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
Trang 1MicroRNAs – micro in size but macro in function
Sunit K Singh1,2, Manika Pal Bhadra3, Hermann J Girschick2and Utpal Bhadra4
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: sunitsingh@ccmb.res.in
(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.
Trang 2let-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-57to 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.
Trang 3and 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-miRmiR-NA) 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
Trang 4Drosha-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 comcom-plex 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) in Drosoph-ila], which probably bridges the initiation and effector
Trang 5steps 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-AGO1-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
Trang 6and 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¢ fi
3¢-exonucle-ase Xrn1p [67,68] Alternatively, transcripts can be
degraded in the 3¢ fi 5¢ 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
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]
Trang 7miRNAs 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
Trang 8regulate 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 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 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
Trang 9been 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 vitroand 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).
Trang 10phosphorodiamidate 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⁄ Pi-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