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Micro RNA- Biogenesis, Mechanism of Action and Applications-A Review

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International Journal of Research in Biotechnology and Biochemistry

Universal Research Publications. All rights reserved

Review article
Micro RNA- Biogenesis, Mechanism of Action and Applications-A Review
Subhabrata Ghosh
Bose Institute, Kolkata-700009, India
Ph-+919903221547
Email-subhabrata09@yahoo.com
subhabrata.boseinstitute@gmail.com
Received 01 November 2011; accepted 14 November 2011
Abstract
MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression post-transcriptionally. After the discovery of
the first miRNA in the roundworm Caenorhabditis elegans, these short regulatory RNAs have been found to be an abundant
class of RNAs in plants, animals, and DNA viruses. The discoveries of RNA interference (RNAi), known as remarkable
process where specific genes are silenced by small noncoding RNAs, have revolutionized our understanding of gene regulation
in plants and other organisms. Recently, Micro RNA is used as a tool for reduction of expression of several genes or to control
de expression of specific genes in eukaryotic genome. The aim of this review is to describe the biology, process of biogenesis
and mechanism of action of Micro RNA and also to underline the most contribution in the ―world of Micro RNA‖ from
biological mechanism to several applications including cancer treatment, neurodegenerative diseases and viral infections like
AIDS, Hepatitis, Swine Flu etc.
© 2011 Universal Research Publications. All rights reserved
Keywords: Micro RNA, Gene regulation, Cancer, Neurodegenarative disease, AIDS

1. Introduction: partially complementary to 7 conserved sites located in the

According to the current convention, a miRNA is defined as a
ssRNA of ~22 nucleotides in length, which is generated by
the RNase-III-type enzyme Dicer from an endogenous
transcript that contains a local hairpin structure1.
1.1 Discovery of MicroRNA:
The founding member of the miRNA family, lin-4, was
identified in C. elegans through a genetic screen for defects in
the temporal control of post-embryonic development16,17.In
C. elegans, cell lineages have distinct characteristics during 4
different larval stages (L1–L4).Mutations in lin-4 disrupt the
temporal regulation of larval development, causing L1 (the
first larval stage)-specific cell-division patterns to reiterate at
later developmental stages18.Opposite developmental
phenotypes — omission of the L1 cell fates and premature
development into the L2 stage — are observed in worms that
are deficient for lin-1419. Even before the molecular
identification of lin-4 and lin-14, these loci were placed in the
same regulatory pathway on the basis of their opposing
phenotypes and antagonistic genetic interactions20.Most
genes identified from mutagenesis screens are protein-coding,
but lin-4 encodes a 22-nucleotide non-coding RNA that is
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
11
3′-untranslated region (UTR) of the lin-14 gene (Figure-1)21,
22
. lin-14 encodes a nuclear protein, down-regulation of
which at the end of the first larval stage initiates the
developmental progression into the second larval
stage,21,23.The negative regulation of LIN-14 protein
expression requires an intact 3′ UTR of its mRNA14, as well
as a functional lin-4gene13. These genetic interactions
inspired a series of molecular and biochemical studies
demonstrating that the direct, but imprecise, base pairing
between lin-4 and the lin-14 3′ UTR was essential for the
ability of lin-4 to control LIN-14 expression through the
regulation of protein synthesis24-26. Through an analogous
mechanism, lin-4 also negatively regulates the translation of
lin-28, a cold-shock-domain protein that initiates the
developmental transition between the L2 and L3 stages27.
Compared with lin-14, lin-28 has fewer lin-4 binding sites,
which might lead to its translational repression being delayed
following lin-4 expression owing to less efficient lin-4
binding28,27.The discovery of lin-4 and its target-specific
translational inhibition hinted at a new mechanism of gene
regulation during development. In 2000, almost 7 years after
the initial identification of lin-4, the second miRNA, let-7,

Figure 1. The molecular hallmarks of lin-4, the founding
member of the microRNA family.
(a) The precursor structure and mature microRNA (miRNA)
sequence of lin-4 (b) Sequence complementarity between lin-4 (red)
and the 3′-untranslated region (UTR) of lin-14 mRNA (blue).lin-4
is partially complementary to 7 sites in the lin-14 3′ UTR; its
binding to these sites of complementarity brings about repression of
LIN-14 protein synthesis.[RISC- RNA-induced silencing complex].
Figure 2. Genomic organization and structure of miRNA
genes.
(a) Intronic miRNA in a protein-coding transcriptional unit (TU). As
an example, miR-10 in HOX4B gene is shown. The green triangle
indicates the location of a miRNA stem-loop and the exons are
shown in yellow.
(b) Intronic miRNAs in a noncoding transcript. The miR-15a-16-1
cluster is shown, which is found in the fourth intron of a previously
defined noncoding RNA gene, DLEU2.
(c) The structure of exonic miRNA in noncoding transcripts, such as
miR-155.
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
12
was discovered, also using forward genetics in worms.let-7
encodes a temporally regulated 21-nucleotide small RNA that
controls the developmental transition from the L4 stage into
the adult stage 29-31. Similar to lin-4, let-7 performs its
function by binding to the 3′ UTR of lin-41and hbl-1 (lin-57),
and inhibiting their translation 29-33. The identification of let-7
not only provided another vivid example of developmental
regulation by small RNAs, but also raised the possibility that
such RNAs might be present in species other than nematodes.
Unlike lin-4, the orthologues of which in flies and mammals
initially escaped bio-informatic searches, and were only
recognized recently34,35,both let-7 and lin-41 are
evolutionarily conserved throughout metazoans, with
homologues that were readily detected in molluscs, sea
urchins, flies, mice and humans36.
1.2 Types and Distribution of small RNAs in Eukaryotic
system:
Apart from microRNAs (miRNAs), several other groups of
small RNAs have been described2: small interfering RNAs
(siRNAs), tiny non-coding RNAs (tncRNAs) and a small
modulatory RNA (smRNA). siRNAs are defined as 21–28-
nucleotide RNAs, which are produced by Dicer from long
double-stranded RNAs (dsRNAs). Small RNAs used in
RNA-interference-mediated experiments are traditionally
referred to as siRNAs, regardless of their origin. Endogenous
siRNAs have also been discovered in various organisms.
Endogenous siRNAs can be further categorized into at least
three distinct sub-classes: trans-acting siRNAs (tasiRNAs),
repeat-associated siRNAs (rasiRNAs) and small-scan RNAs
(scnRNAs). tasiRNAs found in plants are ~21-nucleotide
RNAs that are encoded in intergenic regions, and they act on
mRNAs intrans to induce mRNA cleavage3,4,5. Some
endogenous siRNAs match repetitive elements in the sense or
antisense orientation and are therefore known as rasiRNAs6,7.
Evidence from Schizosaccharomyces pombe and Arabidopsis
thaliana indicates that rasiRNAs might function in
transcriptional gene silencing through methylation of histones
and/or DNA. siRNAs of ~28 nucleotides found in
Tetrahymena thermophila are known as scnRNA because
they might scan for regions in the DNA for genome
rearrangement8-10. The Argonaute family protein TWI1
mediates histone methylation, which is, in turn, required for
DNA elimination8. There are additional small RNAs of which
the biogenesis and function remain less clear, and which
hinders their classification. tncRNAs described in nematode
worms are similar to tasiRNAs11, although their origin from
the long dsRNA precursor needs to be verified. A smRNA of
~20 nucleotides that has been cloned from neural stem cells
modulates transcription possibly by interacting with a
transcription factor 12. The biogenesis of smRNAs is as yet
unknown.
Since the discovery of RNA interference (RNAi),
efforts to identify endogenous small RNAs have led to the
discovery of hundreds of miRNAs in nematodes, fruit flies
and humans. More than 500 different miRNAs have been
Figure 3. Possible mechanisms of actions for Drosha and Dicer.
The ‗single processing centre‘ model applies to both Drosha and Dicer. According to this model, one processing centre is formed in the
interface between two RNase III domains (RIIIDa and b).The processing centre contains two closely located catalytic sites that cleave two
nearby phosphodiester bonds on opposite RNA strands. The catalytic sites are composed of residues that correspond to E40, D44, D107 and
E110 of Aquifex aeolicus RNase III. This ‗single processing centre model‘ differs from the previous model, which assumed the existence of
two processing centres with the second catalytic site being composed of residues E37 and E64 in A. aeolicus RNase III. The double-stranded
RNA (dsRNA) substrate is laid on the cleft between RIIIDa (light orange) and RIIIDb (dark orange). The catalytic cleaves the 5′ strand.
(a) Drosha binds to a primary transcript (pri-miRNA) and introduces a cut at approximately two helical turns, ~22 nucleotides (nt site on the
RIIIDa side cleaves the 3′ strand, whereas another catalytic site on the RIIIDb), from the terminal loop. For simplicity, the domains of Drosha
other than the RIIIDs are not shown. It was proposed that DGCR8/Pasha might help the binding of the complex to RNA and/or orienting the
complex on pri-miRNA.
(b) Dicer cleaves at approximately two helical turns (~22 nucleotides) from the 3′ terminus. The PAZ domain of Dicer is believed to hold the
3′ end of pre-miRNA. The domains of Dicer other than the RIIIDs and the PAZ domain are not shown.

identified in animals and plants, where the number of miRNA
genes is expected to increase to 500–1000 per species, which
would comprise w2–3% of protein-coding genes13.At the
time of writing, the miRNA database (http://www.sanger
.ac.uk/Software/Rfam/mirna/)contained 114 Caenorhabditis
elegans miRNAs, 78 Drosophila melanogaster miRNAs, 369
Danio rerio miRNAs, 122 Gallus gallus miRNAs, 326 Homo
sapiens miRNAs and 117 Arabidopsis thaliana
miRNAs14.Nearly all miRNAs are conserved in closely
related species and many have homologs in distant species.
At least a third of C. elegans miRNAs have homologs in
humans15, suggesting that their functions could also be
conserved throughout the evolution of animal lineages. Large
DNA viruses have also been found to carry miRNA genes:
five in Epstein–Barr virus, 12 in Kaposi sarcoma-associated
herpesvirus, nine in mouse g-herpesvirus, and nine in human
cytomegalovirus.
1.3 Genomic Distribution and gene structure of Micro
RNA:
miRNA genes are scattered in all chromosomes in humans
except for the Y chromosome. Approximately 50% of known
miRNAs are found in clusters37, 38, 39 and they are transcribed
as polycistronic primary transcripts40.The miRNAs in a given
cluster are often related to each other, suggesting that the
gene cluster is a result of gene duplication. A miRNA gene
cluster also often contains unrelated miRNAs. A plausible but
yet-to-be validated possibility is that the clustered miRNAs
are functionally related by virtue of targeting the same gene
or different genes in the same pathway. It was initially
thought that most miRNA genes were located in intergenic
regions. However, recent analyses of miRNA gene locations
showed that the majority (w70%) of mammalian miRNA
genes (161 out of 232) are located in defined transcription
units (TU)41. By combining up-to-date genome assemblies
and expressed sequence tag (EST) databases, Rodriguez et al.
demonstrated that many miRNA genes (117 out of 161) were
found in the introns in the sense orientation, which is more
than previously expected 41. Of these 117 intronic miRNAs,
90 miRNAs are in the introns of protein-coding genes,
whereas 27 miRNAs are in the introns of noncoding RNAs
(ncRNAs). In some cases (14 miRNAs), miRNAs are present
in either an exon or an intron (‗mixed‘) depending on the
alternative splicing pattern. So, miRNA genes can be
categorized based on their genomic locations: intronic
miRNA in protein coding TU; intronic miRNA in noncoding
TU; and exonic miRNA in noncoding TU (Figure-2).
‗Mixed‘ miRNA genes can be assigned to one of the above

International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36

13
groups depending on the given splicing pattern.
Unexpectedly, a large number of miRNAs are found in
introns of protein-coding genes (90 out of 161 miRNAs).
This indicates that previous Bio-informatic searches confined
to intergenic regions might have missed some miRNA genes.
The location of some intronic miRNAs is well conserved
among diverse species. For instance, miR-7 is found in the
intron of hnRNPK in both insects and mammals. Another
interesting example is the miR-106bw25w93 family that is
found in the intron 13 of MCM7 in both humans and mice.
As expected for genes sharing the same promoters, the ‗host‘
transcript and miRNAs usually have similar expression
profiles. MiRNA promoters have been identified
experimentally in numerous studies42-48. Bio-informatic
searches for miRNA-specific promoter elements upstream of
miRNA sequences have not been successful. Instead, the
characterized miRNA promoters contain general RNA
polymerase II (Pol II) transcriptional regulatory elements
previously found in protein coding genes.
2. Biogenesis of Micro RNA:
The current model for miRNA maturation was formulated on
the basis of two simple observations49.First, miRNAs are
transcribed as long primary transcripts that are first trimmed
into the hairpin intermediates (pre-miRNAs) and
subsequently cleaved into mature miRNAs. Second, the
catalytic activities for the first and the second processing are
compartmentalized into the nucleus and the cytoplasm,
respectively (Figure-4). So the nuclear export of pre-miRNA
is necessary for the cytoplasmic processing to occur.
2.1 Nuclear processing by Drosha
Transcription of miRNA genes yields primary transcripts,
pri-miRNAs, that are usually several kilo-bases long and that
contain a local hairpin structure. The stem-loop structure is
cleaved by the nuclear RNase III Drosha to release the
precursor of miRNA (pre-miRNA)50 (Figure-3a) The
remnants (the flanking fragments) are thought to be degraded
in the nucleus, although the ~1.7-kb 3′ flanking fragment of
miR-23a~27a~24-2 has been cloned multiple times in EST
studies and is currently annotated as an
mRNA51.Accordingly, it remains to be seen whether the 5′
and 3′ fragments that surround the stem-loop have their own
functions. Drosha is a large protein of ~160 kDa, and is
conserved in animals52-54. It contains two tandem RNase III
domains (RIIIDs) and a double-stranded RNA-binding
domain (dsRBD) that are crucial for catalysis55. The central
region of the protein, adjacent to the RIIIDs, is also essential
for pri-miRNA processing55.Drosha forms a large complex of
~500 kDa in D. melanogaster 56 or ~650 kDa in humans55,57 In
this complex, which is known as the Microprocessor
complex, Drosha interacts with its cofactor, the DiGeorge
syndrome critical region gene 8 (DGCR8) protein in humans
(also known as Pasha in D. melanogaster and C.
elegans).DGCR8/Pasha is a ~120 kDa protein that contains
two dsRBDs. It also contains a putative WW DOMAIN,
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
14
which is known to be an interaction module for specific
proline-rich sequences. It remains to be determined if this
domain interacts with the proline rich region of Drosha.
Although the biochemical role of DGCR8/Pasha is currently
unclear, it is believed to assist Drosha in substrate
recognition. There are hundreds of different pri-miRNAs in
animal cells, but they do not seem to share any common
sequence motifs. This question cannot be fully answered yet,
but mutagenesis analyses indicate that the tertiary structure of
pri-miRNAs is the primary determinant for substrate
specificity 58, 59. Both the double-stranded stem structure
around the cleavage site and the large terminal loop
(consisting of >10 nucleotides) are vital. Interestingly, it
seems that the Drosha complex can measure the length of the
stem, because the cleavage site is located approximately two
helical turns (~22 nucleotides) from the terminal loop. To
comprehend how the Drosha complex measures the length,
the biochemical roles of the individual domains of Drosha
and DGCR8 need to be delineated.
2.2 Nuclear export by exportin-5
Following nuclear processing by Drosha, pre-miRNAs are
exported to the cytoplasm. Once there, they are subjected to
the second processing step by Dicer (another RNase III
enzyme) to generate the final ~22-nucleotide product. Owing
to compartmentalization of the two processing events, nuclear
export of pre-miRNAs is a crucial step in miRNA biogenesis
60,61
. Nuclear transport occurs through nuclear pore
complexes, which are large proteinaceous channels
embedded in the nuclear membrane62. Members of the
nuclear export receptor family bind cooperatively to a cargo
as well as to the GTP-bound form of the cofactor Ran in the
nucleus. Following export, hydrolysis of GTP to GDP results
in release of the cargo from the export complex. Export of
pre-miRNA is mediated by one of the nuclear transport
receptors, exportin-563-65. When the cells were depleted of
exportin-5, the pre-miRNA level and the mature miRNA
level were reduced in the cytoplasm. Notably, pre-miRNA
does not accumulate in the nucleus subsequent to the
depletion of exportin-5. This indicates that pre-miRNA might
be relatively unstable and also that pre-miRNA might be
stabilized through its interaction with exportin-5. Exportin-5
was originally known as a minor export factor for tRNAs,
because it can transport tRNAs when the primary export
factor, exportin-t, is depleted or overloaded66,67.Considering
that the affinity of exportin-5 to pre-miRNA is much higher
than to tRNA, and that miRNAs are as abundant as 50,000
copies per cell68, pre-miRNAs are probably the main cargo
for exportin-5.
2.3 Cytoplasmic processing by Dicer
Following their export from the nucleus, pre-miRNAs are
subsequently processed into ~22-nucleotide miRNA duplexes
by the cytoplasmic RNase III Dicer 69-73(Figure-3b). Because
Dicer was originally found to function in generating siRNAs,
which are similar in size (21–25 nucleotides) to miRNAs, it
was predicted that Dicer also functions in the processing of

Figure 4. The biogenesis of microRNAs.
~70-nucleotide stem-loop RNAs into mature miRNAs.
Indeed, it was later proven that immuno-precipitated Dicer
generates ~22-nucleotide miRNAs from in vitro synthesized
~70-nucleotide let-7 stem-loop RNAs. Moreover, when the
human homologue and C. elegans homologue of Dicer were
knocked down or knocked out, the ~70-nucleotide pre-
miRNAs accumulated, whereas the ~22-nucleotide mature
miRNAs diminished. Dicer is a highly conserved protein that
is found in almost all eukaryotic organisms such as
Schizosaccharomyces pombe, plants and animals. Some
organisms contain multiple Dicer homologues, in which
different Dicer isotypes are often assigned to take on distinct
roles. For example, D. melanogaster Dicer-1 is required for
pre-miRNA cleavage, whereas Dicer-2 is needed for siRNA
generation. Dicer homologues are multi-domain proteins of
~200 kDa .Apart from two RIIIDs and a dsRBD, Dicer has a
long N-terminal segment that contains a DEAD-BOX RNA
helicase domain, as well as a DUF283 domain and a PAZ
domain. The PAZ domain is also found in a group of highly
conserved proteins known as Argonaute proteins. Structural
and biochemical studies of the PAZ domain from D.
melanogaster AGO1 and AGO2 indicate that the PAZ
domain binds to the 3′ protruding end of small RNAs74-77.
The roles of the other domains in Dicer remain unknown.
Dicer associates with several other proteins including RDE-4
in C. elegans78, R2D279 and FMR180–83 in D. melanogaster 80,
and Argonaute family proteins in various organisms84,85.
These Dicer-interacting proteins do not seem to be required
for the cleavage reaction itself as purified human Dicer and
D. melanogaster Dicer-2 can catalyse the cleavage
reaction80,86, 87. Instead, Dicer-interacting proteins have
various roles in miRNA stability and effector complex
formation and action. For example, one of the Argonaute
family proteins, human AGO2, was recently shown to
function as the ‗slicer‘ enzyme that cleaves target mRNA.
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
15
Figure 5. Mechanisms of miRNA-mediated repression
Mature miRNAs are incorporated into effector complexes
that are known as ‗miRNP‘(miRNA-containing
ribonucleoprotein complex), ‗mirgonaute‘ or ‗miRISC‘
(miRNA-containing RNA-induced silencing complex).On the
other hand, the effector complex that contains siRNA is
known as ‗RISC‘, ‗sirgonaute‘ or ‗siRISC‘.The cleavage
products (~22-nucleotide miRNA duplexes) do not persist in
the cell for long. Usually, one strand of this short-lived
duplex disappears, whereas the other strand remains as a
mature miRNA. Studies on siRNA duplexes indicate that the
relative Thermo-dynamic stability of the two ends of the
duplex determines which strand is to be selected88,89.The
strand with relatively unstable base pairs at the 5′ end
typically remains (for example, G:U pair versus G:C
pair)88,89. The same rule is thought to be applicable to
miRNA. The mechanistic details of strand selection have
been delineated in D. melanogaster. R2D2, which has two
dsRBDs, was shown to sense the differences in
thermodynamic inequalities. R2D2 forms a stable
heterodimeric complex with Dicer-2 and binds to the more
stable end of the siRNA duplex, thereby orienting the
complex on the siRNA duplex. It is not known how the
unselected strand is removed and degraded. It also remains to
be determined if the same machinery acts on the miRNA
duplex and how conserved this machinery is.
2.4 Collaborative actions of biogenesis factors
The multiple steps in miRNA biogenesis seem to be
remarkably well coordinated. Drosha initiates miRNA
processing by the specific cropping of the stem-loop
precursor in the nucleus90. Like other RNase-III-type
endonucleases, Drosha cuts dsRNA to create a short ~2-
nucleotide 3′ overhang90,91. The resulting structure (a short
stem plus a 3′ overhang) seems to be a signature motif for all
dsRNAs that are involved in small-RNA pathways. Exportin-
5 recognizes this signature motif to export pre-miRNA to the
cytoplasm. Following export, pre-miRNA is handed over to
another RNase III Dicer, which has a preference for the
terminus of dsRNAs containing the short 3′ overhang92.
Therefore, Drosha confers specificity to the biogenesis
pathway and facilitates the overall production rate by

Figure 6. MicroRNAs can function as tumour suppressors
and oncogenes
(a) In normal tissues, proper microRNA (miRNA) transcription,
processing and binding to complementary sequences on the target
mRNA results in the repression of target-gene expression through a
block in protein translation or altered mRNA stability (not shown).
The overall result is normal rates of cellular growth, proliferation,
differentiation and cell death.
(b) The reduction or deletion of a miRNA that functions as a tumour
suppressor leads to tumour formation. A reduction in or elimination
of mature miRNA levels can occur because of defects at any stage of
miRNA biogenesis (indicated by question marks) and ultimately
leads to the inappropriate expression of the miRNA-target
oncoprotein (purple squares). The overall outcome might involve
increased proliferation, invasiveness or angiogenesis, decreased
levels of apoptosis, or undifferentiated or de-differentiated tissue,
ultimately leading to tumour formation.
(c) The amplification or over-expression of a miRNA that has an
oncogenic role would also result in tumour formation. In this
situation, increased amounts of a miRNA, which might be produced
at inappropriate times or in the wrong tissues, would eliminate the
expression of a miRNA-target tumour-suppressor gene (pink) and
lead to cancer progression. Increased levels of mature miRNA might
occur because of amplification of the iRNA gene, a constitutively
active promoter, increased efficiency in miRNA processing or
increased stability of the miRNA (indicated by question marks).
ORF,open reading frame.
generating the 3′ protruding ends, which are recognized efficiently
by the downstream biogenesis factors92. In addition, Drosha pre-
determines mature miRNA sequences by precisely generating one
end of the mature miRNA90, 93. The other end is created by Dicer
and measures ~22 nucleotides from the pre-existing terminus of the
pre-miRNA RNA interference (RNAi)-mediated knock-down of
Drosha, exportin-5 or Dicer results in a significant reduction, but not
the full loss, of mature miRNA .This is probably due to the long
half-life of mature miRNA. miRNAs seem to be quite stable once
they enter into the effector complex. But the possibility that there
could be minor alternative biogenesis pathway(s) cannot be
excluded. For example, a small portion of pri-miRNAs might be
exported by mRNA export pathway and become processed in the
cytoplasm.
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
16
2.5 MicroRNA biogenesis in plants
Homologues of Drosha and DGCR8/Pasha have not been
found in plants, which indicate that the Drosha dependent
stepwise processing model applies only to animal cells.
Neither miRNA nor the Drosha homologue has been found in
yeasts. Plant miRNA precursors are quite diverse in structure,
and the stem-loops are usually longer than animal pri-
miRNAs. Genetic studies in A. thaliana showed that DCL1,
one of the four Dicer like proteins in A. thaliana (also known
as CARPEL FACTORY (CAF)), is important for miRNA
accumulation94–95. DCL1 is a nuclear protein, which
indicates that mature ~22-nucleotide miRNA might be
generated in the nucleus in plants96. Although detection of
miRNA precursors has been difficult, presumably owing to
efficient processing and/or rapid turnover of precursors, at
least one study showed that plant miRNAs, similar to animal
miRNAs, might be processed in a stepwise manner97, in
which DCL1 might be solely responsible for all processing
steps. HASTY (HST) is a plant homologue of exportin-5 on
the basis of its amino-acid sequence. An HST mutant showed
pleiotropic phenotypes, which indicates that this protein
might function in miRNA biogenesis too98, 99. A recent
study showed that loss-of-function mutants in HST reduced
the accumulation of most miRNAs, indicating that HST
functions as a nuclear export receptor100. The identity of the
actual cargo for HST-whether it is precursor, miRNA duplex
or single-stranded miRNA is unclear. Additional miRNA
biogenesis factors include HYL1, a two dsRBD-containing
nuclear protein of unknown biochemical function101, 102,
and HEN1, a protein with a dsRBD and a methyl-transferase
domain103, 104. It was recently shown that HEN1
methylates miRNA duplex at the 2′ hydroxyl groups of the 3′-
end nucleotides104.The biochemical role of this 2′-O-methyl
group awaits further investigation.
2.6 Regulation of microRNA biogenesis :
Expression profiling studies indicate that most miRNAs are
under the control of developmental and/or tissue specific
signaling pathways105-115. miRNA expression might be
regulated at multiple steps of RNA biogenesis, although it
remains to be determined which step is controlled and how
this control is achieved. Transcriptional regulation is
probably the main control mechanism. For example, the
temporal regulation of let-7RNA in C. elegans is dependent
on a transcriptional enhancer element, known as the temporal
regulatory element (TRE)116. This element is situated ~1200
bp upstream of the mature let-7 RNA. Electrophoretic
mobility shift assays using nuclear extract indicated that a
~22-nucleotide inverted repeat in the TRE might be a binding
site for a transcription factor that is yet to be identified116.
Some miRNAs seem to be controlled at the
posttranscriptional level. For example, C. elegans miR-38 is
expressed only in the embryo, whereas the precursor (pre-
miR-38) is ubiquitously detected, which indicates that the
maturation of pre-miR-38 might be temporally regulated. It is
possible that pre-miR-38 is retained in the nucleus until
export is triggered by a certain developmental signal.
Alternatively, it might be bound to negative regulatory
factor(s) in the cytoplasm, so that they remain inaccessible to
Dicer until a certain stage.
2.7 Computational tools for discovery of microRNA and
their targets:
Computational predictions have been the mainstay for
discovery of microRNAs and their targets. The algorithms for
microRNA prediction range from custom-made programs to
search for hairpin loops and energetic stability to advanced
algorithms employing machine learning approaches117-119.
Even though the algorithms for microRNA prediction have
improved over time, accurate de novo prediction of
microRNA still remains a challenging task. This is especially
important in the case of viruses as viral microRNAs do not
share close homology even in the same class. We hope the
prediction algorithms will improve with a better
understanding of the sequence and structural components of
precursor hairpins involved in microRNA biogenesis120. The
algorithms for microRNA target prediction also have been
significantly improved from first-generation algorithms
which rely on sequence complementarity rules,
thermodynamic stability and conservation121, 122 by
incorporation of features like target RNA structure123.
2.8 miRNAs are a large family of gene regulators:
miRNAs are non-coding, single-stranded RNAs of ~22
nucleotides and constitute a novel class of gene regulators
that are found in both plants and animals. They negatively
regulate their targets in one of two ways depending on the
degree of complementarity between the miRNA and the
target. First, miRNAs that bind with perfect or nearly perfect
complementarity to protein coding mRNA sequences induce
the RNA-mediated interference (RNAi) pathway. Briefly,
mRNA transcripts are cleaved by ribonucleases in the
miRNA-associated, multi-protein RNAinduced- silencing
complex (miRISC), which results in the degradation of target
mRNAs. This mechanism of miRNA-mediated gene
silencing is commonly found in plants 124-126, but miRNA-
directed mRNA cleavage has also been shown to occur in
mammals127. However, most animal miRNAs are thought to
use a second mechanism of gene regulation that does not
involve the cleavage of their mRNA targets. These miRNAs
exert their regulatory effects by binding to imperfect
complementary sites within the 3′ untranslated regions
(3‘UTRs) of their mRNA targets, and they repress target-
gene expression post-transcriptionally, apparently at the level
of translation, through a RISC complex that is similar to, or
possibly identical with, the one that is used for the RNAi
pathway128-129. miRNAs that use this mechanism reduce
the protein levels of their target genes, but the mRNA levels
of these genes are barely affected. However, recent findings
indicate that miRNAs that share only partial complementarity
with their targets can also induce mRNA degradation, but it is
unclear if translational inhibition precedes destabilization of
the gene targets in these cases130.
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17
3. The mechanism of action of microRNAs:
The mechanism of action of microRNAs is considered to be
by two modes – translational repression and target
degradation (Figure-5). The former is common in mammalian
systems while the latter is found predominantly in plants. The
basic difference in the two mechanisms is thought to be
primarily governed by the levels of complementarity between
microRNAs and their target transcripts. Perfect or near
perfect complementarity as is common in plant microRNAs
and in a small class of eukaryotic microRNAs causes target
cleavage and degradation131analogous to the action of
siRNAs which have perfect complementarity to the target
regions. Evidence suggests that microRNA bound transcripts
are sequestrated into P bodies132, 133 where they are
maintained in a silenced state either by associating with
proteins that prevent translation or possibly by removal of the
cap structure 133.
3.1 Translational inhibition:
The first mechanistic analyses of miRNA function were
carried out using C. elegans: it was found that the abundance
of miRNA-regulated mRNAs was not substantially hanged,
but the abundance of proteins encoded by those mRNAs was
markedly reduced 134,135. Furthermore, the regulated
mRNAs seemed to be present in polysomes134,135 and
engaged with ribosomes capable of subsequent elongation in
vitro135 .These striking observations suggested that miRNA-
mediated suppression of protein production occurred by a
mechanism that operated after the initiation of protein
synthesis. Although subsequent studies have shown that
miRNAs can exert their effects by a variety of mechanisms,
there are now numerous examples in which miRNA-mediated
reduction of protein production is not accompanied by
corresponding changes in mRNA abundance136-140. In most
of these cases, the translational status of regulated mRNAs
(i.e.whether they are associated with polysomes) has not been
determined. Nevertheless, three recent reports provide
evidence for repression of protein production after protein
synthesis has been initiated141-143.In the first study, a
miRNA-regulated mRNA in HeLa cells was found
exclusively in polysomes and associated with elongationally
competent ribosomes141. In the second study, a reporter
mRNA responsive to an endogenous miRNA was also
present in polysomes142. Several lines of evidence showed
that these polysomes were actively translating; however,
nascent polypeptides could not be detected by immuno-
precipitation, suggesting that repression of protein
accumulation resulted from co-translational protein
degradation (i.e. protein degradation concomitant with
translation). In the third study, a reporter constructs
containing a 3‘-UTR with designed target sites for a synthetic
miRNA that has extensive but imperfect complementarity to
those sites was shown to be translationally repressed and
associated with polysomes143. In this case, evidence
suggested that the deficit in protein production resulted from
premature termination of translation (premature ribosome
Table-1: microRNAs and their targets: examples of microRNAs with experimentally validated functions/targets
Micro RNA Target Gene Function Modes of References
Repression
Caenorhabditis elegans
lin-4 lin-14,lin-28 Regulation of developmental transition Translational 173,174,175
between the first two larval stages, L1 repression
and L2
let-7 lin-41, hbl-1 Regulation of developmental transition Translational 176-179
between the last larval stage (L4) and the repression
adult stage
lsy-6 cog-1 Determination of left/right asymmetry of Unknown 180
neuronal development
Drosophila melanogaster
Bantam hid Promotion of cell proliferation and Translational 181
suppression of apoptosis repression
Mus musculus

miR-181 Unknown Promotion of haematopoietic Unknown 183

differentiation towards the B-cell lineage.
miR-196 Hoxb8 Unknown PTGS 184
Arabidopsis thaliana
miR-165, miR- PHB, PHV and Regulation of leaf morphogenesis PTGS 185, 186
166 REV
miR-172 AP2 Regulation of flowering time and floral- Translational 187
organ identity repression
miR-JAW TCP Regulation of leaf development and PTGS 188
transcription embryogenesis
factors
miR-39 SCLfamily Unknown PTGS 189
proteins
Zea mays
miR-166 rid1 Regulation of leaf morphogenesis PTGS 190
AP2, APETALA 2; Hid, head involution defective; Hoxb8, homeobox B8; PHB, PHABULOSA; PHV, PHAVOLUTA; PTGS,
post-transcriptional gene silencing; REV,REVOLUTA; rld1, rolled leaf1; SCL, SCARECROW-LIKE; TCP, teosinte branched
1-cycloidea-PCF.
drop off) 143. By contrast, two other reports have showed that it was impaired143-145. The most
demonstrated miRNA-mediated inhibition at the level of straightforward interpretation of the results obtained so far is
initiation of protein synthesis144, 145. In one study, an that miRNA-mediated inhibition of translation can occur in
endogenous miRNA was shown to reduce polysome an mRNA-specific manner at distinct steps in the process of
association of a reporter construct with appropriate target translation. Nevertheless, the features of individual mRNAs
sites144. In the other study, complete repression of and/or mRNA–miRNA combinations that dictate the
translation required both the 5‘-cap and the 3‘-poly (A)+ tail, mechanism of repression remain to be elucidated.
whereas internal ribosome entry site (IRES)-directed
translation was insensitive to inhibition145. Although the 3.2 mRNA de-adenylation and degradation:
results of these two studies differ in the details [e.g. the role It was originally thought that miRNA-mediated regulation
of the poly (A)+ tail], they both present persuasive evidence was exerted mainly at the level of translation and not at the
that initiation of protein synthesis is impaired by miRNAs at level of mRNA degradation. It is now clear that this idea is
the level of cap recognition. Collectively, the studies of only partially correct. There are numerous examples of
translational inhibition have revealed distinct mechanisms by miRNAs destabilizing their target mRNAs. Indeed, as
which cap-dependent translation can be repressed. detailed in this section, the mechanism by which miRNAs
Interestingly, differences have also been observed in the increase mRNA turnover is perhaps better understood than
ability of miRNAs to suppress translation promoted by the mechanism of translational repression. Early evidence for
miRNA-mediated destabilization came from studies in which
IRESs: two groups found that IRES-directed protein a miRNA was transfected into cells that normally did not
synthesis was unaffected by miRNAs, whereas another group express this miRNA. The presence of such a miRNA was

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18
 Table 2: MicroRNAs that are associated with human cancers
miRNA Gene loci Cancer association Function References
miR-15a, Chromosome Frequently deleted or downregulated in TS 209,210
miR-16-1 13q14 B-cell chronic lymphocytic leukemia;
negatively regulates the antiapoptotic
gene BCL2
miR-143, Chromosome Decreased abundance in colorectal TS 211,212
miR-145 5q32–33 cancer; downregulated in breast,
prostate, cervical and lymphoid cancer
cell lines; miR-145 is decreased in
breast cancer.
miR-21 Chromosome Anti-apoptotic factor; upregulated in OG 211,213,214
17q23.2 glioblastomas and breast cancer.
let-7 Multiple loci Negatively regulate the Ras oncogenes; TS 215,216
family direct cell proliferation and
members differentiation; decreased abundance in
lung cancer
miR-142 Chromosome A t(8;17) translocation that places the N/A 217
17q22 MYC oncogene downstream of the
mir-142 hairpin, resulting in an
aggressive
B-cell leukemia that is due to MYC
overexpression
BIC/miR- Chromosome Upregulated in paediatric Burkitt, OG 211,218-221
155 21q21 Hodgkin, primary mediastinal and
diffuse large-B-cell lymphomas;
upregulated in human breast cancer
miR-17– Chromosome Upregulated by MYC; negatively TS/ OG 222,223
19b cluster 13q31–32 modulates the E2F1 oncogene; loss
of heterozygosity of this cluster is
found in hepatocellular carcinoma;
overexpressed in B-cell lymphomas
N/A-not applicable; OG-oncogene; TS- tumour suppressor.

found to reduce the abundance of many mRNAs, most of
which contained target sites for the miRNA146. In addition,
examination of several miRNA targets in C. elegans revealed
that these also were reduced in abundance in a miRNA-
dependent manner147. The realization that miRNAs can
promote mRNA degradation has enabled the use of mRNA
microarray analysis for the identification of miRNA
targets146, 148-150. Although this approach has been
fruitful, the proportion of the total number of targets that is
identified by these types of analysis remains to be
determined. There is no analogous high throughput method to
identify targets that are regulated strictly at the translational
level. It now seems clear that miRNA-mediated
destabilization is a consequence of de-adenylation followed
by decapping (i.e. removal of the 5‘-cap). Many studies – in
zebrafish embryos, human cells and Drosophila melanogaster
cells – have shown miRNA-dependent de-adenylation149,
151-153. In D. melanogaster cells, de-adenylation is carried
out by the de-adenylase complex CCR4–NOT. Interestingly,
although de-adenylation seems to be a prerequisite for mRNA
decapping and degradation, it does not necessarily lead to
these outcomes. Specifically, in the study in D. melanogaster,
three reporter mRNAs were analyzed. Although all three
were completely de-adenylated, one remained stable, a
second was only partially destabilized, and turnover of a third
was markedly increased. Importantly, the level of regulation
of these reporter mRNAs did not correlate with their stability
in all cases: that is, the stable de-adenylated RNA was still
strongly repressed at the translational level149. Although the
translational repression observed could have resulted from
the loss of the polyA tail, maintaining the poly(A)+ tail (by
knockdown of the de-adenylase) did not relieve the
inhibition149. Results from human cells, showing miRNA-
dependent translational inhibition of a non-polyadenylated
mRNA, are also inconsistent with de-adenylation being the
sole cause of repression of protein synthesis152.
Furthermore, repression of protein synthesis is not
responsible for deadenylation: two groups have shown that
mRNAs that cannot be translated, because of either a strong
stem–loop in the 50-UTR or a defective cap, are still subject
to deadenylation152, 153. These results from the study of
several species and many distinct mRNAs suggest that
increased de-adenylation might be a general consequence of
miRNA–mRNA interactions. However, no change in

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19
poly(A)+ tail length was observed for an miRNA regulated
mRNA in C. elegans, although in this case the tail was short
under all conditions examined. It will be of considerable
interest to determine whether other miRNA-regulated
mRNAs are de-adenylated, particularly those that seem to be
repressed by distinct mechanisms and those for which
miRNA-mediated repression is reversible154.
miRNAs regulate multiple gene targets (Table-1).
The current challenge is to accurately identify targets that are
regulated by miRNAs. Because miRNAs usually bind to their
targets with incomplete complementarity, which allows short
stretches of mismatched base-pairs and G–U base pairing, the
identification of gene targets with a simple BLAST search is
impossible. However, current bio-informatic approaches have
taken advantage of the fact that miRNAs within families have
highest homology at the 5′ end of the mature miRNA. Several
studies have indicated that the 5′ end of the miRNA is crucial
for the stability and proper loading of the miRNA into the
miRISC complex155, 156, and this end is also important for
biological function157–159. Therefore, most bioinformatics
algorithms use a ‗miRNA seed‘ that encompasses the first 2–
8 bases of the mature miRNA sequence to search for
complementarity to sequences in the 3′ UTR of all expressed
genes. These studies have revealed that a single miRNA
might bind to as many as 200 gene targets and that these
targets can be diverse in their function; they include
transcription factors, secreted factors, receptors and
transporters160-169.So, miRNAs potentially control the
expression of about one-third of human mRNAs. However,
many targets are missed using the ‗miRNA seed‘ approach as
there is evidence that other variables besides the first 2–8
nucleotides of the miRNA sequence dictate miRNA
regulation. For example, the complete mature miRNA
sequence of let-7 has been evolutionarily conserved from
worms to humans170, demonstrating the functional relevance
of the 3′ end. Furthermore, mutagenesis studies in one
laboratory have shown that base pairing at both the 5′ and 3′
ends of let-7 is required for down regulation of a target
gene171. Therefore, the number of known miRNA targets
could increase and the challenge will be to determine which
predicted targets are biologically relevant. Sites that are
complementary to several miRNAs have also been identified
within the 3′ UTR of a single gene target, indicating that
complex patterns of combinatorial regulation by miRNAs
exist172. Because miRNAs potentially have a broad
influence over several diverse genetic pathways, the deletion
or mis-expression of these small RNAs is likely to be
pleotropic and contribute to disease, including cancer.
4. MicroRNAs and cancer:
Three important observations early in the history of miRNAs
suggested a potential role in human cancer. Firstly, the
earliest miRNAs discovered in the roundworm C. elegans and
the fruit fly Drosophila were shown to control cell
proliferation and apoptosis191, 192. Their deregulation may
therefore contribute to proliferative diseases such as cancer.
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
20
Secondly, when human miRNAs were discovered, it was
noticed that many miRNA genes were located at fragile sites
in the genome or regions that are commonly amplified or
deleted in human cancer193. Thirdly, malignant tumors and
tumor cell lines were found to have widespread deregulated
miRNA expression compared to normal tissues194-
196(Table-2). The question remained whether the altered
miRNA expression observed in cancer is a cause or
consequence of malignant transformation.
4.1 MicroRNAs as causal cancer genes at genomic
breakpoints :
Five years ago, the first direct evidence for an involvement of
miRNAs in cancer was reported197. Calin et al. studied a
well-known deletion on chromosome13, which is the most
frequent chromosomal abnormality in chronic lymphocytic
leukemia (CLL). This deletion had long been suspected to
contribute to leukemogenesis. However, extensive studies
had failed to identify a causal gene. Calin et al197found that
two miRNA genes, mir-15 and mir-16, were located within
this 30-kb deletion. They subsequently analyzed the
expression of miR-15 and miR-16 in blood samples from
patients with CLL. Both miRNAs were absent or down-
regulated in the majority (68%) of cases when compared to
normal tissue or lymphocytes. This finding suggested that
these two miRNAs were causally involved in the
pathogenesis of chronic lymphocytic leukemia. In 2005, three
reports provided the first mechanistic insight into how
miRNAs might contribute to carcinogenesis. Two
independent studies described the relationship between a
miRNA cluster, mir-17-92, and the Myc oncogenic
pathway198, 199. A third report demonstrated an interaction
between let-7 miRNA and the RAS proto-oncogene200
4.2 MicroRNAs with oncogenic Potential:
A cluster of six miRNAs, the mir-17-92 cluster, was found to
be located within a region on chromosome 13 that is
commonly amplified in human B-cell lymphomas201. He et
al.202demonstrated that the miRNAs from the mir-17-92
cluster were over expressed in lymphoma cell lines carrying
this amplification, and expression levels correlated with gene
copy number of the mir-17-92 locus. Further, the miR-17-92
primary transcript was found to be over expressed in tumor
samples from lymphoma patients. To test their hypothesis
that mir-17-92 actively contributes to lymphomagenesis, the
authors took advantage of a mouse model of human B-cell
lymphoma. These mice develop lymphomas due to an over
expression of the Myc oncogene. The Myc oncogene encodes
the transcription factor c-Myc that regulates cell proliferation,
growth, and apoptosis, and over expression of c-Myc is
common in cancer. He et al. demonstrated that additional
expression of the mir-17-92 cluster accelerated c-Myc-
induced tumorigenesis in mice. The authors therefore
suggested that mir-17-92 was the first potential non-coding
oncogene, referred to as oncomir-1.The cellular function of
miR-17-92 was not identified in these experiments.
Nevertheless, the pathology of the tumors indicated lower
rates of apoptosis as compared to tumors with Myc over
expression alone. Three recent studies contributed towards
our understanding of the oncogenic potential of miR-17-92.
Two reports demonstrated an anti-apoptotic effect of miR-17-
92 through various pathways that promote cell proliferation
and growth203, 204. A third study identified mir-17-92 as a
mediator of angiogenesis in tumors induced by the oncogene
c-Myc205.O‘Donnell et al.206 independently identified the
same cluster of miRNAs, mir-17-92, to be regulated by the
transcription factor c-Myc. The transcription factor Myc
induces expression of E2F1 growth factor. The mir-17-92
cluster which is also induced by c-Myc does, in contrast,
inhibit E2F1 expression. The authors therefore suggested a
novel regulatory mechanism by which c-Myc fine-tunes gene
expression by activating the transcription of target genes and
by simultaneously inducing inhibitory miRNAs that reduce
their translation. The example of the mir-17-92 cluster
highlights that a distinction between oncogenic and tumor
suppressor miRNAs is likely to be an oversimplification. The
same miRNAs may have oncogenic or tumor suppressor
activity depending on the context and the cell type they are
expressed in (Figure-6). A single miRNA may regulate
various unrelated target genes and thereby control opposing
activities such as cellular proliferation and apoptosis. The
ultimate function of a miRNA may depend on the tissue type
they are expressed in and what target genes are present.
4.3 MicroRNAs with tumor suppressor potential :
The let-7 family of miRNAs was the first group of miRNAs
shown to regulate expression of a proto-oncogene, the RAS
protein. RAS proteins are membrane-associated signaling
proteins that regulate cell growth and differentiation. A
miRNA that controls expression of these potentially
oncogenic proteins would be predicted to possess tumor
suppressor activity. Mutations in the RAS oncogene are
present in approximately 15–30% of all human cancers, and
over expression of the RAS oncogene is common in lung
cancer. Johnson et al207 showed that over expression of RAS
protein in lung cancer tissue correlated with reduced
expression of let-7 miRNA. They experimentally confirmed
that let-7 can inhibit RAS expression in human cancer cell
lines. Loss or reduction of let-7 in lung cancer leads to RAS
over expression, thus, promoting cellular growth and
contributing to tumorigenesis. The authors therefore
suggested that let-7 acts as tumor suppressor207. Another
group independently reported reduced expression of let-7 in
lung cancers and found that this correlated with a poor
prognosis 208.
4.4 MicroRNAs in the p53 tumor suppressor network:
Transcriptional networks are often deregulated in cancer cells
and may lead to altered transcription of miRNA genes. Two
recent studies identified a miRNA, miR-34, to be regulated
by the p53 transcription factor224,225. The p53 protein, also
called ―the guardian of the genome‖, regulates the cellular
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21
response to stress and cancer-initiating events such as DNA
damage. He et al.225 found that a miRNA, miR-34, is
directly activated by the transcription factor p53 after DNA
damage. Expression of miR-34 induces cell cycle arrest and
thereby acts together with other effectors of the p53 tumor
suppressor network to inhibit inappropriate cell proliferation.
Another group independently demonstrated that miR-34 is
up-regulated by p53 upon DNA damage and promotes
apoptosis224. Together, these data indicate that altered
expression of miRNAs is not simply a secondary event that
reflects the less differentiated state of cancer cells. In
contrast, at least in some cases, miRNA expression is
specifically driven by tumor suppressors and oncogenes.
4.5 miRNAs and Tumours of the Nervous System :
Several studies found that a high proportion of genomic loci
containing miRNA genes exhibit DNA copy number
alterations in common cancers301 and miRNA mis-
expression has also been described in tumours of the nervous
system and 268–272, 276,302-305. miRNAs have been
shown to act either as tumor suppressors or
oncogenes and, depending on the mRNA target, may
accelerate the oncogenic process302. A suppressor effect was
observed in pituitary adenomas, the most common tumors of
the central nervous system, in which down-regulation of
miR-15a and miR-16 correlates with tumor size 273–275.
Other miRNAs, such as the miR-155 and miR17-92 cluster,
have an oncogenic effect306, 307. The consequence of an
upregulation of miR-21 has been characterized in
glioblastoma tumor cells268, wherein the knockdown of
miR-21 led to increased apoptotic cell death, suggesting that
this miRNA may act as an antiapoptotic player268, 276, 308.
In addition, miRNA profiling in glioblastoma cells has shown
high levels of miR-221, miR-128, miR-181a, and miR-181b
and low levels of miR-181c268, 277, 278.miRNA expression
analysis may also be used for medulloblastoma prognosis.
Down-regulation of miR-9 and miR-125a was observed in
aggressive brain malignancy, which results in the activation
of medulloblastoma cell growth and arrest of apoptosis by
activation of the pro-proliferative truncated TrkC
isoform279.Based on these findings, the potential to
modulate multiple messages at the same time via miRNA
technology would therefore represent an intriguing prospect
for cancer treatment.
4.6 MicroRNAs with a role in tumor invasion and
metastasis:
Transcriptional networks may drive miRNA expression in
cancers. Recent work from Ma et al.226 suggested a model
by which a pleiotropic transcription factor, Twist, induces
expression of a specific miRNA, which suppresses its direct
target and in turn activates a pro-metastatic gene, leading to
tumor cell invasion and metastasis. The expression of miR-
10b induced by the transcription factor Twist promoted cell
migration and invasion in mouse and human breast cancer
cells. Furthermore, the expression level of miR-10b in
 Table-3: miRNAs involved in neurological diseases
microRNA Neurological disease Effect Reference number
miR-29a/b-1 Alzheimer‘s disease downregulation 259
miR-128a Alzheimer‘s disease upregulation 260
miR-298 Alzheimer‘s disease downregulation 261
miR-328 Alzheimer‘s disease downregulation 261
miR-146a Alzheimer‘s disease upregulation 262
miR-133b Alzheimer‘s disease downregulation 263
miR-19 Spinocerebellar ataxia type 1 downregulation 264
miR-101 Spinocerebellar ataxia type 1 downregulation 264
miR-130 Spinocerebellar ataxia type 1 downregulation 264
miR-9 Hungtington‘s disease downregulation 265,266
miR-1 Tourette syndrome deregulation 267
miR-206 Tourette syndrome deregulation 267
miR-21 Glioblastoma upregulation 268
miR-124 Glioblastoma downregulation 269
miR-137 Glioblastoma downregulation 269
miR-124a Medulloblastoma downregulation 270
miR-34a Neuroblastoma downregulation 271
miR-184 Neuroblastoma downregulation 272
miR-15a Pituitary adenoma downregulation 273-275
miR-16 Pituitary adenoma downregulation 273-275
miR-221 Glioblastoma upregulation 268,276-278
miR-128 Glioblastoma upregulation 268,276-278
miR-181a Glioblastoma upregulation 268,276-278
miR-181b Glioblastoma upregulation 268,276-278
miR-181c Glioblastoma downregulation 268,276-278
miR-9, miR-125a Medulloblastoma downregulation 279

primary human breast carcinomas correlated with clinical
progression226. These findings, if confirmed, suggest that
specific miRNAs may have a role beyond the tumor-initiating
event and directly participate in tumor progression and
metastasis.
4.7 MicroRNA profiling—implications for cancer
diagnosis:
Lu et al227 asked the question whether global miRNA
expression profiles could classify human cancer. Micro-RNA
expression profiles clearly differentiated human cancers
according to their developmental origin. Cancers of epithelial
and hematopoietic origin had distinct miRNA profiles. A
subgroup of gastrointestinal tumors, which arise from
endoderm, was distinguished by miRNA expression patterns.
Furthermore, tumors within a single cell lineage such as acute
lymphoblastic leukemia were further differentiated according
to their underlying genetic abnormality into BCR/ABL-
positive tumors, T-cell tumors, and those with MLL gene
rearrangement227. Finally, the authors applied the miRNA
expression profiles they had established to an independent
series of 17 poorly differentiated tumors of unknown origin.
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22
Based on the differential expression of 217 miRNAs, a
correct diagnosis could be established in 12 out of 17 of the
tumors. In contrast, gene expression profiling based on
16,000 messenger RNAs did not accurately classify the
tumors227. This has potential important clinical implications.
If miRNAs prove useful for clinical diagnosis, their key
advantage might be their high stability. In contrast to most
messenger RNAs, they are long-lived in vivo228 and very
stable in vitro229, which might allow analysis of paraffin
embedded samples for routine diagnostic applications.
4.8 Regulation of miRNAs in cancer—who regulates the
regulators?
In few cases, the underlying cause of miRNA deregulation in
cancer is clear.The over-expression of miR-17-92 correlates
with amplification of its gene locus230. Similarly, decreased
expression of miR-15 and miR-16 is associated with a
corresponding chromosomal deletion231. Transcriptional or
epigenetic regulation of miRNAs has been recently reported
232,233.The transcription of a miRNA gene, mir-124a, was
shown to be inactivated by hyper-methylation of its promoter
in various human tumors. This process of epigenetic silencing
is a well-known mechanism to inactivate protein-coding
genes in cancer cells and may similarly apply to miRNAs.
The miRNA gene mir-127 is usually expressed in normal
cells but not in cancer cells. Saito et al.233 demonstrated that
miR-127 was highly induced in cultured human cancer cells
after treatment with demethylating drugs, suggesting that it is
subject to epigenetic silencing through promoter hyper-
methylation. A novel mechanism of miRNA regulation was
suggested by Mayr et al.234 and Lee and Dutta235. They
demonstrated that miRNA function could be regulated
through loss of miRNA binding sites in the target gene. Both
groups independently demonstrated that chromosomal
translocations in a known oncogene, high mobility group A2
(Hmga2), led to loss of the let-7 miRNA binding sites in its
messenger RNA. Disrupted repression of Hmga2 by let-7
promoted oncogenic transformation and growth in
mammalian cells. These two studies provide the first
evidence that disrupting the interaction of a single miRNA
and its target can produce an abnormal phenotype in
mammalian cells. In addition, there is evidence that miRNAs
are regulated indirectly through control of their processing
enzymes. Thomson et al236 showed that a down regulation of
miRNAs in human cancer was not associated with reduced
levels of the primary miRNA transcripts. The authors
therefore suggested regulation of miRNAs during subsequent
processing steps, e.g., through altered function of the enzyme
Drosha236.
5. miRNA and Neurodegenerative Diseases:
Neurodegenerative diseases result from dysfunction,
progressive deterioration, and extensive loss of neurons in the
central and/or peripheral nervous system237,238. In this
regard, Alzheimer‘s disease (AD)239-242,Parkinson‘s
disease (PD)243-245, prion diseases246, amyotrophic lateral
sclerosis (ALS)247-248, and hereditary spastic paraplegia249
may have a genetic or sporadic etiology. Instead,
Huntington‘s disease (HD) 250, 251 and metabolic disorders
with neurological involvement, such as the GM2-
gangliosidoses252-257, can only be genetically transmitted.
There is now compelling evidence that disregulation of
miRNA networks is implicated in the development and onset
of human neurodegenerative diseases258 (Table-3). This, in
turn, may provide the opportunity to elucidate underlying
disease mechanisms and open up novel strategies for
therapeutic applications.
5.1 Alzheimer’s Disease (AD) :
AD is the most common form of dementia. While several
hypotheses have been proposed to explain the disease‘s
etiology, the causes of AD and means of stopping its
progression are still elusive matters 239-242,280-284.
Features of the disease encompass neuronal loss,
intraneuronal neurofibrillary tangles (i.e., aggregates of the
microtubule-associated protein tau following hyper-
phosphorylation), and extracellular deposits of amyloid
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
23
plaques (i.e., deposits of Aβ-peptide) 280-284. Only 10%–
15% of AD cases represent an inheritable disease which
follows an autosomal dominant Mendelian pattern, while the
majority arises sporadically. Apparently, the disease may be
caused by a genetic predisposition, as shown by the
identification of specific DNA mutations in a large number of
families239, 281-284. Despite a variable etiology, a common
pathogenetic cascade resulting from distinct gene defects
and/or unknown environmental factors cannot be ruled out.
For example, accumulation of the Aβ peptide, the cause of
which is unknown, is consistently observed. In approximately
30% of sporadic AD patient samples, the expression of
BACE1 protein, a secretase associated with the formation of
Aβ-peptide, is significantly increased285.In AD, several
miRNAs exhibit abnormal expression levels, suggesting a
dysfunctional orchestration of gene expression 259, 260,
286,287. Interestingly, Boissonneault et al. have found that
miR-298 and miR-328 bind to the 3‘-UTR of BACE1
mRNA, thereby producing a regulatory effect on enzyme
expression in cultured neuronal (N2a) cells. Presence of both
miR-298 and miR-328 in the hippocampus of APPSwe/PS1
mice, a well-documented model for AD, and the observation
that their levels of expression decrease with aging suggest
that altered levels of these miRNAs may deregulate BACE1
and, in turn, lead to increased Aβ formation and disease
progression261. Finally, a recent work from Carrettiero et al.
shows that miR-128a regulates the cochaperone BAG2 and,
in turn, a pathway of degradation for microtubule-associated
tauproteins with a propensity for misfolding. BAG2 would
normally direct tau toward an ubiquitin-independent pathway
and selectively reduce the levels of sarkosyl-insoluble
protein288. Thus, the observation that miR-128a is
upregulated in AD260 may highlight a molecular mechanism
that underlies tau inclusions in neurodegeneration. Taken
together, these findings suggest a mechanistic involvement of
miRNAs in both the amyloid and tau hypotheses for AD
pathogenesis.
5.2 Parkinson’s Disease (PD) :
PD is the second most common neurodegenerative disorder,
characterized by resting tremor, muscular rigidity,
bradykinesia, and impaired balance and coordination243-
245,289-291. Other symptoms include dysautonomia,
dystonic cramps, and dementia. Typical pathological features
are loss of dopaminergic neurons in the substantia nigra (SN)
and presence of Lewy bodies, which consist of intracellular
inclusions affecting surviving neurons in various areas of the
brain289-291. Several gene loci have been implicated in
autosomal, dominant forms of PD. These include PARK1 and
PARK4 (due to a mutation or a triplication of the α-synuclein
gene [SNCA] on 4q21 and 4p15, resp.), PARK3 on 2p13,
PARK5 (due to a mutation in the UCHL1 gene) on 4p14,
PARK8 (due to a mutation in the LRRK2 gene) on 12q12,
PARK10 on 1p, PARK11 on 2q, and PARK13 (due to a
mutation in the HTRA2 gene) on 2p12289-291,292,293. The
implication of miRNAs in PD is intriguing. In murine
models, the competence of embryonic stem cells to
differentiate into midbrain dopamine neurons in vitro was
shown to be disrupted by Dicer deletion and subsequent
suppression of miRNA biogenesis, suggesting a physiological
role for miRNAs in cell differentiation and/or survival. These
results were confirmed in vivo, using mice conditional for
Dicer, which exhibited impaired locomotor activity that
recapitulated motility problems observed in PD
patients.Through a subtractive approach, performed by
comparing miRNA expression profiles in normal human
adult versus PD patients midbrains, it was shown that miR-
133b is specifically missing in PD and that, based on both
over-expression and inhibitory tests in vitro, is likely
implicated in the maturation and function of dopaminergic
neurons294, 263. A markedly reduced expression of miR-
133b was found in Aphakia mice294, a dopaminergic neuron
deficiency model, which lack Pitx3295, a homeobox
transcription factor required for neuron survival and normal
motor activity susceptible to polymorphisms associated with
sporadic PD296. Together, these observations suggest a
relationship between miR-133b and Pitx3, which operate
through a negative feedback loop, wherein Pitx3 promotes
the expression of miR-133b that, in turn, downregulates
Pitx3263. While these results point to a functional role of the
miR-133b/Pitx3 system in ensuring correct dopaminergic
function, miR-133b knock-out mice, which are currently
unavailable, would establish the extent of miR-133b impact
on PD etiology. On the other hand, another study showed that
deletion of Dicer in dopaminoceptive neurons of the murine
striatum led to aberrant anatomical features (smaller brain,
reduced neuron size, astrogliosis) and motor impairments
(clasping and ataxia) but, surprisingly, not
neurodegeneration297. As dysfunction, but not necessarily
loss, of dopaminoceptive neurons was previously implicated
in PD298, these observations, taken together, suggest that the
link between Dicer, miRNAs, and neurodegeneration is
restricted to dopaminergic neurons, thereby pointing to
distinct functional roles in dopaminoceptive cells. Finally,
Wang et al. found that in PD brains and in vitro cell models
disruption of the binding site for miRNA- 433 led to
increased translation of fibroblast growth factor- 20 (FGF20).
Notably, an FGF20 polymorphism at 8p21.3–22 was
previously identified as a PD risk factor correlated with
increased α-synuclein expression, and consequently PD
onset299.
5.3 Huntington’s Disease (HD):
HD is a fatal, neurodegenerative disorder characterized by
involuntary ballistic movements, depression, and
dementia250, 251,300. Hallmarks of HD are progressive
chorea, rigidity, and frequent accurrence of seizures,
emotional problems, loss of cognition, as well as atrophy of
the caudate nucleus. The causal factor of HD is a gene
mutation consisting of abnormally extended repeats of the
CAG sequence within the HTT gene, which translates into a
huntingtin protein containing an excessively increased
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
24
glutamine segment250, 251, 300.Disruption of miRNA
homeostasis, most likely in connection with an aberrant
functionality of the transcriptional repressor REST, was
shown to play a dynamic role in HD. In fact, levels of several
miRNAs with upstream RE1 sites are decreased in HD
patient cortices relative to healthy controls. Interestingly, one
of these, the bifunctional, brain enriched miR-9/miR-9 targets
two components of the REST complex: miR-9 targets REST
and miR-9 targets CoREST265, 266. As a consequence of a
markedly altered miRNA expression, target mRNAs are
subject to dysregulated levels which, in turn, affect the
physiological status of forebrain neurons265, 266
6. MicroRNAs as an antiviral defense mechanism:
Viruses are obligate intracellular parasites and use the cellular
machinery for their survival and replication. The success of
the virus essentially depends on its ability to effectively and
efficiently use the host machinery to propagate itself. This
dependence on the host also makes it susceptible to the host
gene-regulatory mechanisms. Though the gene regulatory
mechanisms involving both host and viral proteins have been
extensively studied, data on small RNA mediated gene
regulation in viral infections is just emerging. Interestingly, it
seems that the cellular microRNAs, in addition to their
normal regulatory roles in cellular gene expression also
double up as fortuitous agents that target foreign nucleic
acids, as in the case of viruses. The inventory of microRNAs
encoded differs between cell types, and thus may contribute
to the tissue tropism of viruses
6.1 Primate Foamy Virus:
Lecellier et al309, for the first time demonstrated that a
mammalian microRNA, mir-32 restricts the accumulation of
the retrovirus primate foamy virus type 1 (PFV-1) in human
cells. PFV is a retro-transcribing virus similar to the Human
Immunodeficiency Virus (HIV), but codes for two additional
proteins, Tas and Bet. Insights into the possible role of
microRNAs came from the observation that cell lines which
express a protein, which interferes with the RNA mediated
silencing machinery, showed higher accumulation of PFV-1.
Disruption of the target site in a mutant of PFV allowed it to
accumulate much faster than the wild type, in infected cells.
The group also demonstrated that Tas could act as a non-
specific suppressor of RNA interference and could
demonstrate that mir-32 related translational block was
indeed higher in Tas(-)cells where Tas was not expressed.
This report not only throws light into the role of microRNAs
in antiviral defense, but also into how viruses have evolved to
offset the effects of RNA interference by encoding
suppressors of interference.
6.2 Human Immunodeficiency Virus:
Five human encoded microRNAs can potentially target the
entire repertoire of accessory genes in HIV, including nef.
The targets were found to be highly conserved in all of the
viral clade sequences with the exception of clade O. The fact
that defective nef is well known to be associated with a long
term non-progressor state led us to speculate that the levels of
the cellular microRNAs would be a decisive factor in
determining the progression of the disease. The targets have
been experimentally validated (unpublished results) by
cloning the target site in the 3'UTR of Green Fluorescent
Protein (GFP) reporter gene. Analysis of previously reported
microarray data310 of these microRNAs in T Cells,
demonstrated that the microRNA levels are indeed variable
among individuals. Although it is believed that HIV encodes
for suppressors of RNAi311 recent microarray data on
microRNA gene expression levels in HIV infected human
cells312 show that above five human encoded microRNAs
are down regulated.
6.3 Mammalian microRNAs as positive regulators
Hepatitis C virus
Jopling et al313 reported an interesting case wherein the
tissue specificity of microRNA expression was exploited by a
virus to establish tissue selectivity. A liver specific micro-
RNA mir-122 was shown to cause accumulation of viral
RNA by binding to the 5' non-coding region of the viral
genome. The authors have also verified the findings by
mutational analysis as well as sequestration of the micro-
RNA. It is possible that this novel mechanism of microRNAs
targeting the 5'UTR of the transcript may be mediated
through a translation controlling switch at the 5'end of the
transcript through changes in RNA secondary structure. This,
by and large, remains the only report of a microRNA
targeting the 5'UTR in a mammalian system and causing
RNA accumulation.
6.4 Influenza virus:
Through computational methods incorporating both
consensus prediction and target accessibility, one group
found that human encoded microRNAs could target critical
genes involved in the pathogenesis and tropism of Influenza
virus A/H5N1 (unpublished results). Two human encoded
microRNAs mir-507 and mir-136 had potential binding sites
in Polymerase B2 (PB2) and Hemagglutinin (HA) genes
respectively. The target regions in the respective genes were
not only found to be conserved across different viral strains,
but were also found to fall in highly accessible regions of the
predicted target RNA structure. Moreover, analysis of
previously reported314 microarray data on microRNA gene
expression in different tissues has shown that mir-136 is
expressed in lung. Both the genes PB2 and HA are known to
be critical for the pathogenicity of the virus. While HA is the
surface glycoprotein involved in direct binding of the virus to
the cell surface, HA in the H5N1 subtype carries a polybasic
site, cleavage at which, by cellular proteases is an essential
step in establishing infection. PB2 is one of the three
components of the Ribonucleoprotein which is responsible
for RNA replication and transcription. Recent evidence, from
recombinant viruses generated by combinations of murine
and avian viruses identified PB2 as one of the two genes
International Journal of Research in Biotechnology and Biochemistry 2011; 1(1) : 11-36
25
associated with virulence. The polymerase activity was
directly correlated with the high virulence of the murine
strain in their cognate host315. Another interesting feature is
that these microRNAs were found to be absent in the chicken
genome, although a large number of human microRNAs (160
of 336 human microRNAs) have homologs in the chicken
genome implicating them in the difference in infectivity and
lethality of the virus in chicken and human
7. MicroRNAs—novel therapeutic targets?
Regulatory RNAs may also have therapeutic applications by
which disease-causing miRNAs could be antagonized or
functional miRNAs restored. The most intuitive choice of
molecules to correct altered miRNA–mRNA interactions are
RNA oligonucleotides. These oligonucleotides need to be
chemically modified to allow for stability in serum and
cellular uptake. Modified antisense oligonucleotides are
already being developed to utilize the intrinsic RNAi pathway
for delivery of gene therapy. If the delivery problem can be
overcome, then miRNA therapies may also be possible. Two
studies have successfully applied 2′-O-Methylmodified
antisense RNAs to inhibit miRNA function in cultured
cells316, 317. Recent work by Krutzfeldt et al.318
demonstrated that modified cholesterol-conjugated antisense
RNAs designated ―antagomirs‖ could effectively inhibit
miRNA function in vivo in the adult mouse. The authors
applied three daily intravenous injections of antagomirs and
achieved effective inhibition of four miRNAs over a period
of weeks in most tissues except brain. A novel approach was
recently reported by Ebert et al 319. They developed miRNA
inhibitors that can be transiently expressed in cultured
mammalian cells.These competitive inhibitors termed
―miRNA sponges‖ derepressed miRNA targets at least as
strongly as chemically modified antisense
oligonucleotides319. A different approach was taken by
Tsuda et al.320. The authors designed synthetic miRNAs to
target overexpressed tumor proteins, such as HER-2 protein.
A synthetic miRNA targeting HER-2 messenger RNA
successfully inhibited HER-2 protein expression in ovarian
cancer cells320. Together, these studies hold some promise of
miRNAs as future therapeutic targets. One limitation of
antisense RNA therapies is the restricted number of cells that
can be targeted. Any approach to knockdown a particular
miRNA with antisense oligonucleotides will only result in
partial knockdown. This may represent a limitation for cancer
therapies. It remains to be seen whether indirectly mediated
bystander effects on cancer cells that have not been directly
targeted may partly overcome this limitation. In contrast, a
partial effect on function may be of therapeutic value in
neurodegenerative diseases, such as Parkinson‘s or
Alzheimer‘s disease. A partial restoration of dopamine
production by antisense therapy might result in a significant
clinical improvement in Parkinson patients. Similarly, a
partial reduction of the disease-causing proteins in
Alzheimer‘s disease may lead to a clinical improvement and
might be achievable by RNA based or miRNA gene therapy.
8. MicroRNAs as biomarkers and therapeutics : complex regulatory networks, thereby mediating many facets
Recently microRNA expression profiles have been analyzed of eukaryotic cell function. As Bartel and Chen proposed, the
for viral infections like HIV321. MicroRNA expression has unique combination of miRNAs expressed in each cell type
been shown to be specific to various stages of infection in might affect or ‗dampen‘ the use of thousands of target
Herpesviruses322 and have been proposed to be associated mRNAs. Supporting this hypothesis, the experimental
with latency in HIV infection323, 324, promising an early introduction of muscle- or brain-specific miRNA into HeLa
biomarker for cancers caused by oncogenic viruses. cells shifted the overall mRNA profile to that of muscle or
MicroRNA profiles have also been explored in a number of brain, respectively. Tantalizing correlations between miRNA
patho-physiological conditions325.Recent reports suggest expression and human diseases have been demonstrated.
that microRNA profiles can be used not only to classify Certain miRNAs are modulated in tumors and might be
different classes of cancers326-328, but could also be used as directly involved in the pathology. Understanding the
biomarkers for diagnosis and prognosis of disease states328. miRNA-guided network will provide a new window for
MicroRNAs and anti-microRNA oligonucleotides (AMOs) diagnostics and therapy of many human diseases.
have been proposed as novel therapeutics329,330. Recent
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