Báo cáo khoa học: MicroRNAs – micro in size but macro in function Sunit K. Singh1,2, Manika Pal Bhadra3, Hermann J. Girschick2 and Utpal Bhadra4 pptx

REVIEW ARTICLE
MicroRNAs – micro in size but macro in function
Sunit K. Singh
1,2
, Manika Pal Bhadra
3
, Hermann J. Girschick
2
and Utpal Bhadra
4
1 Section of Infectious Diseases and Immunobiology, Centre for Cellular and Molecular Biology, Hyderabad, India
2 Section of Infectious Diseases, Immunology and Pediatric Rheumatology, Children’s Hospital, University of Wuerzburg, Germany
3 Centre for Chemical Biology, Indian Institute of Chemical Technology, Hyderabad, India
4 Functional Genomics and Gene Silencing Group, Centre for Cellular and Molecular Biology, Hyderabad, India
Introduction
Small RNAs exhibit a wide spectrum of biological
functions. There are many classes of small RNAs, such
as microRNAs (miRNAs), small interfering RNAs
(siRNAs), repeat associated small interfering RNAs
[1], small nuclear RNA, small nucleolar RNA, Piwi-
interacting RNA [2] and transacting short interfering
RNA [3].
miRNAs are single-stranded RNAs of 19–25 nucleo-
tides in length originating from endogenous hairpin-
shaped transcripts [4]. These miRNAs interact with
their target mRNAs by base pairing, which could lead
to translational repression; decapping, deadenylation
and ⁄ or cleavage of target mRNA. The first known
miRNA, lin-4, was discovered in 1993 by Ambros and
coworkers in the nematode Caenorhabditis elegans
[5,6]. The lin-4 gene plays a role in the developmental

timing of stage-specific cell lineages in C. elegans.
Later on, lin-4 was found to encode a 22-nucleotide
noncoding RNA that negatively regulates the transla-
tion of lin-14. A few years later, another small RNA,
Keywords
Dicer; microRNA; miRNA and cancer;
miRNA and disease; miRNA and
therapeutics; miRNA biogenesis; miRNA
function; miRNA inhibitors; small RNA
Correspondence
S. K. Singh, Section of Infectious Diseases
and Immunobiology, Centre for Cellular and
Molecular Biology, Uppal Road,
Hyderabad 500007, India
Fax: +91 40 27160311
Tel: +91 40 27192523
E-mail:
(Received 30 June 2008, revised 30 July
2008, accepted 1 August 2008)
doi:10.1111/j.1742-4658.2008.06624.x
MicroRNAs (miRNAs) are endogenous small RNAs that can regulate
target mRNAs by binding to their 3¢-UTRs. A single miRNA can regulate
many mRNA targets, and several miRNAs can regulate a single mRNA.
These have been reported to be involved in a variety of functions, including
developmental transitions, neuronal patterning, apoptosis, adipogenesis
metabolism and hematopoiesis in different organisms. Many oncogenes
and tumor suppressor genes are regulated by miRNAs. Studies conducted
in the past few years have demonstrated the possible association between
miRNAs and several human malignancies and infectious diseases. In this
article, we have focused on the mechanism of miRNA biogenesis and the

role of miRNAs in human health and disease.
Abbreviations
AD, Alzheimer’s disease; AGO, argonaute; Ab, amyloid b-peptide; Dcp, decapping enzyme; DCR, Dicer; DGCR8, DiGeorge syndrome critical
region gene 8; dsRBD, double-stranded RNA-binding domain; eIF, eukaryotic translation initiation factor; ES, embryonic stem; Exp-5,
exportin-5; IRES, internal ribosome entry site; KSHV, Kaposi sarcoma herpes virus; LNA, lock nucleic acid; miRISC, microRNA-containing
RNA-induced silencing complex; miRLC, microRNA-containing RNA-induced silencing complex loading complex; miRNA, microRNA; miRNP,
microRNA ribonucleoprotein; P-body, processing body; Pol II, RNA polymerase II; Pol III, RNA polymerase III; pre-miRNA, precursor
microRNA; pri-miRNA, primary microRNA; RISC, RNA-induced silencing complex; RLC, RNA-induced silencing complex loading complex;
RNAi, RNA interference; siRISC, small interfering RNA-containing RNA-induced silencing complex; siRNA, small interfering RNA.
FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS 4929
let-7, was reported as an additional regulator of devel-
opmental timing in C. elegans [7]. Similar to lin-4, let-7
also functions by binding the 3¢-UTR of lin-41 and
lin-57 to inhibit their translation.
To date, 678 human miRNAs have been character-
ized in the Sanger miRBase sequence database [8], and
many more are still to be identified. Approximately
50% of known human miRNAs are found in clusters
[9,10]. The clustered miRNAs are often related to each
other, but can also be unrelated. Clustered miRNAs
may be functionally related in terms of targeting the
same gene or different genes in the same biochemical
pathway. Most mammalian miRNA genes have been
reported to be located in defined transcription units
Fig. 1. MicroRNA biogenesis and function. The miRNA gene is transcribed by Pol II into a pri-miRNA in the nucleus. The pri-miRNA is pro-
cessed into pre-miRNA by the RNase III enzyme Drosha. The pre-miRNA is exported to the cytoplasm with the help of Ran-GTP cofactor
and Exp-5. The miRNA duplex is cleaved from the pre-miRNA by the RNase III enzyme Dicer and TRBP. Helicase unwinds the mature
miRNA duplex. Either each strand of the miRNA pair or only one strand of mature miRNA can be incorporated into miRISC. miRNAs bound
to miRISC mediate the degradation or translational inhibition of their target mRNAs.
MicroRNAs and their roles S. K. Singh et al.

4930 FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS
and intergenic regions [11]. Studies have revealed that
miRNAs have key roles in diverse processes such as
developmental control, hematopoietic cell differentia-
tion, neural development, apoptosis, cell proliferation
and organ development. In this review, we discuss the
mechanism of miRNA biogenesis and the roles of
miRNA during development and different pathological
states.
miRNA biogenesis
miRNA biogenesis includes miRNA transcription in
the nucleus, the export of miRNAs from the nucleus
to the cytoplasm, and subsequent processing and mat-
uration in the cytoplasm (Fig. 1). In most cases, the
transcription of miRNA genes is mediated by RNA
polymerase II (Pol II), resulting in long primary miR-
NA (pri-miRNA) transcripts with a fold-back structure
comprising a stem loop along with flanking segments
[12]. A few recent reports have shown the involvement
of RNA polymerase III (Pol III) in miRNA transcrip-
tion [13]. The sequence of the miRNA remains
embedded in the arms of the stem loop. The pri-miR-
NA contains the 7-methylguanosine cap and a poly(A)
tail, which is unique for Pol II transcripts, similar to
mRNAs [12,14]. However, the cap and poly(A) tail are
removed during miRNA processing. miRNA promot-
ers have been identified in many studies [9,15,16], and
reported to have typical Pol II elements such as a
TATA box [17], although the recent report of Borchert
et al. [13] suggests that members of the human chro-

mosome 19 miRNA cluster (miR-515-1, miR-517a,
miR-517c and miR-519a-1) are interspersed among
Alu repeats and expressed through Pol III. The pro-
cessing of pri-miRNAs into final mature miRNAs
occurs in a stepwise fashion, which is discussed in
detail in subsequent sections of this article.
Enzymatic machinery involved in
miRNA biogenesis and maturation
Drosha
In humans, the generation of precursor miRNA
(pre-miRNA) from the pri-miRNA transcript takes
place exclusively in the nucleus, through the action
of the microprocessor complex, composed of the
RNase III enzyme Drosha and the double-stranded
RNA-binding domain (dsRBD) protein DiGeorge
syndrome critical region gene 8 (DGCR8), into 70–
80 nucleotide pre-miRNAs [18,19]; this is followed
by maturation of miRNA in the cytosol. The stem
loop structure of pri-miRNAs is cleaved in the
nucleus by Drosha during the generation of pre-miR-
NA. This process is known as cropping. Drosha
forms a large microprocessor complex of  650 kDa
along with the dsRBD protein DGCR8 in humans
[20] and a  500 kDa complex along with the
dsRBD protein Pasha in flies (Drosophila melanogas-
ter) [18,21,22]. In contrast to the siRNA pathway,
the miRNA pathway is initiated in the nucleus [23].
The cleavage by Drosha generates a pre-miRNA
hairpin bearing two nucleotide 3¢-overhangs. Precur-
sor miRNAs are exported to the cytoplasm from the

nucleus by exportin-5 (Exp-5) in the presence of
Ran-GTP as a cofactor (Fig. 1) [22,24,25].
It is important to realize that many human miRNA
genes remain located in intronic regions of coding
genes, so their biogenesis remains coupled with mRNA
splicing [12,26]. Drosha releases the pre-miRNA from
the intron shortly before splicing, allowing the genera-
tion of both RNA species at the same time. The preci-
sion of Drosha–DGCR8 cleavage is very important for
miRNA maturation. Any shift in the position of the
Drosha cut, even by a single nucleotide on the pri-
miRNA, will affect the position of Dicer cleavage. A
shift in the Dicer cleavage site could result in different
5¢-ends and 3¢-ends in the mature miRNA. This type
of nucleotide shift may invert the relative stability of
the 5¢-end of the miRNA strand and of the other asso-
ciated strand, which is opposite to the miRNA strand.
Such a shift could result in the selection of the wrong
strand as the mature miRNA. Even if the stability
remains unchanged and the correct strand is loaded
into the RNA-induced silencing complex (RISC), then
the shift in the 5¢-end of the miRNA will change the
position of the seed sequence (2–8 nucleotides of
miRNA, which often match the target mRNA very
closely), which could lead to a change in its target
mRNA [27]. The RISC is a multiprotein complex that
cleaves specific mRNAs, and that is targeted for degra-
dation by homologous dsRNAs during the process of
RNA interference. This complex plays a very impor-
tant role in gene regulation by miRNAs and siRNAs.

There is an interesting mechanism that determines the
precision of cleavage by the Drosha–DGCR8 complex
to generate pre-miRNA transcripts from pri-miRNA
transcripts. Some structural features of the RNA have
been shown to be involved in determination of the
Drosha cleavage site [27].
The ssRNA segments flanking the base of the stem
loop are crucial for Drosha cleavage [28]. The deletion
of single-stranded regions or their conversion to
dsRNA greatly impairs the conversion of pri-miRNA
to pre-miRNA [28]. In a recent report, Davis et al. [29]
have shown the role of SMAD protein in Drosha-
S. K. Singh et al. MicroRNAs and their roles
FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS 4931
mediated miRNA maturation. Transforming growth
factor-b and bone morphogenetic protein signaling
have been reported to promote the rapid increase in
expression of mature miR-21 by promoting the pro-
cessing of primary transcripts of miR-21 (pri-miR-21)
into precursor miR-21 (pre-miR-21) by the Drosha
(also known as RNASEN) complex [29]. Transforming
growth factor-b-specific and bone morphogenetic pro-
tein-specific SMAD signal transducers are recruited to
pri-miR-21 in a complex with RNA helicase p68 (also
known as DDX5), a component of the Drosha micro-
processor complex [29]. Thus, SMAD protein plays an
important role in Drosha-mediated miRNA matura-
tion [29].
Export and import of miRNAs between the
nucleus and cytoplasm

Exp-5 is a member of the karyopherin family of nucle-
ocytoplasmic transport factors, and plays a role in the
export of miRNAs from the nucleus to the cytoplasm
[30]. The function of Exp-5 is dependent on the GTP-
bound form of Ran cofactor for specific binding to its
export substrate in the cell nucleus. This process
involves the hydrolysis of Ran-GTP to Ran-GDP by
the cytoplasmic Ran GTPase-activating protein [31].
The role of Exp-5 in nucleocytoplasmic transport was
verified by using RNA interference (RNAi). In the
event of Exp-5 depletion by RNAi, the level of mature
miRNAs goes down but pre-miRNA does not accumu-
late in the nucleus. The lack of accumulation could be
due to instability of pre-miRNA. This suggests the
possibility that interaction of pre-miRNA with Exp-5
is required for the stability of pre-miRNA [32]. Exp-5
has been reported to recognize the ‘minihelix motif’ of
pre-miRNA, which consists of a > 14 bp stem and a
short 3¢-overhang.
Hwang et al. recently reported that a hexanucleo-
tide element directs the process of nuclear import
rather than export of miR-29b. In contrast to most
of the animal miRNAs, miR-29b has been reported
to be predominantly localized in the nucleus [33].
The special hexanucleotide terminal motif (AGU-
GUU) acts as a transferable nuclear localization ele-
ment of miR-29b, and is responsible for the nuclear
enrichment of miR-29b. These RNAs may prove to
be useful tools for manipulation of gene expression
in the nucleus. It is supposed that miR-29b could

have a role in regulation of the transcription or
splicing events of target transcripts. This role of
miR-29b is quite unique and is different from the
routine translational regulatory functions performed
by other miRNAs [33].
Role of Dicer in miRNA maturation
Dicer is an ATP-dependent multidomain enzyme of
the RNase III family, and has been reported to be
involved in cleavage of double-stranded siRNA and
miRNA. Dicer was initially identified in Drosophila
[34] and has been subsequently reported in humans,
plants and fungi. The mechanism of recognition of the
pre-miRNA by cytoplasmic Dicer is not known [35].
In the cytoplasm, the pre-miRNAs are processed into
 22-nucleotide duplex miRNAs by the RNase III
enzyme Dicer (Fig. 1). Some organisms have a single
Dicer gene [36–39], whereas others have many [40,41].
In species with several Dicers, different homologs are
required for different functions [40,42,43]. Two Dicer
homologs (DCR1 and DCR2) have been reported in
Drosophila. DCR1 processes pre-miRNA, whereas
DCR2 processes long dsRNA in Drosophila [43–45].
The only Dicer gene in C. elegans, DCR1, is required
for the processing of both the long dsRNA and
pre-miRNAs.
Dicer cleavage results in the release of a duplex with
mature miRNA in one of the strands of the stem loop.
Both arms of the pre-miRNA stem loop structures are
imperfectly paired, containing G:U wobble pairs and
single nucleotide insertions. These imperfections cause

one strand of the duplex to be less stably paired at its
5¢-end [27]. The conversion from dsRNAs to ssRNAs
is a complex process, involving several RNA–protein
and protein–protein interactions. RISC loading com-
plex (RLC) is an RNA–protein complex that initiates
the formation of the RISC. The RLC puts a small
RNA duplex in the correct orientation for subsequent
RISC assembly [35]. The small RNAs (siRNAs and
miRNAs) in the RLC remain ready to be unwound
for functional RISC assembly. The siRISC loading
complex (siRLC) of Drosophila contains a DCR2–
R2D2 heterodimer and an siRNA duplex. R2D2 has
been reported to be a DCR2 stabilizer as well as the
asymmetric sensor for setting the siRNA orientation
for RISC assembly [35]. Detailed information on miR-
ISC loading complexes (miRLCs) is not available. In a
recent report, MacRae et al. [46] have demonstrated
the assembly of human RLC in vitro from purified
components without any cofactors or chaperones.
They demonstrated that reconstituted RLC maintains
the endogenous RLC functional activities of dicing,
slicing, guide-strand selection and argonaute (AGO)2
loading [46].
Dicer interacts with the dsRNA-binding protein part-
ner, the TAR RNA-binding protein (TRBP), in humans
[RDE4 in C. elegans and Loquacious (Loqs)inDrosoph-
ila], which probably bridges the initiation and effector
MicroRNAs and their roles S. K. Singh et al.
4932 FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS
steps of miRNA action [47–49]. DCR binds with high

affinity to the ends of dsRNAs bearing two-nucleotide
3¢-overhangs, which results in unwinding of duplexes.
The thermodynamic properties of siRNA–miRNA
duplexes play a critical role in determining molecular
function and longevity [50]. The unwinding of the
duplex strands starts at the ends with the lowest thermo-
dynamic stability. The relative stabilities of the base
pairs at the 5¢-ends of two strands determine the fate of
the strand, which has to participate in the RNAi path-
way [51]. Along with the thermodynamic stabilities, a
role of proteins such as R2D2 has also been reported
during the strand selection process. The orientation of
the DCR2–R2D2 protein heterodimer on the siRNA
duplex determines the siRNA strand, which has to asso-
ciate with the core RISC protein AGO2 in Drosophila
[52]. The exact mechanism by which R2D2 guides the
asymmetric assembly of the RISC in Drosophila is not
known. Dicer has an RNA helicase domain to cleave the
dsRNA.
In general, the miRNA strand, which has its 5¢-ter-
minus at the lowest thermodynamic stability, acts as
the mature miRNA (guide strand), and the other
strand (passenger strand) is degraded. However, a
recent report has shown that both strands could be
coaccumulated as miRNA pairs in some tissues, and
subjected to strand selection in other tissues [53].
Ro et al. [53] also reported that both strands of the
miRNA pair can target equal numbers of genes, and
were able to suppress the expression of their target
genes. This study provided evidence for a novel mecha-

nism involved in tissue-dependent miRNA biogenesis
and miRNA target selection [53]. Mature miRNAs are
incorporated into the effector complexes, known as
miRNP (microRNA ribonucleoprotein), mirgonaute,
or miRISC. The identification of the target by the
RISC is based on the complementarity between mature
miRNA and the mRNA. The degree of complementar-
ity decides whether the complex has to undergo endo-
nucleolytic cleavage of target mRNA or translational
repression.
In contrast to miRNAs, siRNAs are often synthe-
sized in vitro or in vivo from viruses or repetitive
sequences. siRNAs have been reported to be involved
in antiviral defense, and also in protecting the genome
against disruption by transposons. The presence of the
selective AGO protein family is one of the several
common features of siRISC and miRISC.
AGO proteins in the RISC
AGO proteins are well conserved in diverse organisms
[54], and constitute a large family involved in develop-
mental regulation in eukaryotes. Several AGO homo-
logs have been reported in eukaryotic organisms, such
as eight in humans [55], five in Drosophila [54], 27 in
C. elegans [56] and only one in fission yeast [36,56].
These homologs are characterized by the presence of
two domains, PAZ (Piwi ⁄ Argonaute ⁄ Zwille) and
PIWI. The PAZ domain of AGO proteins binds to
the 3¢-end of the ssRNA, possibly by recognizing the
3¢-overhangs [57,58].
AGO proteins are the core components of the RISC

in different organisms. Different AGO proteins specify
distinct RISC functions. Cofractionation studies in
Drosophila have shown that AGO2 cofractionates and
remains functionally associated with DCR2, whereas
AGO1 remains functionally associated with DCR1
[44,59]. These observations verify that DCR1 is
involved in miRNA maturation, whereas DCR2 is
involved in initiation of RNAi in Drosophila [43,44].
Although miRNAs and siRNAs have distinct biogene-
sis pathways in Drosophila, they have a common
sorting pathway, which partitions them into AGO1-
containing or AGO2-containing effector complexes
[60].
In contrast to Drosophila, humans and C. elegans
contain only one Dicer, which initiates the formation
of both siRISCs and miRISCs. In the case of humans,
different AGO proteins (AGO1 to AGO4) have been
reported to be involved during RISC assembly, but
only AGO2-associated RISCs have been reported to
be involved in the cleavage of target mRNA. There-
fore, AGO2 is also called slicer argonaute [61,62].
Slicer activity has been reported in the PIWI domain
of AGO proteins, on the basis of mutagenesis studies
[61]. Specific amino acid residues of the PIWI domain
of AGO2 are essential for slicer activity in AGO2
proteins of human and Drosophila [35].
Processing bodies (P-bodies) and their
biological function
It was thought that once mRNAs finish their job,
enzymes in the cytoplasm simply break them down.

Several groups reported that most of this degradation
occurs in P-bodies (processing bodies) or glycine-tryp-
tophan or decapping enzyme (Dcp) bodies. P-bodies
are found as discrete cytoplasmic bodies in yeast and
mammals. The conservation of P-bodies from yeast to
mammals suggests their important role in the cytoplas-
mic function of eukaryotic mRNA. P-bodies include
the Dcp1p ⁄ Dcp2p, activators of decapping, Dhh1p
(referred to as RCK in mammals), Pat1p, Lsm1-7p,
Edc3p and the 5¢–3¢-exonuclease Xrn1p [63–66].
P-bodies have been reported as the sites for decapping
S. K. Singh et al. MicroRNAs and their roles
FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS 4933
and degradation of mRNAs. In yeast, the major path-
way of mRNA turnover is initiated with the shortening
of a 3¢-poly(A) tail, by a process called deadenylation.
Deadenylated transcript acts as a substrate for the
Dcp1p ⁄ Dcp2p decapping complex, for removal of
the 5¢-cap structure. The decapping process exposes
the transcript to degradation by the 5¢fi3¢-exonucle-
ase Xrn1p [67,68]. Alternatively, transcripts can be
degraded in the 3¢fi5¢ direction following deadenyla-
tion in exosomes, by a conserved complex of 3¢-to-5¢-
exonucleases. Several observations suggest that the
processes of mRNA decapping and translation are
inversely related. Mutations in different translation
initiation factors result in decreased rates of translation
along with increases in the rate of mRNA decapping
[69,70].
Recent reports have demonstrated that mRNA

subjected to miRNA repression accumulates in P-
bodies. P-bodies contain untranslated mRNAs and
can serve as sites of mRNA degradation. This sug-
gests that RISC proteins direct the mRNA to
P-bodies, possibly for storage. So, the P-bodies do not
just degrade mRNA, but also temporarily sequester
them away from the translation machinery. Parker
and coworkers have recently revealed the localization
of AGO proteins in mammalian P-bodies. They
found that mRNAs targeted for translational repres-
sion by miRNAs become concentrated in P-bodies in
an miRNA-dependent manner [71]. This study pro-
vides a strong link between miRNAs and P-bodies,
and suggests that translation repression by the RISC
delivers mRNAs to P-bodies [71]. Other studies have
also demonstrated that about 20% of let-7-repressed
reporter mRNAs and 20% of fluorescently labeled
microinjected let-7 miRNA colocalized with visible
P-body structures [72,73]. The involvement of P-
bodies in miRNA-based repression requires further
investigation to determine the fraction of translation-
ally repressed mRNAs, and the miRNAs localized in
P-bodies.
Ways to handle translational activities
An miRISC represents an effector complex that medi-
ates miRNA functions inside cells. The guide miRNAs
are perfectly complementary to either the coding
region or 3¢-UTR of target mRNA in plants [74]. In
most cases, plant miRISCs can mediate mRNA degra-
dation. The perfect complementarity between mature

miRNA and target mRNA has not been reported in
animals and humans, except for the HoxB8 gene in
mice, which can be cleaved by miR-196 despite imper-
fect sequence identity [75]. Nucleotides 2–8 of miRNA,
known as the ‘seed region’, do often match very
closely to the target mRNA, and are considered to
comprise the most critical region for selecting targets.
The miRNAs sharing common seed sequences are
grouped into miRNA families. These miRNAs possibly
have overlapping targets and are considered to be
redundant [33]. miRNAs handle the translational activ-
ities by mediating pretranslational, cotranslational or
post-translational gene silencing.
In eukaryotic translation, the step of initiation
starts with the recognition of the 5¢-terminal cap
structure (m7Gpp) of mRNA by the eIF4E subunit
of the eukaryotic translation initiation factor (eIF),
eIF4F and eIF4G [76]. The interaction of eIF4G
with polyadenylate-binding protein 1 and eIF4E
results in stimulation of translational initiation [76].
However, some cellular and viral mRNAs initiate
translation without the involvement of the m7G cap
and eIF4E. In such cases, the 40S ribosomes are
recruited to mRNA through the internal ribosome
entry site (IRES) [76,77]. Several reports have dem-
onstrated that translation of m7G-capped mRNAs,
but not of mRNAs containing the IRES, is repressed
by miRNAs [72,76]. In such cases, AGO2 and
related proteins might compete with eIF4E for m7G
binding and thus prevent the translation of capped,

but not IRES-containing, mRNAs [78]. However,
other reports demonstrate the interaction of miRNA
with the ORFs of genes whose translational activities
are governed by IRES-mediated translational events
[79,80]. MiRISCs can repress translational events at
both initiation and postinitiation levels. MiRISCs are
also known to increase cotranslational degradation
of nascent proteins, reduce the elongation rate of
translation, and increase the rate of mRNA deadeny-
lation [72,73,80–82]. It is not well understood
whether miRNAs always target the same or different
steps of translational events under various physiolog-
ical conditions [73].
In recent reports, it has been shown that the miR-
NA-mediated repression can be effectively reversed or
prevented [83–85], and miRNPs can act as transla-
tional activators [86]. Cationic amino acid transporter-1
(CAT 1) mRNA has been reported to be translation-
ally repressed by the liver-specific miRNA miR-122 in
human hepatoma cells, and accumulates in cytoplasmic
P-bodies. However, amino acid starvation results in
the release of CAT 1 mRNA from P-bodies and its
recruitment to polysomes [76]. APOBEC3G (apolipo-
protein B mRNA editing enzyme catalytic polypeptide
like 3G) has also been reported to interfere with
miRNA action by altering the distribution of target
messages between P-bodies and polysomes [87].
MicroRNAs and their roles S. K. Singh et al.
4934 FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS
miRNAs as developmental regulators

miRNAs play important roles in the regulation of
stem cells, organ differentiation and developmental
timings [88]. Dicer mutational and knockout studies
have shown a defect in miRNA biogenesis. Muta-
tions in Dicer genes result in reduced levels of miR-
NAs. Knockout of mouse dcr1 results in depletion
of embryonic stem (ES) cells. Dicer-deficient ES cells
are viable, but do not form mature miRNAs, and
fail to differentiate in vitro and in vivo [89,90]. Mouse
and human ES cells express a specific set of miR-
NAs, which are downregulated upon differentiation
into embryoid bodies [91]. Dicer mutant zebra fish
embryos have been reported to develop normally for
about 8 days postfertilization, but the process of
development ceases after 8 days, when embryos run
out of maternal Dicer.
Giraldez et al. generated maternal zygotic Dicer
mutants of zebra fish by a germ cell replacement tech-
nique to eliminate the maternal contribution of the
Dicer gene. In maternal zygotic DCR mutants of Dicer
knockout zebra fish, the pre-miRNAs are not processed
into mature miRNAs [92], and show morphogenesis
defects during gastrulation, brain formation and neural
differentiation. Loss of Dicer leads to the defects in posi-
tioning as well as defasciculation of axons. These obser-
vations suggest that miRNAs are not only essential for
cell fate determination and early patterning, but are also
essential for subsequent later steps in early embryonic
development in zebra fish [92].
The differential pattern of miRNA expression

has also been reported during different stages of
development. Most of the miRNAs show highly tissue-
specific expression during the late stages of develop-
ment [25,93]. Injection of miR-430 into maternal zygo-
tic Dicer mutant zebra fish embryos rescues the brain
morphogenesis defects and to some extent the other
neuronal defects, indicating the importance of miR-430
in regulation of morphogenesis in the zebra fish [92].
The Dicer knockout studies have provided much
strong evidence regarding the role of miRNAs in dif-
ferent species, but these results should be interpreted
with caution, due to the role of Dicer in other func-
tions, such as heterochromatin formation and chromo-
some segregation.
miRNAs in health and disease
miRNAs have already been implicated in a number of
diseases, and both miRNA inhibition and activation
show great promise in the treatment of various types
of cancer, and viral and metabolic diseases. Aberrant
gene expression is the main reason for miRNA dys-
function in cancer, which results in abnormal
mature ⁄ precursor miRNA expression in tumor samples
[94]. MicroRNA germline and somatic mutations or
polymorphisms in the protein-coding mRNAs targeted
by miRNA also contribute to cancer predisposition,
initiation or progression [94] The expression patterns
of different miRNAs in various types of human tumor
have been studied extensively [95]. Significant downre-
gulation of most of the miRNAs has been reported in
various tumors as compared to normal tissues [95].

Amplification, rearrangement and deletions have been
reported among various miRNA location sites in
cancer patients. This provides a clue about the associa-
tion between miRNA and cancer pathogenesis [96].
The dysregulation of miRNA expression has been
reported in many types of cancer, including Burkitt’s
lymphoma [97], colorectal cancer [98], lung cancer [99],
breast cancer [100] and glioblastoma [101]. MiR-143
and miR-145 miRNAs are downregulated in colon
cancer tissue [98]. Let-7 miRNAs are downregulated in
several lung cancers [99].
Overexpression of miRNAs with antiapoptotic activ-
ity has been reported in cancer cells. The miR-17 clus-
ter (miR-17-5p, miR-17-18, miR-17-18a, miR-17-19b,
miR-17-20 and miR-17-92) of miRNAs, located on
human chromosome 13q31, has been shown to be
associated with antiapoptotic activity. This region of
the chromosome (chromosome 13q31) has often been
associated with several types of lymphoma and solid
tumor [95,102,103]. He et al. [104] also reported a
higher level of expression of miR-17 cluster miRNAs
in B-cell lymphoma samples. The lymphomas express-
ing the miRNAs of the miR-17 cluster show a high
mitotic index without extensive apoptosis. The high
mitotic index without apoptosis suggests that miR-17
cluster miRNAs suppress cell death [104]. It is worth
mentioning here that the individual miRNAs of the
miR-17 cluster could not accelerate tumor formation
individually. This suggests that the oncogenic effect
requires a cooperative interaction between the miR-

NAs in the cluster. The miRNA miR-21 with an anti-
apoptotic function was found to be overexpressed in
breast cancer tissue [100], glioblastoma tumor tissues
and cell lines [101]. Inhibition of miR-21 in a glioblas-
toma cell line resulted in caspase activation and
enhanced apoptosis [95,101].
miRNAs with proapoptotic activity are likely to
function as tumor suppressor genes, and have been
reported to be underexpressed in cancer cells. The fam-
ily of let-7 miRNAs falls into this category. RAS gene
dysregulation has been reported among lung cancer
patients. The let-7 miRNA has been demonstrated to
S. K. Singh et al. MicroRNAs and their roles
FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS 4935
regulate the RAS oncogenes. The level of RAS protein
was inversely correlated with let-7 miRNA levels in
lung cancer samples, without any change in RAS
mRNA levels [95,105].
miR-15a and miR-16-1 are reported to have tumor-
suppressing activity in B-cell chronic lymphocytic
leukemia. MiR-15a and miR-16-1 are located on
human chromosome 13 in a region that is frequently
deleted in B-cell chronic lymphocytic leukemia. The
conserved target site for miR-15a and miR-16-1 has
been found in the 3¢-UTR of the antiapoptotic gene
bcl2. Overexpression studies of miR-15a and miR-16-1
have shown reduced expression of Bcl2 protein in a
leukemic cell line [95,100,106]. Ma et al. recently
reported the association of miRNA with cancer cell
invasiveness and the ability to metastasize. Investiga-

tors named the miR-10b prometastatic miRNA, due to
its ability to promote tumor cell invasion [107].
Fragile X syndrome is one of the commonly inher-
ited mental retardation syndromes. The gene responsi-
ble for fragile X syndrome, FMR1 (fragile X
retardation 1), is located on human chromosome 10.
This syndrome is caused by loss of an RNA-binding
protein called familial mental retardation protein,
which has been reported to be regulated by miRNAs
[108]. Tourette’s syndrome is another neuropsychiatric
disorder among humans in which the role of miRNAs
has been reported [109]. The 3¢-UTR of the SLITRK1
gene contains the binding site of miR-189, which is
mutated in some Tourette’s syndrome patients [110].
In situ hybridization of SLITRK1 mRNA and
miR-189 revealed coexpression in the neuroanatomical
circuits most commonly implicated in Tourette’s syn-
drome. This demonstrates how an miRNA can be
involved in the establishment of a disease phenotype
[110].
miRNA expression profiles have been reported to be
altered in sporadic Alzheimer’s disease (AD). Small,
soluble oligomers of amyloid b-peptide (Ab) have been
reported to have a role in the molecular basis for
memory failure in AD. Ab oligomeric ligands (also
known as ADDLs) are known to be potent inhibitors
of hippocampal long-term potentiation. In a recent
study, Hebert et al. [111] reported the interaction of
miRNAs with BRACE-1 ⁄ b secretase genes. BRACE-
1 ⁄ b secretase is a rate-limiting step for Ab production,

and its increased expression has been reported among
AD patients. Hebert et al. [111] reported that miR-
29a, miR-29b-1 and miR-9 can regulate BRACE-1
expression in vitro. They found that expression of the
miR-29a ⁄ b-1 cluster is significantly decreased in AD
patients, which results in abnormal accumulation of
high BRACE-1 protein and Ab levels among AD
patients [111]. The altered expression of miRNAs has
also been reported in postmortem samples of cerebellar
cortex from autism patients [112].
The roles of miRNAs have also been reported in
various viral infections. Some viruses perturb miRNA
expression of the host cells, for their survival, and oth-
ers encode their own miRNAs, which target various
host genes [113]. Viruses have been reported to encode
miRNAs [114], but the functions of most of them are
not known. Herpes viruses such as Epstein–Barr virus
or Kaposi sarcoma herpes viruses (KSHVs) have been
reported to express miRNAs. Epstein–Barr virus
induces cellular miRNAs such as miR-21, miR-155 or
miR-146a during its infection cycle. Out of these, miR-
146 has been reported to be upregulated in various
tumors [115]. In a recent report, 12 miRNA genes were
identified within the genome of KSHV, and these miR-
NAs affect the expression of large number of cellular
genes during KSHV infection [116]. The miR-28, miR-
125b, miR-150, miR-223 and miR-382 cluster of cellu-
lar miRNAs have been reported to contribute to the
maintenance of HIV-1 latency in resting primary
CD4

+
T-lymphocytes [117]. Hepatitis B virus also
encodes viral miRNA as a means of regulating its own
gene expression [118]. Hepatitis C virus utilizes the
liver-specific host miRNA miR-122 as a positive regu-
lator of its own replication [119,120]. It is now time to
study the function of virus-encoded miRNAs by utiliz-
ing bioinformatics and molecular biology tools.
Irrespective of diseases, miRNAs are also involved
in many other physiological functions. The expression
of miR-375 takes place in murine pancreatic islets cells
and plays an important role in regulation of the myo-
trophin gene and thereby glucose-stimulated insulin
exocytosis [121]. Higher expression levels of miR-375
have been reported in pituitary glands of zebra fish
embryos [25], which indicates its possible involvement
in neuroendocrine activities [90]. MiR-122 and miR-1
play roles in mammalian liver development and cardio-
myocyte differentiation, respectively [90,122]. The role
of miRNAs is well known in ES cell differentiation,
lineage specification and organogenesis, especially neu-
rogenesis and cardiogenesis [123]. The miR-1 gene has
been reported to be a direct transcriptional target of
muscle differentiation regulators, including serum
response factors, myogenic differentiation factor D,
and myocyte-enhancing factor 2 [124]. The higher level
of miR-1 results in a reduction in the number of prolif-
erating ventricular cardiomyocytes in the developing
heart. This suggests that miR-1 modulates the effects
of critical cardiac regulatory proteins to control the

balance between differentiation and proliferation during
cardiogenesis [125]. miR-1, miR-133 and miR-206 have
MicroRNAs and their roles S. K. Singh et al.
4936 FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS
been reported to be involved in proliferation, differen-
tiation and regeneration of skeletal muscles [126].
Recently, Chim et al. [127] have reported the exis-
tence of placental miRNA in maternal plasma, which
opens up new possibilities of using the miRNAs
as molecular markers for pregnancy monitoring. Four
placental miRNAs (miR-141, miR-149, miR-299-5p and
miR-135b) were found to be present at higher levels in
maternal plasma during the predelivery period than
after delivery. The measurement of miRNA in maternal
plasma for prenatal monitoring and diagnosis would be
an interesting future research direction [127].
Clinical implications of miRNA
research
The widespread role of miRNAs in the biological system
makes them valuable targets for therapeutic interven-
tion. The base pair interaction between miRNAs and
their target mRNAs is key for miRNA function.
Modified synthetic antisense oligonucleotides act as
potential inhibitors of the miRNAs. Antisense
oligonucleotides against miRNA pair with the miRNAs,
occupying their binding sites and leaving their target
mRNA in the unbound state [128]. Antisense oligonu-
cleotides are useful tools for the inhibition of specific
miRNAs. These have the potential to develop into a
new class of therapeutic agents [129]. An abnormal phe-

notype might appear through aberrant suppression of
any specific mRNA, due to the induction of its corre-
sponding miRNA. In such cases, antisense oligonucleo-
tides complementary to either the mature miRNA or its
precursors can be designed (Fig. 2) to release the sup-
pressive effect on mRNA [129,130]. Boutla et al. [131]
demonstrated the inhibition of miRNA in Drosophila
embryos by using antisense modified oligonucleotides
against miRNAs through microinjection. Modified oli-
gonucleotides have previously been shown to be effec-
tive inhibitors of both coding and noncoding RNAs
in vitro and in vivo, and some of them, such as a 20-mer
Fig. 2. Interference in the miRNA pathway by modified antisense synthetic oligonucleotides. Inhibition of miRNA can be achieved by intro-
ducing antisense synthetic oligonucleotides against miRNAs in the cytoplasm (shown as continuous lines). The possible targets of antisense
synthetic oligonucleotides against miRNAs in the nucleus are pri-miRNA and pre-miRNA (shown as dotted lines).
S. K. Singh et al. MicroRNAs and their roles
FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS 4937
phosphorodiamidate morpholino oligomer targeting c-
Myc, are currently under investigation in human clinical
trials [129,132]. The most important property of such
oligonucleotides is specificity and high binding affinity
for RNA. Two independent groups have transiently
transfected 2¢-O-methyl-modified antisense RNAs
directly in cultured cells, and have shown miRNA-spe-
cific inhibition [133,134]. These in vitro studies look
promising, but the real challenge is in vivo use of modi-
fied miRNA inhibitors. In-depth studies are required to
develop precise strategies for the pharmacological
delivery of small RNAs into their target cells, for
utilization of the potential of miRNAs as therapeutics.

Several studies have been conducted to determine
the role of RNAi in the suppression of disease-asso-
ciated molecular pathologies in various animal mod-
els of disease [135]. The role of miR-122 in
regulating cholesterol biosynthesis and in maintaining
the adult liver phenotype, its association with he-
patocarcinogenesis and its role in hepatitis C virus
replication make it an invaluable target with which
to expand our knowledge of the pathophysiology of
diverse liver diseases [136]. Recently, it has been
shown that inhibition of miR-122, a liver-enriched
miRNA, has therapeutic potential in mice. Krutzfeldt
et al. synthesized a 23-nucleotide RNA molecule (an-
tagomir) complementary to miR-122 in such a way
as to stabilize the RNA and protect it from degra-
dation. They conjugated this small nucleotide with
cholesterol molecules for their easy delivery into liver
cells. This group successfully demonstrated inhibition
of endogenous miR-122 in mice after injecting this
small nucleotide complex through the tail vein [137].
The silencing of miR-122 by antagomir-122 resulted
in a 44% decrease in plasma cholesterol levels in
mice. Investigators expected that miR-122 might
downregulate any repressor of the genes associated
with the cholesterol biosynthetic pathway. Antago-
mir-122 may enhance the level of expression of the
possible repressor, after binding with miR-122, which
in turn results in inhibition of the transcription of
cholesterol-synthesizing enzymes. Approximately 11
genes involved in cholesterol biosynthesis were

reported to be downregulated by antagomir-122
[135,137]. Although there are many reports demon-
strating the silencing of specific miRNAs by the use
of miRNA inhibitors in mice, Elmen et al. [138] have
recently demonstrated the silencing of miR-122 by a
lock nucleic acid (LNA)-based miRNA inhibitor
(LNA-antimiR). They demonstrated that delivery of
NaCl ⁄ P
i
-formulated LNA-antimiR inhibited the
expression of miR-122 in the liver of nonhuman
primates [138].
Krutzfeldt et al. [137] have developed new methods
for the effective delivery of antisense oligonucleotide
against miRNAs. These include modification in the
RNA backbone, at each nucleotide, by an O-methyl
moiety at the 2¢-ribose position. The terminal nucleo-
tides at both ends are also modified with a phosphoro-
thioate linkage, in contrast to the standard
phosphodiester linkage in RNA and DNA. Unmodi-
fied oligonucleotides were used to inhibit the expres-
sion of let-7 miRNA in C. elegans, but this strategy
was not effective, due to the unstable nature of
unmodified oligonucleotides in vivo [133]. Therefore,
modifications of synthetic antisense oligonucleotides
against miRNAs are required to make them thermo-
stable and nuclease-resistant, which protects them once
they are exposed to serum and cellular nucleases. The
third modification is cholesterol functionality at the
3¢-end of the nucleic acid. This improves pharmacoki-

netic properties by increasing binding to serum pro-
teins, and improving stability and half-life in serum
and cellular uptake [128,139]. Pharmaceutical com-
panies such as Regulus and Santaris have focused
their drug discovery research on the development of
miRNA-based therapeutics for viral infectious diseases
and metabolic disorders.
Problems in therapeutic application
Although there have been successful attempts at deliv-
ery of antisense RNAs to cells and tissues, successful
delivery of small RNAs to the brain is one of the
major challenges in the development of small RNA-
based neurotherapeutics. Appropriate access of
plasmid–lipid complexes or viral vectors to the desired
tissues and cells of neural origin is a critical issue.
Several modifications and improvements in delivery
methods are ongoing, but they still need precision. The
blood–brain barrier is a big hurdle in the treatment of
neurological diseases, because it inhibits the passive
entry of therapeutic molecules from the peripheral
circulation into the brain. The study conducted by
Kumar et al. [140] suggests that the short peptide
derived from rabies virus glycoprotein potentiates the
transvascular entry of siRNAs into the brain. They
demonstrated that rabies virus glycoprotein-9R-bound
antiviral siRNA provided effective protection against
viral encephalitis in mice, without any induction of
inflammatory cytokines and antibodies against pep-
tides [140]. The intracellular concentrations of the
target RNA and the small RNA-based drugs will

determine the extent and duration of suppression.
Therefore, there is a need to conduct studies on dose
optimization and modes of delivery of miRNAs, in
MicroRNAs and their roles S. K. Singh et al.
4938 FEBS Journal 275 (2008) 4929–4944 ª 2008 The Authors Journal compilation ª 2008 FEBS
order to utilize their broad potential of miRNA-based
therapeutic drugs.
Conclusion
The field of miRNA is still in the development phase,
and many questions have to be resolved. The technol-
ogy for identification of the miRNA target genes and
the functions of the predicted miRNAs using bioinfor-
matics prediction tools still need refinement. In order
to explore the regulatory mechanisms mediated by
miRNAs, it is important to understand how miRNA
genes are regulated. Further studies are required to
identify the in vivo targets, to elucidate the regulatory
networks and to develop methods for the cell-specific
delivery of miRNAs. Understanding the function of
miRNAs in the complex molecular network regulating
the development and function of various cells and
tissues will increase our knowledge about the potential
role of miRNAs and their involvement in gene regula-
tion. The field of miRNAs has great potential to help
in our understanding of the development and diffe-
rentiation processes along with the development of
miRNA-based therapeutics.
Acknowledgements
The authors are thankful to Praveen Singh B. Hajeri
and Deepankar Pratap Singh for their help in drawing

the figures associated with this article. There are no
financial conflicts among the authors related to
this article. The authors are grateful to Lalji Singh,
Director, CCMB Hyderabad, for providing all the
necessary support.
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