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Birth Defects Research (Part C) 78:180 –189 (2006)
MicroRNAs: Fundamental Facts and Involvement
in Human Diseases
Gianpiero Di Leva, George A. Calin, and Carlo M. Croce*
MicroRNAs (miRNAs) are a group of small noncoding RNAs that have been
identified in a variety of organisms. These small, 18 –22-nucleotide (nt)
RNAs are transcribed as parts of longer molecules called pri-miRNAs,
which are processed in the nucleus into hairpin RNAs of 70 –100 nt, called
pre-miRNAs, by the double-stranded RNA (dsRNA)-specific ribonuclease
Drosha. The function of most miRNAs is not known, but for a few members
the participation in essential biological processes for the eukaryotic cell is
proven. In this review, we summarize how miRNAs were discovered, their
biological functions, and importance in animal development, highlighting
their function in proliferation, apoptosis, and cell differentiation.
Furthermore, we discuss the deregulation of miRNAs in human diseases
and their involvement in tumorigenesis. Birth Defects Research (Part
C) 78:180 –189, 2006. © 2006 Wiley-Liss, Inc.
DISCOVERY OF microRNAs
The discovery of microRNAs (miRNAs) began in early 1981 when
Martin Chalfie et al. (1981), during
a loss-of-function study in C. elegans, revealed that mutations in
the lin-4 gene lead to continued
synthesis of larval-specific cuticles.
At that time, together with lin-14,
lin-29, and lin-28, these genes
were classified as heterochronic
genes, capable to control the timing
of specific postembryonic developmental events in C. elegans. Seven
years later, Victor Ambros (1989)
described, in hypodermal cells, an
interaction hierarchy of heterochronic regulatory genes to coordinate the “larva-to-adult switch.”
These experiments have shown
that, in the early stages of development, lin-14 and lin-28 inhibit lin29, preventing the switch; next,
lin-4 inhibits lin-14 and lin-28, triggering the activation of lin-29 and
the following switch in the L4 larval
stage (Ambros, 1989).
Lin-14 has been shown to encode
a nuclear protein that is normally
present in most somatic cells of late
embryos and L1 larvae, but not in
the later larval stages or adults.
Gary Ruvkun et al. (1991) found
that two lin-14 gain-of-function
mutations lead to an abnormal protein accumulation in the later larval
stages; these mutations delete the
3⬘ untranslated region (UTR) of
lin-14 mRNA, highlighting a regulatory element in the UTR that controls the temporal gradient of the
protein. Since lin-4 was described
to downregulate the temporal levels of lin-14 protein, the authors
proposed that the lin-4 gene product could be the trans-acting factor,
capable of binding the 3⬘UTR of
lin-14 and negatively regulating it.
Finally, in 1993, two independent
studies, published in the same issue of Cell by Lee et al. (1993) and
Wightman et al. (1993), presented
the real nature of the lin-4 gene and
its ability to regulate heterochronic
gene expression. After cloning the
lin-4 gene, they demonstrated that
the potential open reading frame of
lin-4 does not encode for a protein;
they identified two small lin-4 transcripts of approximately 22 and 61
nt and found that the 3⬘UTR of
lin-14 mRNA contains sequences
complementary to lin-4. These data
suggested that the temporal regulation of lin-14 is driven by lin-4
RNA through antisense RNA-RNA
interactions, involving the small
RNA lin-4 and the 3⬘UTR of lin-14,
whose translation was inhibited.
Seven years later, Reinhart et al.
(2000) showed that the let-7 gene
is another heterochronic switch
gene coding for a small 21-nt RNA,
with complementary sequence to
the 3⬘UTR of lin-14, lin-28, lin-41,
lin-42, and daf-12; they proposed
that the sequential stage-specific
expression of let-7 and lin-4 RNAs
was capable through an RNA–RNA
interaction with the 3⬘UTR of the
target genes to trigger the temporal cascade of regulatory heterochronic genes specifying the timing
of C. elegans developmental events
(Reinhart et al., 2000).
At that time, these discoveries
were considered as a new piece in
the complicated gene expression
regulation puzzle restricted to the
small temporal RNA (stRNA) let-7
and lin-4 in worms. This idea was
completely changed when independent groups tried to investigate
whether RNAs similar to stRNA
Gianpiero Di Leva, George A. Calin, and Carlo M. Croce are from the Department of Molecular Virology, Immunology, and Medical
Genetics and Comprehensive Cancer Center, Ohio State University, Columbus, Ohio.
Grant sponsor: National Cancer Institute Program Project Grants (to C.M.C); Grant sponsor: Kimmel Foundation Scholar award (to
G.A.C.); Grant sponsor: CLL Global Research Foundation (to G.A.C).
*Correspondence to: Carlo M. Croce, Ohio State University, Comprehensive Cancer Center, Wiseman Hall Room 385K, 400 12th Avenue,
Columbus, OH 43210; E-mail: [email protected]
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bdrc.20073
© 2006 Wiley-Liss, Inc.
MicroRNAs: FACTS & INVOLVEMENT IN HUMAN DISEASES 181
could play a more general role in
gene regulation (Lagos-Quintana et
al., 2001; Lau et al., 2001; Lee and
Ambros, 2001). Cloning the RNA
from different organisms and cellular systems, by using the same
strategy applied to clone small interfering RNA (siRNA) processed
from exogenous double-stranded
RNAs (dsRNAs) in an embryo lysate, researchers in three laboratories were able to isolate a new class
of RNAs with the same characteristics of lin-4 and let-7, providing evidence for the existence of a large
class of small RNAs with potential
regulatory roles. Because of their
small size, the authors referred to
these novel RNAs as microRNAs
(miRNAs), a new abundant class of
riboregulators that could regulate
the gene expression at posttranslational level by base-pairing the
3⬘UTR of mRNA targets. After this
discovery, a new challenge for the
researchers was to define the biological function and the potential
target genes of these new genes.
miRNAs: GENOMICS,
BIOGENESIS, AND
MECHANISM OF ACTION
How Many miRNAs Are
Enough?
At the beginning, the majority of
miRNAs were identified by direct
cloning of small RNAs (Bartel,
2004); this approach had constriction because it permitted just the
detection of abundantly expressed
miRNAs. Three observations suggested that miRNAs identification
could be facilitated using computational approaches. First, miRNAs
are produced from a precursor
transcript of 70 –100 nt with a extended stem-loop structure. Second, miRNAs are usually highly
conserved between the genomes of
related species. Third, miRNAs display a characteristic pattern of evolutionary divergence. In accordance with these criteria, many
computational procedures have
been developed to predict miRNAs
in the genome of different organisms, such as miRNAscan (http://
genes.mit.edu/mirscan) in humans,
miRNaseeker (http://www.fruitfly.
org/seq_tools/miRseeker.html)
in
Drosophila, or others in C. elegans
(for review see Bentwich, 2005;
Brown and Sanseau, 2005). The sensitivity of these bioinformatic approaches was demonstrated by the
presence of a high percentage of previously experimentally identified
miRNAs within the top predicted candidates and by confirmation using
Northern blotting analysis and a
more sensitive PCR method (Schmittgen et al., 2004). The estimate of
miRNA genes in the metazoan genome represents nearly 1% of the
predicted genes, a fraction similar to
that seen for other very large gene
families with regulatory roles, such
as those encoding transcription-factor proteins.
In 2003, the rapid growth of the
number of miRNA genes led Sam
Griffiths-Jones from the Wellcome
Trust’s Sanger Institute to create a
comprehensive and searchable database of published miRNA sequences via a web interface: The
miRNA Registry (http://microrna.
sanger.ac.uk/sequences)
(Griffiths-Jones, 2004). The primary
aims of this registry are two-fold.
The first is to avoid inadvertent
overlap by assigning unique names
to distinct miRNAs. The miRNAs are
annotated with numerical identifiers based on sequence similarity;
for example, if a standard name is
“miR-15,” the next miRNA without
similarity will receive the name
“miR-16” For homolog miRNAs in
different organisms, it is usual to
assign the same name on the similarity of the 22-nt mature sequence. Identical mature forms are
assigned the same name and, if
they are produced from different
genomic loci, they are differentiated by suffixes such as “miR-16-1”
and “miR-16-2.” Differences in one
or two bases are identified by suffixes, such as “miR-181a” and
“miR-181b.” If an miRNA hairpin
precursor gives rise to two mature
miRNAs, one from each arm, the
rule is to denote miRNAs in the form
“miR-142-5p” (5⬘ arm) and “miR142-3p” (3⬘ arm) until the data will
confirm which form is predominantly expressed; in such cases,
the species less expressed will be
identified by using an asterisk (such
as “miR-191*”). The second aim of
the miRNA registry is to provide a
database for all miRNAs sequences,
including the stem-loop structure,
with the highlighted miRNA in red,
genomic location, homologous sequences, and possible target predictions.
At the beginning of 2005, a phylogenetic shadowing study on miRNAs in primate species revealed a
characteristic conservation profile
of miRNAs genes that the authors
have used to efficiently detect 83%
of the known miRNAs and predict
an extensive set of novel miRNAs,
opening the possibility that as
many as 1000 miRNAs may exist in
the human genome (Berezikov et
al., 2005). In version 8 (February
2006), the miRNA Registry contained 3518 entries from 40 organisms including viruses and mammals; there were 332 human
miRNAs and the number is continuously expanding (Griffiths-Jones et
al., 2006).
Genomics of miRNAs
Almost 50% of mammalian miRNAs
are located in introns of protein encoding genes or long noncoding
RNA (ncRNA) transcripts, whereas
the remaining part is considered as
independent transcription units
with specific promoter core elements and polyadenylation signals
(for review see Pasquinelli, 2002;
Cullen, 2004; Kim and Nam, 2006).
Among the intragenic miRNAs,
40% are found in introns of protein
encoding genes, whereas ⬃10%
are located in introns of long ncRNA
transcripts. The vast majority of
miRNA clusters are single transcription units or overlapped in the same
host transcripts, within exons or introns, and in some cases depend on
alternative splicing of the host
gene, implying that they are polycistronic transcripts. Additionally,
many miRNAs overlap with two or
more transcription units transcribed on opposite DNA strands.
The analysis of the genomic loci
of miRNAs demonstrates that host
genes encoding proteins are involved in a broad spectrum of biological functions, ranging from embryonic development to the cell
Birth Defects Research (Part C) 78:180 –189, (2006)
182 DI LEVA ET AL.
cycle and physiology. When the
miRNA host genes are classified for
gene ontology (GO) “biological process,” the two most commonly
identified biological processes are
“metabolism” and “cellular physiological process,” whereas the classification for GO “molecular function” identifies “purine nucleotide
binding” and “DNA binding” proteins. Remarkably, several host
genes are involved in human disease: the Chloride Channel Protein
5 (CCP5) gene is involved in Dent
disease and nephrolithiasis, an Xlinked recessive disorder (Frymoyer et al., 1991); this gene hosts
miR-188, but a causative role in
this disease for miR-188 has not yet
been explored.
In addition to the miRNAs located
in protein coding genes, a large
group of miRNAs resides in transcripts that lack a significant protein-coding potential, classified as
long ncRNAs. These types of ncRNA
transcripts are sometimes referred
to as mRNA-like ncRNAs (mlncRNA)
because they are spliced, polyadenylated, and also spatio temporally
expressed. Deleted in Leukemia 2
(DLEU2) and BIC are host-genes
mlncRNAs, for miR-15a/16-1 cluster and miR-155, respectively (Calin et al., 2002; Eis et al., 2005).
The maturation of miRNAs is a
very complex process, and in the
following sections we will try to illustrate the machinery that the cell
needs to activate the intricate multistep processing from nucleus to
cytoplasm, required for the production of miRNAs.
Transcription and
Maturation of miRNAs
Initially, the researchers believed
that miRNAs were transcribed by
RNA polymerase III like other small
RNAs, and as some transfer RNAs
(tRNAs). However, numerous evidence supported the possibility of a
transcription mediated by RNA
polymerase II. Several polyadenylated transcripts, long kilobases and
comprising microRNAs, have been
identified in expressed sequence
tag (EST) analysis (Tam, 2001;
Smalheiser, 2003; Bratcht et al.,
2004), and the expression profiles
of miRNAs and host genes evidenced an elaborate expression
control, typical for genes transcribed by RNA polymerase II. In
2004, three direct pieces of evidence were reported to evaluate
the strict correlation between miRNAs and polymerase II: 1) the
miRNA transcripts are capped and
polyadenylated; 2) the transcription of miRNAs is sensitive to
␣-amanitin at the specific concentration for polymerase II inhibition;
and 3) the promoter region, responsible for miRNA transcription,
is associated with the polymerase II
complex (Kim and Nam, 2006; Lee
et al., 2004).
Animal miRNAs are identified as
part of an 80-nt RNA with stemloop structures (pre-miRNA) that
are included in several hundred or
thousands of nucleotide-long miRNAs precursor, named primary
miRNA precursor (pri-miRNA) (Fig.
1). Until now, a few different primiRNA precursors have been isolated and characterized, three from
human, one from C. elegans, and
one from plants. They are all
capped, polyadenylated, and apparently noncoding: the human
cluster miR-23a⬃27a⬃24-2 primary precursor is an unspliced
⬃2.2 kb RNA; in contrast, the primiRNA for human miR-155 (BIC)
includes two introns and two alternative polyadenylation sites capable of producing two spliced primiRNAs of 0.6 and 1.4 kb (Cullen,
2004).
The production of miRNAs from
pri-miRNA is a complex and coordinated process where different
groups of enzymes and associated
proteins, located in the nucleus or
cytoplasm, operate the multistep
maturation of these tiny RNAs.
Principally, the maturation process
of miRNAs can be resumed in three
important steps: cropping, export,
and dicing.
In the cropping step, the primiRNA is converted to pre-miRNA
through the cleavage activity of the
Drosha enzyme, a nuclear Ribonuclease III endonuclease capable of
cropping the flank regions of primiRNA, in turn to liberate the 60 –
70-nt pre-miRNA (Bartel, 2004).
Different structural requisites are
Birth Defects Research (Part C) 78:180 –189, (2006)
needed to achieve efficient precursor maturation by Drosha: first, a
large terminal loop (⬎10 nt) in the
hairpin and a stem region one turn
bigger than the pre-miRNA; second, 5⬘ and 3⬘ single-stranded RNA
(ssRNA) extensions at the base of
the future miRNAs (for review see
Filipowicz et al., 2005; Tomari and
Zamore, 2005). It has been proposed that Drosha may recognize
the primary precursor through the
stem-loop structure, and then
cleave the stem at a fixed distance
from the loop to liberate the premiRNA. How the enzyme is capable
of discriminating the pri-miRNA
stem-loop structure with respect to
the stem-loops of other cellular
RNAs is not clear, but probably proteins associated with Drosha confer
specificity to this process. In fact,
Drosha has been found as a part of
a large protein complex of ⬃650
kDa, which is known as the “Microprocessor,” where Drosha interacts
with its cofactor, the Di George syndrome critical region gene 8
(DGCR8) protein in human and Pasha in Drosophila melanogaster
(Landthaler et al., 2004). The Microprocessor appears to represent
a heterotetramer consisting of two
Drosha and two DGCR8 molecules;
because DGCR8 contains two consensus dsRNA binding domains,
this protein may play an important
role in substrate discrimination and
binding.
The resulting product of cropping, the pre-miRNA, presents a 5⬘
phosphate and 3⬘ hydroxy termini,
and two or three nucleotides with
single-stranded overhanging ends,
classic characteristics of RNase III
cleavage of dsRNAs. After the Microprocessor nuclear activity, the
produced pre-miRNA is exported
to the cytoplasm by Exportin-5
(Exp5)/RanGTP (Kim, 2004). Exp5
forms a nuclear heterotrimer with
RanGTP and the pre-miRNA from
Drosha processing. This interaction, which is dependent on RNA
structure but independent of sequence, stabilizes the nuclear premiRNA and promotes the export to
the cytoplasm. In the export step,
once the Exp5-RanGTP-pre-miRNA
complex has reached the cytoplasm
through the nuclear pore, the
MicroRNAs: FACTS & INVOLVEMENT IN HUMAN DISEASES 183
Figure 1. miRNA biogenesis and mechanisms of action. For details, see text pages 182 and 183.
RanGTP is hydrolyzed to RanGDP
and the pre-miRNA is released.
Following arrival into the cytoplasm, the pre-miRNA is processed
into 18 –22-nucleotide miR duplexes by the cytoplasmic RNase III
Dicer and, in humans, its partner
TRBP. The PAZ domain of Dicer is
thought to interact with the 3⬘ overhang nucleotides present in the
pre-miRNA hairpin, while the
dsRNA binding domain binds the
stem and defines the distance of
cleavage from the base of premiRNA. The cleavage products of
22-nt-long miRNA duplexes have a
reduced half-life. Normally, one
strand of this duplex is degraded,
whereas the other strand accumulates as a mature miRNA. Studies
on siRNAs have highlighted that the
selection of the right strand is related to the thermodynamic stability of the duplex, and the strand
with relatively unstable base pairs
at the 5⬘ end usually represents the
mature miR.
MiRNA in Action: RISC and
Gene Target Inhibition
In the RNA duplex produced from
the Dicer activity, the mature
miRNA is only partially paired to the
miRNA*, the small RNA that resides
on the opposite pre-miRNA stem.
From the miRNA-miRNA* duplex,
only the miRNA enters preferentially in the protein effector complex, the RNA-induced silencing
complex (RISC) or miRgonaute,
which mediates the degradation or
translation inhibition of mRNAs target gene (Tang, 2005).
Animal miRNAs are imperfectly
paired to the 3⬘ UTR of target mRNA
and inhibit the protein production
by an unknown and very controversial mechanism; in some cases, the
miRNAs show nearly precise complementarity to their target and trigger
mRNA degradation as siRNA in the
RNA interference process (Tang,
2005).
Several proteins have been identified as essential components of
RISC, but only a few have been
functionally characterized in the
posttranslational regulation. The
core components of RISC are members of the Argonaute (Ago) protein
family, whose members present a
central PAZ domain like Dicer and a
carboxy terminal PIWI domain.
This domain binds the miR/miR*
duplex to the 5⬘ end, whereas the
PAZ domain binds to the 3⬘ end of
ssRNAs; moreover, structural and
biochemical studies have suggested that the Ago proteins are the
target-cleaving endonucleases of
RISC, and in this activity the complex is helped and coordinated by
other proteins whose functions are
Birth Defects Research (Part C) 78:180 –189, (2006)
184 DI LEVA ET AL.
TABLE 1. Examples of functions of miRNAs in animals.
miRNA
Species
Function
Reference
lin-4
C. elegans
Regulation of life span (proportionally with expression)
MiR-273
MiR-1
C. elegans
Drosophila
Controls laterality of the chemosensory system
Control of muscle during larval growth
Human
Drosophila
Regulation of cardiogenesis
Suppress embryonic apoptosis by posttranscriptional
repression of proapoptotic factors
(Boehm and Slack,
2005)
(Chang et al., 2004)
(Sokol and Ambros,
2005)
(Zhao et al., 2005)
(Leaman et al., 2005)
Drosophila
Normal development of head and posterior abdominal
segments
Promotes photoreceptor differentiation
miR-2, miR-6, miR11, miR-13, miR308
MiR-2, miR-13
MiR-7
Drosophila
MiR-14
MiR-278
Drosophila
Drosophila
bantam
Drosophila
MiR-1-1
Mouse
MiR-122
miR-142s
Mouse
Mouse
MiR-181
Mouse
MiR-196
Mouse
miR-200a, miR-141,
miR-429, miR-199a
MiR-223
Mouse
Limb development, acting upstream of Hoxb8 and Sonic
hedgehog
Skin morphogenesis
Mouse
Myeloid differentiation
MiR-15a, miR-16-1
Human
Human
Regulation of granulopoiesis
Regulation of B lymphocytes survival
MiR-375
Human
Regulation of insulin secretion
Suppression of cell death; normal fat metabolism
Control of energy homeostasis by influencing insulin
production and adipose-tissue glycogen stores.
Controls cell proliferation and prevents apoptosis
Control of balance between differentiation and
proliferation during cardiogenesis
Regulator of cholesterol and fatty-acid metabolism
B lymphocyte differentiation;
Myeloid differentiation
B lymphocyte differentiation
Myoblast differentiation by targeting Hox-11
not really understood, such as RNAbinding protein VIG, the Fragile-X
related protein in Drosophila, the
exonuclease Tudor-SN, and many
other putative helicases (Nelson et
al., 2003).
In human cells, after transfection of miRNAs by vectors or
miRNA precursors, and subsequent activation of RISC activity,
the core component of RISC, together with the triggering miRNA
target mRNA, is concentrated in
cytoplasmic foci known as Processing bodies (P-bodies) or GWbodies. In accordance with this
triggered RISC localization, the
researchers thought that the miRNAs, in association with AGO proteins, might be capable of re-
pressing the translation at the
ribosomal level and to relocalize
the mRNA targets to the P-bodies
(Liu et al., 2005).
The most important characterization of the function of miRNAs is the
identification of the mRNA targets.
Because animal miRNAs have a 5⬘
end-restricted complementarity to
the mRNA target (only five to eight
nucleotides of perfect complementary, an RNA sequence named
“seed region”), the miRNAs are predicted to regulate a large number of
animal genes. Different algorithms
have been developed to predict the
animal miRNA targets; they are
based on different criteria, resulting from the analysis of targets
demonstrated in vivo: 1) perfect or
Birth Defects Research (Part C) 78:180 –189, (2006)
(Boutla et al., 2003)
(Li and Carthew,
2005)
(Xu et al., 2003)
(Teleman and Cohen,
2006)
(Brennecke et al.,
2003)
(Zhao et al., 2005)
(Esau et al., 2006)
(Chen et al., 2004)
(Chen et al., 2004)
(Naguibneva et al.,
2006)
(Hornstein et al.,
2005)
(Yi et al., 2006)
(Chen et al., 2004)
(Garzon et al., 2006)
(Fazi et al., 2005)
(Calin et al., 2002;
Cimmino et al.,
2005)
(Poy et al., 2004)
nearly perfect base-paring at the
seed region and thermodynamic
stability of the duplex miRNAmRNA; 2) phylogenetic conservation of the seed region; 3) multiple
target sites in a single target by the
same or different miRNAs; and 4)
absence of strong secondary structures at the miR-binding site of the
target. Several computational procedures are available to predict
miRNA targets, such as DianaMicroT
(http://www.diana.pcbi.
upenn.edu/cgi-bin/micro_t.cgi),
TargetScan (http://genes.mit.edu/
targetscan/), and miRanda (http://
www.microrna.org/miranda_new.
html) (Enright et al., 2003; Kiriakidou et al., 2004; Lewis et al.,
2005).
MicroRNAs: FACTS & INVOLVEMENT IN HUMAN DISEASES 185
TABLE 2. Examples of miRNAs involvement in human diseases.
Cancer types
miRNA involvement
Reference
Human leukemias and
carcinomas
⬇ 50% of miRNAs located in minimal
LOH/Amplified regions; miRNAs expression
profiles classify human cancers
Deletions and down regulation of miR-15a and
miR-16-1
(Calin et al., 2004b)
(Lu et al., 2005)
miRNA profiles associate with survival and
clinical parameters; Germline and somatic
mutations in miRNA genes; miR-15 and miR16 target BCL2
High expression of precursors for miR-155/BIC
(Calin et al., 2004a) (Calin et
al., 2005a) (Cimmino et
al., 2005)
Accumulation of miR-155 and BIC RNA; BIC and
miR-155 are highly expressed
Overexpression of pri-miRNA-17-92 cistron
miRNA profiles associate with survival and
clinical parameters
miRNA profiles associate with survival and
clinical parameters
Germline polymorphisms with possible
functional consequences in c-KIT oncogene
interactions sites with miRNAs.
Polymorphisms with possible functional
consequences in SLITRK1 interaction site with
miRNAs.
Loss of expression of the MPR protein, that
interacts with members of the miRNA pathway
Reduced expression of SMN protein, involved in
RNP complexes containing miRNAs
Loss of expression of DGCR8 protein, a Drosha
interactor in human cells
(Eis et al., 2005) (Kluiver et
al., 2005)
(He et al., 2005b)
(Yanaihara et al., 2006),
(Takamizawa et al., 2004)
(Iorio et al., 2005)
B cell chronic
leukocytic leukemia
(CLL)
Pediatric Burkitt’s
Lymphoma
B cell lymphomas
Human lymphomas
Lung cancers
Breast cancers
Thyroid cancers
Tourette’s syndrome
Fragile X syndrome
Spinal muscular
atrophy (SMA)
Di George syndrome
(Calin et al., 2002)
(Metzler et al., 2004)
(He et al., 2005a)
(Abelson et al., 2006)
(Jin et al., 2004)
(Mourelatos et al., 2002)
(Landthaler et al., 2004)
*NR - not reported; LOH - loss of heterozygosity; BIC gene - noncoding RNA transcript located at chromosome 21q21.3.
pre-miRNA155 is likely processed from a transient spliced or unspliced nuclear BIC transcript; FMPR - fragile X mental
retardation protein; SMN - Survival of Motor Neurons protein; RNP - ribo-nucleo-proteins; DGCR8 - Digeorge syndrome
critical region gene 8.
miRNAS: FUNCTIONS IN
NORMAL AND DISEASE
STATES
With the discovery of new members of the miRNA family on a daily
basis, it becomes evident that
these small genes must be involved in normal cellular homeostasis (Ambros, 2004; Bartel,
2004; Sevignani et al., 2006).
Furthermore, with the development of new techniques for genome-wide screening of miRNA
expression, abnormal levels of
miRNAs were identified in malignant cells with respect with
normal counterparts (EsquelaKerscher and Slack, 2006; Hammond, 2006; Calin and Croce,
2006).
The functions of miRNAs, initially
a “shadow” area of research, re-
vealed a general participation in every functional aspect of normal cells
in organisms with different degrees
of complexity. For example, in Drosophila, miR-14 suppresses cell
death and is required for normal fat
metabolism (Xu et al., 2003), while
bantam encodes a developmentally
regulated miRNA that controls cell
proliferation and regulates the proapoptotic gene hid (Brennecke et
al., 2003). As shown in Table 1,
participation of miRNAs in essential
biological processes has been consistently proven, such as cell proliferation control (miR-125b and let7), hematopoietic B-cell lineage
fate (miR-181), B-cell survival
(miR-15a and miR-16-1), brain
patterning (miR-430), pancreatic
cell insulin secretion (miR-375),
and adipocyte development (miR-
143) (for reviews see Harfe, 2005;
Miska, 2005; Hwang and Mendell,
2006).
As a consequence of extensive
participation in normal functions, it
is quite logical to ask the question if
abnormalities in miRNAs should
have importance in human diseases. The answer to this fundamental question is built on many
recent investigations, obtained
mainly from the study of human
cancers. As shown in Table 2, miRNAs and/or proteins involved in the
processing of miRNAs are involved
in various types of human diseases.
miRNAs can act both as tumor
suppressors and oncogenes (Esquela-Kerscher and Slack, 2006;
Hammond, 2006; Calin and Croce,
2006). Homozygous deletions (as
is the case for miR-15a/miR-16a
Birth Defects Research (Part C) 78:180 –189, (2006)
Figure 2. miRNAs as cancer players: mechanism of alterations. For details, see text pages 185 and 187. Modified with permission
from Calin et al. (2004b).
186 DI LEVA ET AL.
Birth Defects Research (Part C) 78:180 –189, (2006)
MicroRNAs: FACTS & INVOLVEMENT IN HUMAN DISEASES 187
cluster), and the combination mutation plus promoter hypermethylation or gene amplification (as is
the case of miR-155 or the cluster
miR-17-92) appear to be the main
mechanisms of inactivation or activation, respectively (He et al.,
2005b; Lu et al., 2005; O’Donnell et
al., 2005). Because of the small
size of miRNAs, loss-of-function or
gain-of-function point mutations
represent rare events (Calin et al.,
2005a). miRNAs activity can be influenced either by the reposition of
other genes close to promoters/
regulatory regions of miRNAs (as is
the case of miR-142s – c-MYC
translocation), or by the relocalization of an miRNA near other regulatory elements. The overall effects
in the case of miRNA inactivation is
the overexpression of target mRNAs, while miRNA activation will
lead to downregulation of target
mRNAs involved in apoptosis, cell
cycle, invasion, or angiogenesis
(Fig. 2).
To date, only a few miRNA::mRNA
interactions with importance for
cancer pathogenesis have been
proven (Calin et al., 2005b; Calin
and Croce, 2006). It was elegantly
demonstrated that the let-7 miRNA
family regulates RAS oncogenes
and that let-7 expression is lower in
lung tumors than in normal lung tissue, while RAS protein has an inverse variation (Johnson et al.,
2005). Furthermore, enforced expression of the miR-17-92 cluster
from chromosome 13q32–33, in
conjunction with c-myc, accelerates tumor development in a
mouse B-cell lymphoma model (He
et al., 2005b). Two miRNAs from
the same cluster, miR-17-5p and
miR-20a, negatively regulate the
E2F1 transcription factor, a gene
shown to function as a tumor suppressor in some experimental systems (O’Donnell et al., 2005).
Recently, an unexpected mechanism of miRNAs involvement in
human disease was identified.
Tourette’s syndrome (TS) is a neurologic disorder manifested particularly by motor and vocal tics, and
is associated with behavioral abnormalities. Sequence variants of a
candidate gene on chromosome
13q31.1 named SLITRK1 (Slit and
Trk-like 1) were identified in patients with TS. One of them, named
var321, found in two unrelated patients, was located in the 3⬘UTR
binding site for the miR-189 and
might affect SLITRK1 expression
(Abelson et al., 2006). This mechanism of abnormal miRNA::mRNA
interaction seems to be a general
one, as it was shown also in the
case of oncogene c-KIT. Three of
the highly overexpressed miRNAs
in thyroid cancers, miR-221, miR222, and miR-146, are predicted to
interact with the KIT oncogene
mRNA at two different sites. Tumors in which the upregulation of
these miRNAs was the strongest
showed dramatic loss of KIT, and in
half of the cases the downregulation was associated with germline
single nucleotide polymorphisms
(SNP) in the two recognition sites in
KIT for these three miRNAs (He et
al., 2005a).
One important proof for the functional importance of such abnormalities is represented by the reproduction of similar diseases in
mouse models with abnormal miRNAs expression. Recently, the first
example of a transgenic miRNA
mouse was published: as expected
by the overexpression of miR-155
in human lymphomas (Eis et al.,
2005; Jiang et al., 2006), the miR155 transgenic mice overexpressing the gene only in B cells, exhibit
a preleukemic pre-B cell proliferation in spleen and bone marrow,
followed by frank B cell malignancy
(Costinean et al., 2006). These
findings indicate that the role of
miR-155 is to induce polyclonal expansion, favoring the capture of
secondary genetic changes for full
transformation. This is an exciting
proof that deciphering miRNA alterations is important, and that miRNAs, as small as they are, represent
big culprits in human diseases.
ACKNOWLEDGMENTS
We thank Cinzia Sevignani for the
assistance with figures and Dimitri
Iliopoulos for the helpful comments
and discussions. We apologize to
many colleagues whose work was
not cited due to space limitations.
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