Disease Genes and Gene Regulation by microRNAs
Robert Roberts & Clifford J. Steer
Received: 31 March 2010 /Accepted: 5 April 2010 /Published online: 22 April 2010
# Springer Science+Business Media, LLC 2010
The approach to biology and genetics has been markedly
influenced by recent discoveries, namely, non-protein-
coding RNAs , the annotation of single nucleotide
polymorphisms (SNPs) by the HapMap Project , and
the assembly of these SNPs onto computerized chips
(microarrays) to pursue genome-wide association studies
, This issue discusses the application and progress of
these new discoveries as they relate to the biology and
genetics of disease.
The application of genetics to the pursuit of genes
responsible for single-gene disorders received a major boost
in the 1980s. The work of Nakamura et al.  and Murray
et al.  made available hundreds of highly informative
DNA markers consisting of short repeats that span the
human genome. These markers coupled with polymerase
chain reaction greatly accelerated mapping the chromosomal
location (locus) of genes responsible for disease. The past
two decades have been the golden age for single-gene
disorders. It is estimated there are 6,000 inherited single-
gene disorders, of which the genes for more than 2,000 have
been identified . However, single-gene disorders are rare
and have a prevalence of less than one tenth of 1%. In
contrast, diseases such as coronary artery disease (CAD),
the number one killer, are common yet have a large genetic
component. The technology to pursue the mapping of loci
associated with common diseases would have to wait.
These common diseases are polygenic with multiple genes
contributing to their genetic predisposition; thus, each gene
exerts only minimal effect on the phenotype. Thus, several
genes acting in concert are required to induce the
phenotype. To map the chromosomal locus of a gene with
minimal effect on the phenotype requires not hundreds of
DNA markers but hundreds of thousands of DNA markers.
It also requires thousands of unrelated cases and controls
analyzed for gene frequency in cases versus controls,
referred to as a case-control association study. The preferred
approach is to genotype with hundreds of thousands of
markers selected to span the whole genome. Then, in an
unbiased fashion, one can determine which markers are
more common in cases (risk variant) or more common in
controls (protective variant). In 2005, the technology
arrived in the form of a microarray with 500,000 SNPs
 and the necessary high throughput platform . In
2007, two groups simultaneously mapped the first risk
variant, 9p21, for CAD  and myocardial infarction .
The pursuit was intense, and in just 5 years, more than 400
chromosomal loci have been mapped to be associated with
The case-control association study method and its
genome-wide application referred to as genome-wide
association study (GWAS) has been a remarkable success.
The GWAS method has been dissected and analyzed in
three reviews of this issue. The authors analyzed the
advantages and disadvantages of the GWAS and provide a
progress report on their application to cardiovascular
disease in particular, CAD and hypertension. The ultimate
application of these loci in the prevention, diagnoses and
treatment of disease will require identification of the
causative sequence and its function. We have already been
The John & Jennifer Ruddy
Canadian Cardiovascular Genetics Centre,
Ottawa, ON, Canada K1Y 4W7
C. J. Steer (*)
Department of Medicine,
University of Minnesota Medical School,
Minneapolis, MN 55455, USA
J. of Cardiovasc. Trans. Res. (2010) 3:169–172
surprised, namely, most loci were not as expected in
protein-coding DNA sequences but rather in noncoding
regulatory sequences and regions of non-protein-coding
RNAs. The 9p21 locus which exhibits the most robust
association for CAD encodes a non-coding RNA referred to
as ANRIL .
The primary role of RNA in the cell has traditionally
been considered in the context of protein expression,
limiting RNA to its function as mRNA, tRNA, and rRNA.
The discovery of a diverse array of noncoding RNA
(ncRNA) genes that functions as RNAs to regulate DNA
replication, transcription, RNA processing, translation and
stability of mRNAs, and even protein stability and
translocation has changed this view profoundly [12, 13].
MicroRNAs (miRNAs) are a recently discovered class of
small ncRNAs that modulate gene expression. Mature
miRNAs are produced from sequential processing of primary
transcripts via specific RNAses. These 18- to 24-nt-long
miRNAs down-regulate protein expression of specific
mRNA by either translational inhibition or mRNA degrada-
tion . Numerous reports have now shown that miRNAs
are differentially expressed in many types of human cancers
[15, 16]. In addition, altered miRNA expression has also
been found in most disease states unrelated to cancer .
The molecular signaling pathways modulated by miRNAs
have become a major focus of study in both normal and
abnormal cell function.
Mature miRNAs are evolutionarily conserved small
ncRNAs, which in general are transcribed by RNA
polymerase II as primary transcripts (pri-miRNAs). More
than 70% of miRNAs are located in either introns or exons
of protein-coding genes and the remainder found in inter-
genic regions. A significant number of pri-miRNAs are
expressed as polycistronic (transcript containing multiple
miRNAs) RNAs . The pri-miRNAs are subsequently
processed by Drosha, an RNAse III enzyme to a ∼70-
nucleotide-long stem-loop structure called precursor
miRNA (pre-miRNA). The pre-miRNAs are exported to
the cytoplasm by exportin-5 and then processed as mature
double-stranded miRNAs by Dicer, another RNAse III
enzyme; and then bound to miRNA induced silencing
complex (RISC) to form mature miRNAs 18–23 nucleo-
tides in length. The key proteins in the complex are
Argonaute 2 and transactivation-responsive RNA-binding
protein. In the RISC complex, the active strand is retained
and the passenger strand is selectively degraded. The
mature miRNA along with RISC binds to complementary
sites in the mRNA transcripts and (typically) down-
regulates gene expression [14, 19]. miRNAs that bind to
mRNA targets with imperfect complementarity regulate the
target gene at the level of protein translation, while
miRNAs that bind to their mRNA targets with perfect
complementarity induce degradation [20, 21].
The discovery of miRNAs has dramatically changed our
view of gene expression and its regulation, adding a new
layer of complexity to an already intricate process. miRNAs
were first discovered in 1993 in the nematode Caenorhab-
ditis elegans. The lin-4 gene, which was known to regulate
the timing of larval development in C. elegans, was found
to express two small RNA transcripts, 22 and 61 nucleo-
tides long. These small RNA molecules were shown to
have antisense complementarity to the 3′UTR of the lin-14.
In addition, it was shown that the negative regulation of
lin-14 gene was due to lin-4-mediated posttranscriptional
regulation, which resulted in decreased lin-14 protein,
without noticeable changes in mRNA levels [22, 23]. This
pivotal discovery was not fully appreciated until 7 years
later, when the 21-nucleotide let-7 RNA was discovered,
again displaying complementarity to the 3′UTR of several
genes . Importantly, let-7 is critical for C. elegans
developmental timing regulation and is conserved in
Since these initial findings, a monumental research effort
has been underway to unravel the basis of miRNA
biogenesis, mechanisms of action, and regulatory impact
on normal and pathogenic cellular processes. It is now clear
that miRNAs are abundant, evolutionary conserved, and
endogenously encoded small ncRNA molecules. This has
lead to the identification of more than 700 human miRNAs
and many more in other mammals, worms, fish, and plants
. Nevertheless, this growing miRNA class is predicted
by bioinformatics to include more than 1000 members ,
which may turn out to be significantly underestimated .
In addition, miRNAs were predicted to posttranscriptionally
regulate more than 30% of human genes, which again may
be a real underestimate. In fact, it was recently suggested
that miRNAs might regulate more than 60% of human
There is growing evidence to support the role of
miRNAs in the regulation of many crucial biological
processes including human cancer . Nevertheless, the
biological function of the vast majority of miRNAs remains
unknown, despite the rapid rate of information that is being
generated for individual miRNAs and their targets. Sys-
temic analysis of the spatial–temporal expression of
miRNAs has shown that many of these small ncRNAs
have strong tissue specificity together with tight temporal
expression regulation starting from early phases of em-
bryogenesis. This fine modulation has been shown to play
an important role in cell lineage commitment and embry-
onic tissue development by temporal activation/inactivation
of specific mRNA targets.
In the cardiovascular field, miRNAs are now acknowl-
edged as fundamental in regulating the expression of genes
that govern physiological and pathological myocardial
adaptation to heart disease [1, 30, 31]. Numerous studies
170J. of Cardiovasc. Trans. Res. (2010) 3:169–172
have documented the implications of miRNAs in nearly
every developmental and pathologic process of the cardio-
vascular system, including cardiac arrhythmia, hypertrophy,
fibrosis, ischemia, heart failure, and atherosclerosis. In this
review we summarize the key miRNAs that can solely
modulate the cardiovascular pathological process and
discuss the mechanisms by which they exert their function
and act as novel therapeutic targets and/or diagnostic
markers. We will examine their role in ischemic heart
disease; as diagnostic markers and therapeutic targets;
critical modulators of cell differentiation, migration, prolif-
eration, and apoptosis; their own transcriptional and
posttranscriptional regulation; their critical role in cardiac
remodeling; the function they play in regulation of vascular
smooth muscle differentiation and response to injury; and
the importance of a systems biology approach to under-
standing their involvement in the regulation of gene
expression at every level of cardiovascular study.
A major obstacle in establishing miRNA-based clinical
therapies is in the efficient delivery of miRNA mimics and
inhibitors to target organs. There is little doubt that
miRNAs play a significant role in cardiovascular devel-
opment and disease, regulating cardiomyocyte self-renewal
and differentiation, and normal cardiac structure. Of the
30 or more miRNAs that appear to be key factors in
apoptosis, their potential gene network goes far beyond
that small number . miRNAs have complex gene
regulation mechanisms, in part determined by the presence
or absence of the target genes in a given cell type.
Balancing and maintaining the threshold levels of these
miRNAs is critical as a cell confronts its destiny to live or
die. It is essential to identify the cross-talks between
miRNA and other noncoding small RNAs such as Piwi-
interacting RNA and repeat-associated small interfering
RNA in understanding their multifaceted roles. miRNAs
are critical effectors of cell regulation; and we are just
beginning to understand their role as gatekeepers of both
survival and cell death.
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