions as an oncogene in glioma and glioblastoma multiforme by
regulating PTEN, the molecular antagonist of the Akt pathway, resulting in the inhibition of
RB1 and MAP3K2/MEKK2 expression and JNK-dependent apoptosis.66,78 miR-26 is also
thought to function as a tumor suppressor as its expression was either downregulated or its
downregulation resulted in increased anaplasia or metastasis in various neoplasms (eg,
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hepatocellular carcinoma, breast cancer, squamous cell carcinoma, thyroid cancers,

rhabdomyosarcoma, Myc-induced lymphomas) (reviewed in Gao and Liu49). More recently,
miRNA expression in cancers was shown to be controlled by epigenetic mechanisms such as
DNA methylation. In primary lung tumors, methylation of CpG islands in genes for
miR-9-1, miR-9-3, miR-34b/c, and miR-193a resulted in their downregulation with a
concurrent increase in the expression of their target genes, RAR-β2 and NKIRAS1.77 Other
examples include hypermethylation of miR-91, miR-124a-3, miR-148, miR-152, and
miR-663 in human breast cancer and aberrant DNA methylation and downregulation of
miR-127 in prostate and bladder cancers.98,148 In the latter example, chromatin-modifying
drugs successfully restored miR-127 expression and downregulated its predicted target Bcl6,
a proto-oncogene, thereby highlighting the therapeutic potential of chromatin-modifying
drugs in modulating miRNA expression.

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High-throughput techniques such as miRNA microarrays, which provide unique miRNA

expression signatures (miRNomes) of different cancers and their subclassifications, are now
being used for both diagnostic and classification purposes. miRNomes have proved to be
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more reliable than mRNA profiles in some tumor subclassifications wherein arrays of ~200
miRNAs provided a better classification of tumors by type and source than a collection of
>15 000 mRNAs.108 Furthermore, miRNAs are more stable than mRNAs in both body
fluids and routinely collected formalin-fixed, paraffin-embedded tissues.108 Recent studies
have also detected their presence in plasma and serum of patients (discussed later), thus
providing a more convenient and noninvasive approach for miRNA profiling. These
developments, coupled with the unique properties of miRNAs, have opened up exciting new
avenues for the classification, diagnosis, prognosis, and treatment of various cancers.

miRNAs in infectious diseases—miRNAs can influence the manifestation and

pathogenesis of infectious diseases in a multitude of ways. These include modulating the (1)
pathogenicity of individual pathogens, (2) the efficiency of host innate and adaptive immune
response, and (3) the magnitude and resolution of inflammatory responses. Providing an
extensive coverage of the broad range of regulatory roles played by miRNAs in immunity
and inflammation is beyond the scope of this review, and the readers are referred to a
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number of excellent reviews† that have dealt with this topic in greater detail. However, it is
relevant to note that changes in miRNA expression during inflammation/immune response
are controlled not only by altered transcriptional levels but also by interaction of products of
inflammatory/immune responses with the structural and functional components of the
miRNA biogenesis pathway.

Studies that have focused on viral pathogenesis pathways revealed that viral infections can
alter the levels of host miRNAs that specifically regulate antiviral mechanisms, viral
latency, and lytic cycles. These miRNAs either directly target the viral RNA or the host cell
factors vital for their replication. Several viruses encode their own miRNAs (v-miRNAs)
that in turn regulate their production in host cells.136 v-miRNAs target and downregulate
specific host genes, thereby creating a cellular environment that is permissive for virus
replication. Also, from an evolutionary standpoint, it is advantageous and faster to generate
a miRNA complementary to a new target gene than produce a regulatory protein that
performs the same function. Unlike eukaryotic miRNAs, v-miRNAs are generally not
conserved.151 Pioneering studies done in Epstein-Barr virus (EBV) latently infected B cells
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have shown that several unique v-miRNAs originated from the Bam H1 fragment H open
reading frame 1 (BHRF1) and the Bam H1-A region rightward transcript (BART) present in
the viral genome.136 Some of these v-miRNAs are thought to target lytic genes, suppress
viral proliferation, and sustain latency. EBV miRNAs have also been detected in various
types of lymphomas that tested positive for EBV. Interestingly, EBV is also known to
increase its copy numbers in latently infected cells by hijacking the functions of the host
cellular miRNAs, particularly miR-155.82,189 Other miRNAs that were reported to be
upregulated during latency include miR-21, miR-23a, miR-24, miR-27a, and miR-34a, while

†References 14, 30, 50, 140, 151, 161, 162, 184.

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miR-96 and miR-128a/b were downregulated in lymph nodes of patients with EBV-positive
classic Hodgkin lymphoma.22,123
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Viruses such as human immunodeficiency virus 1 (HIV-1) have also been reported to
suppress miRNA-mediated silencing during replication in host cells.169 Components of the
host miRNA processing machinery—namely, Dicer and Drosha—were found to be essential
in inhibiting virus replication both in HIV-1–infected peripheral blood mononuclear cells
and latently infected cells. In this study, when Dicer and Drosha were knocked down using
small interfering RNAs (siRNAs), virus replication kinetics was enhanced compared with
cells transfected with a nonfunctional siRNA. Furthermore, HIV-1 also actively suppressed
the expression of the polycistronic miRNA cluster miR-17/92, which enabled efficient viral
replication by upregulating its target histone acetyltransferase Tat cofactor, named PCAF.
Along similar lines, the HIV-1 Tat protein, a transcriptional activator with a basic RNA
binding domain that can inhibit interferon response, actively suppressed miRNA-siRNA
processing by interfering with Dicer activity.11 In addition, cellular miRNAs such as
miR-28, miR-125b, miR-150, miR-223, and miR-382, can inhibit HIV-1 replication by
binding to complementary sites located within the viral genome.65,170 These anti-HIV
miRNAs were found to be enriched in resting CD4+ T cells and were thought to contribute
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to the development of proviral latency. Interestingly, these miRNAs were also found to be
differentially expressed between monocytes and macrophages, providing an explanation as
to why macrophages and not monocytes are permissive to HIV infection and replication.177

Other examples of cellular miRNAs modulating viral expression include targeting of (1)
influenza virus replication by miR-323, miR-491, miR-654, and let-7c; (2) primate foamy
virus 1 by miR-32; (3) vesicular stomatitis virus by miR-24 and miR-93; (4) hepatitis B
virus by miR-125a-5p, miR-199a-3p, and miR-210; and (5) hepatitis C virus (HCV) by
miR-196, miR-296, miR-351, miR-431, and miR-448.‡ A unique and unexpected finding in
HCV was that a liver-specific miRNA, miR-122, directly targeted the viral RNA sequence
to upregu-late virus replication.72 This finding is very intriguing as it represents a major
deviation from the usual norm that host miRNAs generally repress viral replication.80

Given the dependence on miR-122 for HCV replication, blockade of miR-122 function
using an experimental DNA-based drug SPC3649 (locked nucleic acid–modified antagomir
to miR-122) successfully inhibited HCV replication in chimpanzees.89 SPC3649
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administration at the highest dose produced prolonged suppression of HCV viremia in blood
and liver without the emergence of escape mutants. Furthermore, the drug successfully
alleviated HCV-induced liver pathology and produced no side effects in treated animals.
This drug is currently being evaluated in phase II clinical trials in humans and, due to the
lack of any detectable side effects, has great potential to become the first miRNA-based
therapeutic intervention for HCV infection.

Various bacterial diseases also cause marked alterations in the expression of miRNAs,
especially in cells involved with the immune response. Recent studies have characterized
changes in host miRNA expression following infection with a wide array of bacterial

‡References 80, 92, 131, 135, 138, 160, 197.

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organisms that include both extracellular (eg, Helicobacter pylori) and intracellular (eg,
Salmonella enterica) pathogens. Dysregulated miRNAs have also been demonstrated to
affectthe severity of sepsis that follows bacterial infections. Upregulation of miR-155 and
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downregulation of miR-146a resulted in increased severity of sepsis in murine models of

endotoxemia. miR-155 repressed SHIP1 and SOCS1, 2 negative regulators of inflammation,
while repression of miR-146a led to upregulation of its targets IRAK1 and TRAF6, 2
proinflammatory signaling proteins associated with the TLR/IL-1R pathways.4,16,79
Upregulation of miR-155 has also been shown to potentiate the immune response against
Salmonella typhimurium.144

In Mycobacterium tuberculosis infections, a novel host evasion mechanism mediated by

miR-99b has been described wherein miR-99b expression was high in infected dendritic
cells and macrophages, and its blockade resulted in significantly reduced bacterial
growth.158 This study also found that knockdown of miR-99b resulted in the upregulation of
proinflammatory cytokines such as interleukin (IL)–6, IL-12, and IL-1b and augmented
tumor necrosis factor–α (TNF-α) and TNFRSF-4 production. A second study showed that
miR-21 can be induced after Bacillus Calmette-Guérin (BCG) vaccination via NF-κB
activation.188 miR-21 suppressed IL-12 production by targeting IL-12p35, which in turn
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impaired anti-mycobacterial T-cell responses and promoted dendritic cell apoptosis by

targeting Bcl-2. An analysis of differentially expressed miRNAs that might play a role in the
preferential development of a lepromatous form of leprosy over the tuberculoid form found
significant enrichment of miR-21 in Mycobacterium leprae–infected monocytes in the
lepromatous form.107 In this study, miR-21 directly downregulated TLR2/1-induced
CYP27B1 and IL-1b expression and indirectly upregulated IL-10. The end result of these
changes was the inhibition of expression of genes encoding 2 vitamin D–dependent
antimicrobial peptides, CAMP and DEFB4A, thus providing an effective mechanism for the
bacteria to escape from the vitamin D–dependent antimicrobial pathway. This ability of M.
leprae to upregulate miR-21 is thought to aid in the progression from the self-limiting
tuberculoid form to the progressive lepromatous form of leprosy. The miRNA expression
profiles of various infectious diseases, including fungal pathogens, are now available, and
the functional implications of these profiles, especially in understanding the pathogenic
mechanisms and in developing miRNA-based antimicrobial therapeutics, are aggressively
pursued areas of research.
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miRNAs in other noninfectious diseases—Dysregulation of miRNA expression has

been not only associated with the manifestation of developmental defects in various
organisms and organ systems as discussed earlier but also implicated in a wide array of other
noninfectious diseases, including autoimmune diseases, metabolic disorders, and genetic
diseases. miR-155 and miR-326 are overexpressed in human multiple sclerosis
(MS).35,73,74,127 In vivo silencing of these miRNAs in a mouse model of MS—namely,
experimental autoimmune encephalomyelitis—demonstrated that their functional relevance
could be attributed to their ability to enhance Th17 response and modulate T-cell
developmental pathways by affecting the expression of a complex array of targets.73 Excess
Th17 response is thought to play a key role in the manifestation of various autoimmune
diseases. Multiple studies have looked at miRNA expression profiles in various tissues from

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 Bhaskaran and Mohan Page 12

patients with systemic lupus erythematosus (SLE) or animal models of SLE with
considerable variation in the miRNA signatures reported between studies. Most notably,
miR-146a was significantly decreased in patients with SLE and was strongly associated with
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clinical disease activity and activation of the type I interferon (IFN) pathway.134,156
Reduced expression of miR-146a resulted in aberrant accumulation of its target proteins
(STAT1, IRF5, TRAF6, and IRAK1), leading to a long-lasting hyperactivation of the IFN
pathway and disease manifestation.167 Patients with SLE are known to display aberrant
DNA hypomethylation, and elevated expression of 2 miRNAs, miR-21 and miR-148, has
been proposed to contribute to the development of SLE through their effects on inhibiting
DNA methylation in T cells.156,167

The role of miRNAs as key regulators of metabolism and the therapeutic implications of
these miRNAs in treating metabolic disorders are also being investigated. miRNAs are
known to play a major role in controlling glucose homeostasis and insulin signaling.
Aberrant miRNA expression in cardiometabolic disorders such as obesity, fatty liver
disease, insulin resistance, type 2 diabetes, and coronary artery disease highlights their roles
in the manifestation of their respective pathologies. miR-122, expressed primarily in the
liver, was the first miRNA to be linked to metabolic control.100 Early studies showed that
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miR-122 played a significant role in cholesterol and lipid regulation. Even partial silencing
of miR-122 in mice resulted in a ~25% to 30% reduction in plasma cholesterol levels,
decreased hepatic cholesterol and fatty acid biosynthesis, and an increase in fatty acid β-
oxidation.43 The silencing of miR-122 also resulted in decreased hepatosteatosis in mice fed
a high-fat diet. Similar effects of miR-122 silencing on circulating cholesterol levels were
also observed in African green monkeys.40 However, the exact mechanisms underlying
cholesterol lowering in response to miR-122 silencing remains unknown as genes in liver
that are involved in cholesterol and lipid metabolism do not seem to be direct targets of
miR-122. miR-33a is another miRNA known to control cholesterol homeostasis by
cooperating with SREBP2, a cholesterogenic transcription factor, to boost intracellular
cholesterol levels (reviewed in Rottiers and Naar146). Other miRNAs that play important
roles in metabolic disorders include (1) miR-29, which activates insulin secretion, and
miR-9 and miR-375, which inhibit insulin secretion via their effect on monocarboxylate
transporter 1, one cut homeobox 2, sirtuin 1, phosphoinositide-dependent kinase 1, and
myotrophin; (2) modulation of insulin signaling in adipose tissue by miR-103 and miR-107
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by targeting caveolin 1 and miR-29; and (3) miR-34a in humans and rodent models of
hepatic metabolic diseases, including obesity, type 2 diabetes, nonalcoholic fatty liver
disease, and nonalcoholic steatohepatitis, most likely through an intricate regulatory network
of miR-34a, SIRT1 (a key sensor and regulator of metabolic states), farnesoid X receptor,
and p53 (reviewed in Rottiers and Naar146). Furthermore, miR-375 and miR-223 are also
present in circulating high-density lipoproteins, indicating that disease-associated miRNAs
can be transported in lipoprotein particles, thereby exerting their regulatory effect in a
paracrine fashion on distant target tissues.175

The importance of miRNAs in genetic diseases is evident from their roles in development
and organogenesis described earlier, wherein mutation of relevant miRNAs or target genes
is associated with manifestation of a defective phenotype. However, only a few studies have
established a clear link between miRNAs and specific genetic disorders. An excellent

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example of point mutations in the mature miRNA sequence playing an etiopathogenic role
in a Mendelian disease includes 2 different nucleotide substitutions in the seed region of
miR-96 (13G>A and +14C>A) in 2 Spanish families affected by an autosomal dominant
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form of deafness, DFNA50.116 A single base change (A>T) in the seed region of miR-96 in
mice also resulted in a progressive hearing loss phenotype, providing convincing evidence
that loss of miR-96 target gene regulation arising from point mutations in its seed region
results in hearing loss phenotype in both human and mouse.102 Similarly, mutations and
subsequent sequence variations can also occur in the 3′ UTR of mRNAs, thereby altering
miRNA recognition/binding sites. Such a mechanism has also been demonstrated in an
autosomal dominant form of hereditary spastic paraplegia (SPG31), in which 2 different
point mutations in the predicted miR-140 binding site on the 3′ UTR of the REEP1 gene
resulted in failure to regulate its putative target gene expression.10,201 At least a couple more
studies have looked at human genetic diseases characterized by mutations in genes involved
in miRNA processing/biogenesis. Mutations in the DGCR8 gene, a component of the
Drosha complex, is present in DiGeorge syndrome, and loss of function of the FRM1 gene
that codes for an RNA binding protein occurs in fragile X syndrome.104,157

miRNAs in Veterinary Medicine

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Most miRNA studies are conducted in laboratory animals and cell lines in which the
primary objective is to understand their roles in development and diseases specific to
humans. Although studies have documented organ- and breed-specific miRNA signatures in
various animal species through computational analysis and polymerase chain reaction/
microarray profiling, studies that have directly addressed diseases/disorders specific to food,
companion, avian, and exotic animals are extremely limited. The handful of studies in this
direction is mostly limited to screening of miRNAs in specific conditions, tissues, or species
and have not explored deeper into their mechanism of action and target validation. At least 2
miRNAs, miR-17-5p and miR-181a, have been described to be significantly upregulated in
canine B- and T-cell lymphomas.120 Similarly, in canine osteosarcoma samples, an inverse
correlation between decreased expression of miR-134 and miR-544 originating from the
14q32 locus and aggressive tumor growth characteristics was observed.150 These findings
are in agreement with those reported in human osteosarcoma. In canine mammary cancers,
especially in tubular papillary carcinomas, miR-29b and miR-21 were found to be
significantly upregulated.15 miRNA expression profiling in canine oral melanoma tissues
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found that decreased expression of miR-203 was associated with a shorter survival time,
thereby highlighting its potential as a new prognostic marker for this disease.125 The same
study also showed marked downregulation of miR-203 and miR-205 in canine and human
melanoma cell lines. Over-expression of miR-205 significantly inhibited the growth of
canine and human melanoma cells in vitro by targeting erbb3, a member of the epidermal
growth factor receptor (EGFR) family of receptor tyrosine kinases. A recent study compared
miRNA expression profiles in the serum of Doberman Pinschers with dilated
cardiomyopathy to healthy controls.164 The study detected the expression of a total of 404
miRNAs in serum, of which 22 showed differential expression between the 2 groups.
Although none of the differentially expressed miRNAs attained statistical significance, the
study certainly highlights the potential of miRNAs to serve as disease biomarkers in
veterinary medicine. Another study along similar lines performed in cats detected a

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significant increase in miR-122 (>40-fold) and miR-139b (>14-fold) in the serum of newly
diagnosed diabetic cats when compared with healthy lean cats and cats in diabetic
remission.48
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An interesting study in Texel sheep, renowned for their exceptional meatiness, identified a
new class of regulatory mutations that offer some novel insights into the heritability of
complex traits.29 In this study, the GDF8 allele that encoded for the myostatin gene was
found to contain a G to A transition in its 3′ UTR, which in turn resulted in the creation of a
target site for miR-1 and miR-206, 2 highly expressed miRNAs in skeletal muscle. This
resulted in translational inhibition of the myostatin gene, thereby contributing to the
development of muscular hypertrophy in this breed. Similar studies to investigate the role of
miRNAs in economically important traits such as mam-mary gland development and
skeletal muscle and adipose tissue development, which defines meat quality, are being
pursued in multiple species and breeds of production livestock.56,113

miR-181, a well-known positive regulator of immune response, was shown to directly

impair or even inhibit porcine reproductive and respiratory syndrome virus (PRRSV)
infection.58 Synthetic miR-181 mimics significantly inhibited PRRSV replication in vitro in
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a specific and dose-dependent manner by binding to a highly conserved region downstream

of open reading frame 4 (ORF4) of the viral genomic RNA. Studies that have profiled
miRNAs in lung tissues of pigs infected with Actinobacillus pleuropneumoniae and porcine
epithelial cells infected with pseudorabies have identified several miRNAs that might play
roles in immune and inflammatory responses specific to both pathogens.137,187

miR-146a was elevated in the blood of ferrets, horses, and in a human cervical carcinoma
cell line (HeLa) infected with Hendra virus (paramyxovirus, genus Henipavirus).165
Blockade of miR-146a function significantly reduced Hendra virus replication in vitro,
suggesting a role for this miRNA in Hendra virus replication. This effect was mostly
mediated through its target, ring finger protein, a member of the A20 ubiquitin editing
complex that negatively regulates NF-κB activity. Increased NF-κB activity is thought to aid
in activating and sustaining Hendra virus replication. A study that looked at specific miRNA
profiles of myopathic horses with polysaccharide storage myopathy or recurrent exertional
rhabdomyolysis in 2 different breeds indicated that it might be possible to distinguish one
form from the other based on unique miRNA profiles.8 Interestingly, these miRNA profiles
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were also found to be breed specific.

In birds, several virus-encoded miRNAs were found to be conserved among different field
strains of oncogenic Marek's disease virus (MDV1), and their expression has been detected
in both lytic and latent infections, including MDV1-derived tumors.19 This study found that
even though each avian herpes-virus had unique sequences, all originated from similar
locations on the viral genome. Hence, these miRNAs tended to be clustered in the rapidly
evolving repeat regions of the viral genomes. MDV1 and herpesvirus of turkeys (HVT)
encode homologs of the host miRNA, miR-221, which targets a gene important in cell cycle
regulation. MDV1 also encodes another miRNA (mdv1-miR-M4), which, together with
Kaposi sarcoma–associated herpesvirus-encoded miR-K12-11, was verified as functional
orthologs of miR-155, a well-characterized miRNA previously linked to lymphoid

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malignancies and modulation of immune responses.200 Novel miRNAs were also found to
be encoded by duck enteritis virus, the functions of which remain to be determined.191 In
zebrafish, miR-142-3p was found to be essential for hematopoiesis and affected the cardiac
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cell fate.124

Circulating miRNAs
Recent studies have shown that a significant number of miRNAs are present extracellularly
in various body fluids that include serum, plasma, urine, saliva, and other body
fluids.60,180,196 The origin and function of these miRNAs are not well understood. There are
indications that they might play a role in cell-to-cell communication and can be exported
from one cell and recognized, taken up, and used by another cell, thereby drawing parallels
with the endocrine system.45,83,175,176 Termed circulating miRNAs, they are highly stable
and resistant to RNase activity and extreme pH and temperatures, unlike conventional RNA
molecules.26,117 Since body fluids contain ribonucleases, it is suggested that they are
protected from degradation by being packaged in lipid vesicles (microvesicles and
exosomes), in complexes with RNA binding protein or both.45,51,171 More importantly, they
can be readily detected in serum and plasma, and their expression patterns have correlated
positively with various diseases conditions.
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In a study that looked at miRNAs in the serum of patients with diffuse large B-cell
lymphoma (DLBCL), expression levels of 3 tumor-associated miRNAs—miR-155,
miR-210, and miR-21—were significantly upregulated in patient sera.91 Also, patients with
DLBCL with high miR-21 serum levels showed increased relapse-free survival but not
overall survival. A comprehensive analysis of miRNAs in serum to characterize blood
miRNA profiles from healthy individuals and patients found that patients with lung cancer,
colorectal cancer, and diabetes had unique serum miRNA profiles.26 Patients with oral
squamous cell carcinoma had higher levels of miR-31 in their plasma that decreased
significantly following tumor removal, a trend that was also observed in patient saliva.105 In
patients with rheumatoid arthritis and osteoarthritis, plasma miR-132 levels were
significantly higher than those in healthy controls and correlated well with disease
activity.122 Other studies have shown significant correlation between expression levels of
specific circulating miRNAs and their expression in specific tissues with a few exceptions
(reviewed in Zen and Zhang196). These findings also suggest that the levels of circulating
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miRNAs strongly correlate with the extent of tumor development.196 These properties and
the opportunity to detect their expression by noninvasive means have led to numerous
investigations geared toward developing miRNAs as reliable bio-markers. However, there
are a few stumbling blocks in this direction. For instance, the specificity of biomarkers
based on a single miRNA or a very small group of individual miRNAs is relatively poor,
which might be due to the complex nature of certain malignancies, thus highlighting the
need to discover and validate signature miRNA panels in complex malignances.196 Since all
biological fluid samples are essentially cell free, a lot more needs to be done to optimize
several key steps such as RNA extraction, normalization, quantitation, and analysis of serum
miRNA profiles before they can be used as reliable biomarkers of specific disease
conditions.

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Future Implications and Concluding Remarks

The discoveries and current developments in miRNA biology have essentially
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revolutionized the biological landscape and have created renewed interest within the
scientific community to reevaluate and significantly modify the conventional perceptions
about gene expression, gene regulation, and RNA functionality. miRNA discovery
effectively challenges the “central dogma of molecular biology,” in which RNA was
assigned the role of a mere intermediary to the flow of information between the genetic
material (DNA) and the end product (protein). The discovery and validation of miRNA
function also refute the faulty “junk DNA hypothesis,” which stated that more than 80% of
our DNA serves no biological purpose and is “junk” that has accumulated over time as
evolutionary fossils.142 The implications of the insights coming out of studies in miRNA
biology are far reaching. Many knockout or overexpression studies performed in the past to
determine the function of individual genes invariably included deletion of introns. Since a
significant number of miRNAs are now known to be present in introns, the inadvertent
deletion of any miRNA coding sequences contained in these regions might also be a
contributing factor to the phenotypic consequences observed in these individual knockout/
overexpression studies that were attributed solely to the protein coding gene. Data in this
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direction indicate that many knockout studies need to be reexamined to determine if loss of a
miRNA contributed to the phenotype, warranting careful planning of knockout studies in
future.130

Besides regulating gene expression, very recent studies have identified novel functions for
miRNAs as ligands capable of signal transduction by binding to cell surface receptors. In
this regard, tumor-secreted miR-21 and miR-29a were recently shown to bind TLR7 and
TLR8 in immune cells, resulting in the activation of a TLR-mediated prometastatic
inflammatory response that enhanced tumor growth and metastasis.46

Since a single miRNA can regulate a number of genes and a single gene can be regulated by
multiple miRNAs, loss of function of 1 or more miRNAs may not be evident at a functional
level due to the inherent ability of the biological system to substitute 1 molecule with
another capable of performing similar functions. Also, there is growing consensus that
miRNAs, in many cases, are “fine-tuners” of gene expression, and hence, unlike genes
encoding proteins, changes in spatial and temporal expression of miRNAs can be fleeting,
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can fluctuate rapidly, and might involve minimal changes in their expression levels. Hence,
the ability to observe these changes is limited by the sensitivity and sophistication of the
detection system. Although a lot of progress has been made in addressing these bottlenecks,
more work is needed to better understand their biological function.

Therapeutic implications of miRNAs are enormous. Individual miRNAs important in the

development of diseases can be specifically targeted using anti-miRNAs, which are
antisense oligonucleotides with specific modifications.145 In cancer therapeutics, a class of
cholesterol-conjugated (to facilitate better cellular uptake and serum protein binding) anti-
miRNAs, termed antagomirs, was used to block oncomirs.145,172 Other approaches that
efficiently inhibit miRNAs in vivo include locked nucleic acid (LNA) oligos or 2′-O-
methoxyethyl phosphorothioate (MOE) modification.172 In African green monkeys, mature

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miR-122 was effectively and stably silenced using intravenously administered LNA–anti-
miRNA-122 with no adverse effects.40 Many studies in this direction have yielded
promising results. In diseases associated with downregulation of miRNAs (eg, tumor
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suppressors), a novel approach that involves delivering synthetic RNA duplexes that mimic
the relevant miRNA duplexes, which can then be recognized by the RISC complex and
processed into mature miRNAs, is being actively pursued. Although achieving stability and
efficiency of the in vivo delivery of miRNAs is a major challenge, progress made in this
direction indicates the rapid emergence of an entirely new field of small RNA-based
therapeutics. These developments, together with their role as potential biomarkers
(discussed previously), make them invaluable tools in probing multiple aspects of a disease
process that includes diagnosis, classification, and treatment.

In conclusion, the aim of this review was not to comprehensively analyze the role of
individual miRNAs in a specific disease or development process but rather to present the
reader with the broad spectrum of capabilities that these small RNAs possess using some
well-established examples. The discovery of miRNAs adds a previously overlooked and
important variable that can substantially influence the development, manifestation, and
progression of disease processes. Although most of the present studies in this direction are
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done in laboratory animals and aim to tackle human diseases, the knowledge and progress
made in this field should be translatable into veterinary medicine in the near future.

Acknowledgement
We thank Drs Andrew A. Lackner and Chad J. Roy from Tulane National Primate Research Center (TNPRC) for
their constructive criticism and advice on the manuscript.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of
this article: This work was supported by National Institutes of Health grants OD011104 (formerly RR00164),
R01DK083929 (to MM), and T32-OD011124 (MB).

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Figure 1.
Canonical microRNA (miRNA) biogenesis pathway: miRNAs are initially transcribed as
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long, variable-length hairpin RNA substrates called primary miRNAs (pri-miRNAs) directly
off the DNA template in the nucleus by RNA polymerase II (A). In the nucleus, these pri-
miRNAs are again processed by the Microprocessor, a large complex that includes the
RNase type III enzyme called Drosha and the RNA binding protein DGCR8, into ~70- to
120-nucleotide-long premature (pre) hairpin precursor forms called pre-miRNAs (A). The
pre-miRNAs are then exported to the cytoplasm by exportin 5 (B), where the loop region of
the pre-miRNA is cleaved by the RNAse type III enzyme called Dicer into ~18- to 23-
nucleotide-long mature miRNAs (C). A cellular protein called transactivation response RNA
binding protein (TRBP) (D) facilitates the entry of the Dicer-miRNA complex into the
RNA-induced silencing complex (RISC) that contains Argonaute 2 (Ago2), protein kinase
RNA activator (PACT), trinucleotide repeat-containing gene 6A (TNRC6A), and other RNA
binding proteins (E). After incorporation of the mature miRNAs into the RNA-induced

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silencing complex (RISC), the “passenger” strand is degraded, and the “guide” strand is
guided to the 3′ UTR of the target messenger RNA (mRNA) to either degrade (in case of
perfect base complementarity) or bring about translational inhibition (in case of multiple
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sequence mismatches between the target mRNA and the miRNA) (F). Mounting evidence
suggests that transcripts bound by miRNA-incorporated Ago2 are channeled into structures
called P-bodies, resulting in translational repression (G). (Loosely adapted and redrawn from
Yeung et al.192)
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