MicroRNAs (miRNAs) regulate gene
expression by binding to and modulating the translation of specific
messenger RNAs. Here, the expression levels of all known miRNAs are
evaluated using a proprietary microarray detection system. These
high-throughput analysis tools are critical to identifying miRNA
function both in normal tissues and during tumorigenesis.
| Mar
31, 2005 |
| By:
David
Brown, Emmanuel
Labourier, Sapna
Chacko |
| Pharmaceutical
Discover |
|
MicroRNAs (miRNAs) are an important class of small (21–23 nt),
single-stranded RNA molecules expressed in animals and plants that
specifically regulate the translation of messenger RNAs (mRNAs).
Although these evolutionarily conserved, non-coding molecules first were
identified in a genetic screen more than 10 years ago (1), the depth of
the miRNA gene class was recognized comparatively recently. As would be
expected for molecules that regulate gene expression, miRNA levels have
been shown to vary between tissues and developmental stages.
Characterization of a number of miRNAs indicates that they influence
processes such as early development (2), cell proliferation, cell death
(3), apoptosis and fat metabolism (4).
Figure 1. A schematic of miRNA
processing and activity. MicroRNAs are transcribed as parts of
primary RNA molecules as long as 1000 nt. Two dsRNA-specific
ribonucleases, Drosha and Dicer, digest the long RNA into the
precursor hairpin miRNA (70–100 nt) and the mature miRNA
(19–23 nt), respectively. The mature miRNA is bound by a
complex similar to the RNA-induced silencing complex (RISC) that
participates in RNA interference. (Image courtesy of ambion
inc.)
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MicroRNAs are transcribed as parts of RNA molecules as long as 1000 nt.
These longer RNAs are processed in the nucleus into hairpin RNAs of
70–100 nt by the dsRNA-specific ribonuclease, Drosha (Figure 1). The
hairpin RNAs are transported to the cytoplasm (via a transportin-5
dependent mechanism), where they are digested by a second,
double-stranded RNA (dsRNA)-specific ribonuclease, Dicer. The resulting
19 to 23mer mature miRNA is bound by a complex similar to the
RNA-induced silencing complex (RISC) that participates in RNA
interference (RNAi). In animals, the complex-bound, single-stranded
miRNA binds specific mRNAs through sequences that are significantly —
though not completely — complementary to the mRNA. By a mechanism that
is not fully understood, but which does not involve mRNA degradation as
in RNAi, the bound mRNA remains untranslated, resulting in reduced
expression of the corresponding gene.
MicroRNAs as Regulators of Global Gene
Expression Several hundred
miRNAs have been cloned and sequenced from mouse, human, Drosophila,
Caenorhabditis elegans and Arabidopsis samples. Estimates
suggest that 200–300 unique miRNA genes are present in the genomes of
humans and the mouse. The sequences of many of the miRNAs are homologous
among organisms, suggesting that miRNAs represent a relatively old and
important regulatory pathway (5). In animals, miRNAs are partially
homologous to their mRNA targets. Using similar target selection
criteria, a number of laboratories has predicted the genes miRNAs bind
to and consequently regulate (6, 7). Three points are worth noting about
miRNAs:
1. Each miRNA appears to regulate
the translation of multiple genes, and many genes appear to be regulated
by multiple miRNAs. This could explain why at least some miRNAs have
such broad functionality and also might point to complex translational
control of some genes.
2. It has been predicted that the
expression of as many as 10% of mammalian genes are regulated by miRNAs.
3. If miRNAs indeed regulate the
translation — but not the stability — of mRNAs, this might at least
partially explain why gene expression profiles based upon mRNA analysis
do not always correlate with protein expression data. As more is learned
about the mRNA targets of different miRNAs, it will be possible to more
accurately assess gene expression for a given sample by combining the
profiles of mRNA and miRNA expression.
Potential Importance of MicroRNAs as
Disease Markers Given the
apparent importance of miRNAs in regulating development and
differentiation, it is likely that mutations that affect miRNA
expression and activity might contribute to oncogenesis. Indeed, at
least five reports have correlated aberrant miRNA expression and miRNA
gene locations with cancer:
1. Chronic lymphocytic leukemia (CLL)
patients commonly exhibit a chromosomal abnormality at 13q14. The genes
for the miRNAs miR-15 and miR-16 are located here and appear to be
deleted in the majority of CLL cases (8).
2. Twenty-eight different miRNAs
were identified in human colorectal mucosa; two of these proved to be
significantly down-regulated in 12 adenocarcinoma samples and
precancerous adenomatous polyps, compared to matched, normal tissues
(9).
3. Sequence analysis of tumor cells
from 11 Burkitt lymphoma patients revealed that all had a chromosome
rearrangement in the miR-155 gene, compared to control (10).
4. Comparison of the chromosomal
locations of 186 miRNA genes and cancer-associated genomic regions
revealed that more than half of the miRNAs (98 of 186) are in common
break-point regions, fragile sites, minimal regions of loss of
heterozygosity and minimal regions of amplification (11).
5. The let-7 miRNA negatively
regulates the expression of the oncogenic RAS protein and is
significantly down-regulated in lung tumors. This miRNA may act as a
tumor suppressor in the lung (12).
While these data are preliminary,
they suggest that down-regulation of miRNAs could directly or indirectly
affect the expression of some oncogenes and contribute to the diseased
state.
Figure 2. Detection of miRNAs
across different tissues with the mirVana™ system. miR-200b
and miR-16 miRNAs were detected by solution hybridization in 1
mg of total RNA from 12 different human tissues. let-7 miRNA
expression was analyzed by Northern blot (4 mg of total RNA); 5S
and 5.8S rRNAs were used as loading control. Total RNA
isolation, solution hybridization assays and probe preparation
were performed using the mirVana™ kits and reagents.
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Challenges of MicroRNA Analysis
MicroRNA analysis is not trivial. The size, and sometimes the low
expression levels, of miRNAs can make their analysis difficult. The
small size of these molecules alone precludes the use of standard
detection methods such as reverse transcriptase polymerase chain
reaction (RT-PCR) or microchips. Furthermore, most RNA isolation
procedures have been optimized to recover long (greater than 500 nt)
mRNA while ignoring smaller molecules. As a result, conventional RNA
extraction methods can result in the loss of substantial amounts of the
small RNA fraction from samples. Currently, most researchers are
analyzing expression patterns of miRNAs by Northern blot analysis with
polyacrylamide gels — a technique that is relatively insensitive and
labor intensive. As shown in Figure 2, the use of improved kits and
reagents, optimized for miRNA isolation and detection, provide valuable
and highly reproducible data to identify tissue-specific miRNAs or to
analyze their distribution across tissues. These tools also have proven
to be invaluable to characterize miRNA processing and mechanism of
action, and they extend to the analysis of other important non-coding
small RNAs, such as siRNAs.
Figure 3. An overview of the miRNA
array procedure. miRNA isolation, labeling, clean-up and array
hybridization are performed with the flashPAGE™ Fractionator
and mirVana™ miRNA analysis systems. Representative results
from a hybridized microarray are shown on the right.
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Global MicroRNA Analysis
The global expression patterns of all known miRNA genes were evaluated
by a novel, high-throughput, nonisotopic detection method. In a recent
article (13), an oligonucleotide microchip was shown to reproducibly
report specific miRNA expression signatures in human and mouse tissues.
The method relies upon direct reverse transcription of total RNA samples
with random biotinylated primers, followed by hybridization on glass
slide microarrays and detection by streptavidin-dye conjugates. However,
in this complex mixture of labeled nucleic acid, the biologically
relevant species (i.e., the mature 21–23 nt long miRNAs processed from
longer miRNA precursors) represent less than 0.01% of the sample. To
reduce the opportunity for non-specific cross hybridization of longer
mRNA, ribosomal RNA (rRNA) or precursor miRNA species to individual
miRNA-specific oligonucleotide probes, Ambion (Austin, Texas, USA)
scientists have developed an miRNA microarray analysis system that
interrogates only the mature and functionally active miRNAs (Figure 3).
The miRNA array analysis procedure comprises four steps and relies upon
many of the processes and instrumentation that are used for standard
mRNA microarray analysis:
Step 1. Total RNA is
isolated from a tissue or cell sample using the mirVana™ miRNA
Isolation Kit (Ambion), optimized for quantitative recovery of small
RNAs.
Step 2. RNA species smaller
than approximately 30 nt are further purified by a rapid column gel
electrophoresis method (flashPAGE™ Fractionator [Ambion].
Step 3. miRNAs are 3´
labeled with poly(A) polymerase, amine-modified nucleotides and
amine-reactive Cy™ dyes (GE Healthcare, Little Chalfont, United
Kingdom).
Step 4. The fluorescently-labeled
miRNAs are hybridized to a glass slide arrayed with miRNA-specific
probes. This mirVana™ miRNA Array Probe Set is processed using
standard array scanners.
The method was fully evaluated and
optimized for high reproducibility. For example, a direct comparison
between human colon and prostate samples resulted in an average
correlation of 98% for six independent replicates (Figure 4). Out of the
164 different miRNAs interrogated, 15 had an average expression level
two-fold higher in the prostate than in the colon (e.g., let-7), seven
were expressed two-fold higher in the colon than in the prostate (e.g.,
miR-200b) and 38 did not show significant variations (e.g., miR-16). The
results correlated perfectly with data obtained by Northern blot or
solution hybridization assay (compare with Figure 2).
Figure 4. An example of miRNA
array analysis. Expression profiles for 164 miRNAs in the human
normal colon vs. prostate were compared in six independent
experiments. The average standard deviation of LogRatio across
replicates was 0.15; the average correlation between the
replicates was 98% (ranging from 96.7%-99.8%). Shown are the
results for let-7a, let-7c, miR-16 and miR-200b miRNAs.
"Red" illustrates higher expression in prostate, while
"green" depicts higher expression in colon. LogRatio =
Log2(prostate/colon).
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The miRNA array analysis procedure has
been used to determine miRNA expression signatures in various mouse and
human cell lines, in organs, whole blood, white blood cells and in tumor
and normal adjacent tissues from patients with lung, colon, breast,
prostate, bladder, thyroid or pancreas cancer. Each time, the
quantitative data from the miRNA array experiments were validated using
independent methods of miRNA detection, demonstrating that
oligonucleotide microchips are robust tools for high-throughput analysis
of miRNA expression profiles.
Outlook
miRNAs represent an interesting new class of biomolecules that regulate
gene expression at the level of translation. High-throughput analysis
tools such as the miRNA array system will help better decipher their
complex spatial and temporal expression patterns during tissue
development, as well as their alteration in disease states. These data,
in turn, will be invaluable to understand how these genes are regulated,
which genes they target and to unravel new pathogenic and therapeutic
pathways.
References
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3. J. Brennecke et al., Cell 113,
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6. B.P. Lewis et al., Cell 115,
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12. S.M. Johnson et al., Cell (2005).
In press.
13. C.G. Liu et al., PNAS 101,
9740–9744 (2004).
David Brown and Emmanuel
Labourier are senior scientists and Sapna Chacko is a
technical writer/editor at Ambion Inc. The authors can be reached at
Ambion, Inc., Research and Development, 2130 Woodward Street, Austin,
Texas 78744-1832 USA. Tel. 512-651-0200; fax 512-651-0102; e-mail dbrown@ambion.com
; elabourier@ambion.com
; schacko@ambion.com
.
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