J. Biomedical Science and Engineering, 2011, 4, 666-676
doi:10.4236/jbise.2011.410083 Published Online October 2011 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online October 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Next generation sequencing for profiling expression of
miRNAs: technical progress and applications in drug
development
Jie Liu1, Steven F. Jennings2, Weida Tong3, Huixiao Hong3*
1University of Arkansas at Little Rock/University of Arkansas for Medical Sciences Bioinformatics Graduate Program, Little Rock,
USA;
2Department of Information Science, University of Arkansas at Little Rock, Little Rock, USA;
3National Center for Toxicological Research, US Food and Drug Administration, Jefferson, USA.
Received 28 July 2011; revised 1 September 2011; accepted 19 September 2011.
ABSTRACT
miRNAs are non-coding RNAs that play a regulatory
role in expression of genes and are associated with
diseases. Quantitatively measuring expression levels
of miRNAs can help in understanding the mechani-
sms of human diseases and discovering new drug
targets. There are three major methods that have been
used to measure the expression levels of miRNAs: real-
time reverse transcription PCR (qRT-PCR), microar-
ray, and the newly introduced next-generation sequen-
cing (NGS). NGS is not only suitable for profiling of
known miRNAs as qRT-PCR and microarray can do
too but it also is able to detect unkno wn miRNAs which
the other two methods are incapable of doing. Pro-
filing of miRNAs by NGS has progressed rapidly and
is a promising field for applications in drug develo-
pment. This paper reviews the technical advancement
of NGS for profiling miRNAs, including comparative
analyses between different platforms and software
packages for analyzing NGS data. Examples and
future perspectives of applications of NGS profiling
miRNAs in drug development will be discussed.
Keywords: miRNAs; Next-Generation Sequencing;
Expression; Data Analysis; Drug Development
1. INTRODUCTION
miRNAs (also known as microRNAs) are endogenous,
non-coding ribonucleic acids (RNAs) approximately
twenty-one nucleotides in length. First encountered in
1993 at Harvard University [1], a rapid increase in
published articles referring to miRNA ensued as shown
in Figure 1. A testament to the significant and increasing
scientific interest that is driven by research needs and
fostered by rapid advances in technologies for measuring
expression levels of miRNAs, it is now clear that
miRNAs play a fundamental role in normal tissue
development. With an important regulatory role in
expression of genes, they are associated with diseases
involving multiple changes in gene expression, including
cancer and viral infections. Therefore, quantitatively
measuring expression levels of miRNAs facilitates the
understanding of mechanisms of human diseases and the
discovering of new drug targets.
Primarily, there are three methods that are used to
measure the expression levels of miRNAs: real-time
reverse transcription PCR (qRT-PCR), microarray hybri-
dization, and, more recently, next-generation sequencing
(NGS). NGS not only profiles known miRNAs as do the
more traditional methods (e.g., qRT-PCR and microar-
rays), but it is also able to identify unknown miRNAs
which are beyond the capabilities of these traditional me-
thods. Profiling miRNAs by NGS has progressed rapidly
Figure 1. Annual number of publications related to miRNAs
from 2001 to 2010 based on a keyword search in PubMed.
Keyword used: microRNA. (Search was conducted on Jul. 11,
2011).

J. Liu et al. / J. Biomedical Science and Engineering 4 (2011) 666-676 667
and is a promising field for applications in drug deve-
lopment. This paper will summarize the technical advan-
cement of NGS for profiling miRNAs, including com-
parative analyses between different platforms, experi-
mental protocols, algorithms for matching short reads to
known miRNA sequences, strategies for quantitatively
measuring expression levels, and methods for detecting
unknown miRNAs. Different pipelines and software
packages for analyzing NGS data for profiling of
miRNAs will be reviewed. Examples and future perspec-
tives of applications of NGS profiling miRNAs in drug
development will be discussed.
2. BIOLOGY OF miRNAs
miRNAs are on average only twenty-one nucleotides
long, expressed from longer transcripts encoded in ani-
mal, plant and virus genomes. miRNAs are transcribed
as long precursors, pri-miRNAs, and then cleaved to
~65nt hairpin-shaped, precursor pre-miRNAs by the
RNase III enzyme Drosha and its cofactor DGCR8 (Di-
George syndrome critical region gene 8 or Pasha) (Fig-
ure 2). Pre-miRNAs are further processed to generate
mature miRNAs. miRNAs are post-transcriptional regu-
lators that bind to complementary sequences on target
messenger RNA transcripts (mRNAs), usually resulting
in translational repression and gene silencing [2,3].
The first miRNA was discovered in 1993 during a
study of the gene lin-14 in C. elegans development [1].
However, miRNAs were not recognized as a distinct
class of biologic regulators with conserved functions
until the early 2000’s when a second miRNA (let-7) was
characterized. Since then, researches have revealed mul-
tiple roles of miRNAs in negative regulation (e.g., tran-
script degradation and sequestering and translational
suppression) and possible involvement in positive regu-
lation (e.g., transcriptional and translational activation).
Currently, there are over 16,000 miRNAs from over one
hundred species in the miRNA registry, miRBase [4],
including about 1400 human miRNAs [5].
miRNAs repress their target genes’ expression by rec-
ognition of the eight nucleotides (seed sequences) on the
3’ UTR of the genes [6]. As such, the relationship be-
tween miRNAs and their target genes are many-to-many:
a single miRNA may target multiple genes and a single
gene may contain recognition sites for multiple miRNAs.
Most human genes are the conserved targets of miRNAs
[7]. miRNAs are involved in all major biological process,
including the regulation of most physiological processes:
cell proliferation [8], cell apoptosis [9,10], metabolism
[11], and development and morphogenesis [12,13].
The breadth and importance of miRNA-directed gene
regulation are coming into focus as more miRNAs and
their regulatory targets and functions are discovered.
Figure 2. miRNA biogenesis and action.
Given the ability of miRNAs to target multiple genes
and key biological processes, these molecules have re-
ceived intensive research interest both as biomarkers and
as therapeutic agents [14-16].
miRNAs appear to be involved in many diseases [17],
including diabetes [18], cardiomyopathies [19], psychia-
tric disorders including schizophrenia [20,21], and can-
cer [22]. In cancer, miRNAs may exert oncogenic func-
tions by inhibiting tumor suppressor genes or may act as
tumor suppressors by inhibiting oncogenes [23,24].
3. EXPRESSION OF miRNAs
The key question for miRNA research is which miRNAs
are active under a given set of experimental conditions
and how does that pattern change in the dynamic cellular
environment. The technical challenge is to precisely
measure miRNA expression levels. Northern blotting
was the earliest technique that attempted to systema-
tically profile miRNA expression in various experi-
mental systems [25]. The primary methods currently
used for measuring the expression levels of miRNAs are
qRT-PCR [26,27], microarray hybridization [28,29], and
next generation sequencing [30].
qRT-PCR is a laboratory technique used to generate
multiple copies of a DNA sequence by using a pair of
primers that are complementary to the sequence on each
of the two strands of the cDNA. It consists of three
major steps: reverse transcription (RT), denaturation, and
DNA extension. RT transcribes RNA to cDNA using
reverse transcriptase. The denaturation step then sepa-
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Table 1. Some popular miRNA microarray platforms.
Company Platform miRBase Species
Agilent Human miRNA
Microarray 8x60K 16.0 (2010) Human
Agilent Mouse miRNA
Microarray 8x60K 16.0 (2010) Mouse
Agilent Rat miRNA Microarray
8x15K 16.0 (2010) Rat
Affymetrix GeneChip® miRNA 2.0
Array 15.0 (2010) multiple
Illumina MicroRNA Expression
Profiling Assay 12.0 (2008) multiple
Exiqon miRCURY LNA™
microRNA Array 16.0 (2010) multiple
Life Tech-
nologies
NCode™ Human
miRNA Microarray V3 10.0 (2007) Human
rates the strands and the primers can bind again at lower
temperatures and begin a new chain reaction. Finally,
DNA extension from the primers is done with thermo-
stable Taq DNA polymerase. qRT-PCR is a sensitive me-
thod for measuring expression levels of precursor or ma-
ture miRNAs. It requires low amounts of starting mate-
rial. With the design of RT primers with high specificity
towards individual mature miRNA species, multiple
primers could be potentially combined in a single pool
that would enable much higher throughput profiling than
is currently possible with individual sample analyses.
qRT-PCR is suitable for quantification of a few known
miRNAs in a large number of samples.
miRNA microarray hybridization is a popular method
for measuring miRNA expression levels because a large
number of miRNAs can be measured simultaneously
[29]. Several companies offer miRNA microarray plat-
forms which are designed and developed based on the
same miRNAs database, miRBase [4]. Therefore, the
major difference among the miRNA microarray plat-
forms is the miRNAs on the arrays. Because this field is
evolving very rapidly, users should select a platform
based on the version of miRBase which contains the
most relevant information for their samples. Companies
frequently update their contents to reflect a newer
miRBase release. Table 1 lists some popular miRNA
microarray platforms.
4. NGS
Rapid determination of DNA sequence base pairs, first
reported by Sanger [35,36], provided a tool to decipher
genes. However, low throughput and—more impor-
tantly—high cost hindered using this sequencing tech-
nology for deciphering the human genome. A break-
through came in 2005 when the sequencing-by—syn-
thesis technology developed by 454 Life Sciences was
published [37]. Since then, several NGS platforms, such
as Illumina Genome Analyzer (Illumina, Inc., San Diego,
CA, USA) and SOLiDTM (Life Technologies Corpora-
tion, Carlsbad, CA, USA), have been developed and
applied to various fields of biological and medical re-
search, including measuring expression levels of known
miRNAs and detecting unknown miRNAs.
4.1. NGS Platforms
Different NGS platforms use divergent sequencing
chemistries. Table 2 compares the main features of three
NGS platforms, but it is important to note that these
values are constantly changing as newer models are re-
leased. Both Illumina Genome Analyzer system and
SOLiD use short-read sequencing technologies. The
Roche 454 Genome Sequencer has the advantage of
longer sequence reads and is the best choice for denovo
sequencing of new genomes.
4.1.1. Illumina Genome Analyzer
The Illumina Genome Analyzer system currently is the
most widely-used, short-read sequencing platform. It
applies the sequencing-by-synthesis method. miRNAs
are first reversely translated to DNA and the DNA sam-
ples are randomly sheared into fragments, then ligated to
oligonucleotide adapters at both ends. Single-stranded
DNA fragments are attached to reaction chambers and
are extended and amplified by bridge PCR amplification
with fluorescently-labeled nucleotides for sequencing.
The Genome Analyzer is widely used by many genome
sequencing projects for its consistent data quality and
proper read lengths. The new Illumina HiSeq 2000 has
dramatically increased throughput.
4.1.2. Roche 454 Genome Sequencer FL X
The 454 Genome Sequencer uses the principle of pyro-
Table 2. Comparison of NGS platforms.
Platform
Illumina Roche 454 SOLiD
Sequencing-
by-synthesis Pyrosequencing Ligation-based sequencing
ApplificationBridge PCR Emulsion PCR Emulsion PCR
Read length35 - 150 bp ~400 bp 75/35 bp
Paired ends/
separation Yes/200 bp Yes/3000 bp Yes/3000 bp
Mb/run 1300 Mb 100 Mb 3000 Mb
Time/run
(paired ends)4 days 7 hours 5 days
Comments Most widely
used
Longer reads, fast
run, higher cost
Good data
quality
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sequencing. Sheared DNA fragments are ligated to speci-
fic oligonucleotide adapters and are amplified by emul-
sion PCR on the surfaces of agarose beads. The current
maximum read length of the 454 platform is 600 bp
which is the longest short-read among all of the NGS plat-
forms. Thus, the 454 Genome Sequencer FLX is best
suited for applications requiring longer reads, such as
RNA isoform identification in RNA-seq and de novo
assembly of microbes in metagenomics [38].
4.1.3. Applied Biosystems SOLiD Sequencer
The Applied Biosystems SOLiD sequencer uses the
sequencing-by-ligation approach and may offer the best
data quality. It amplifies sheared DNA fragments by an
emulsion PCR approach with small magnetic beads. But
the DNA library preparation procedures prior to sequenc-
ing currently take five days which is both tedious and
time consuming.
4.2. Sequence Alignment
A NGS experiment generates huge amount of sequence
data. Consequently, there is a high demand for bioin-
formatics tools to cope with these large amounts of
sequencing data. The key process in NGS data analysis
is to align the huge amount of short reads to a given
genome. A variety of algorithms and software packages
have been specifically developed for dealing with mil-
lions of NGS short-read alignments (Table 3).
Bowtie (http://bowtie.cbcb.umd.edu/) is an ultrafast
and efficient alignment program for aligning short
sequences to large genomes [39]. Bowtie indexes the re-
ference genome using a scheme based on the Burrows-
Wheeler index to keep its memory footprint small. It
does not use an exact matching algorithm, which is quite
common in other tools, because exact matching does not
directly allow for sequencing errors or genetic variations.
The two key algorithmic strategies that make Bowtie ex-
tremely fast in alignment of short reads are backtracking
and double indexing. Backtracking allows mismatches
and favors high-quality alignments, while the double in-
Table 3. Short-read sequence alignment tools.
Name Description
Bowtie Uses a Burrows-Wheeler transform to create a per- manent, reusable index of the genome; faster run for
short sequence alignment to reference genome
BWA Slower than bowtie but allows indels in alignment
MAQ performs only ungapped alignments and allows up to
three mismatches
SeqMap Up to 5 mixed substitutions and insertions/deletions.
Various tuning options and input/output formats.
SOAP Allow up to 3 gaps and mismatches. SOAP2 uses
bidirectional BWT to build the index of reference and
increases the running speed.
TopHat Splice junction mapper for RNA-Seq reads
dexing avoids excessive backtracking.
BWA (http://maq.sourceforge.net/) is a software pack-
age for aligning short sequencing reads against a large
reference sequence such as the human genome [40]. This
alignment algorithm is based on a backward search with
the Burrows-Wheeler Transform (BWT) of the reference
genome. It allows mismatches and gaps for single-end
reads. BWA also supports paired-end mapping. It ge-
nerates a mapping quality index and gives multiple hits
if requested.
MAQ (http://maq.sourceforge.net/) is a software pack-
age for rapidly mapping shotgun short reads to a re-
ference genome and using quality scores to derive geno-
type calls of the consensus sequence of a diploid genome
[41]. MAQ searches for the ungapped match with lowest
mismatch score. For each alignment, a quality score is
assigned to measure the probability that the true align-
ment is not the one found by MAQ. Using MAQ, users
can map reads, call consensus sequences including single
nucleotide polymorphisms (SNPs) and indel variants,
simulate diploid genomes and read sequences, and post-
process the results in various ways.
SeqMap (http://www.stanford.edu/group/wonglab/jiangh/
seqmap/) is a tool for aligning a large number of short
sequences to a reference genome [42]. It explores the
whole reference genome for each short read se- quence
from NGS data. Multiple substitutions and inser-
tions/deletions are allowed in SeqMap. It accepts FASTA
input format and output results in various formats.
Parallel computing with SeqMap on a cluster of com-
puters is supported. This program is fast, usually taking
just a few hours on a desktop PC for a typical alignment
of NGS data.
SOAP (http://soap.genomics.org.cn/) is a program for
efficient gapped and ungapped alignment of short read
sequences onto reference sequences [43]. It adopts a
seed-and-hash look-up table algorithm to accelerate its
alignment process. First, short reads and the reference
sequences are converted to a numeric data type using a
2-bits-per-base encoding system. Then the look-up table
is checked to determine how many bases are different
between a short read and a reference sequence. SOAP is
a command-driven program and supports multi-threaded,
parallel computing. It accepts FASTA format for refer-
ence and both FASTA and FASTQ formats for input
short reads. With SOAP, users can do single-read or pair-
end resequencing, small RNA discovery, and mRNA tag
sequence mapping.
TopHat (http://tophat.cbcb.umd.edu/) is designed to
align RNA-Seq reads and to create a view of the
junctions, or to align to a known set of junctions [44].
The TopHat pipeline consists of multiple steps. Initially,
all the short reads are aligned to the reference genome
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using Bo wtie . The short reads that have not been aligned
to the genome are set aside as “initially unmapped
reads”. The aligned short reads are then assembled to
generate possible splices between neighboring exons se-
quences flanking potential donor/acceptor splice sites
with neighboring exons joined together to generate po-
tential splice junctions. Then, the “initially unmapped
reads” are aligned with these potential splice junction
sequences.
4.3. NGS for Profiling miRNAs
4.3.1. miRNA Databases
NGS short reads are aligned to a known reference
sequence database. These miRNA databases are the repo-
sitory for known miRNA sequence and annotation data.
They are the core of NGS data analysis for expression
profiling of miRNAs. The most important and popular
miRNA databases include, but not limited to: miRBase
[4], deepBase [45], microRNA.org [46], miRGen 2.0
[47], miRNAMap [48], and PMRD [49].
The miRBase database (http://www.mirbase.org) is an
online database repository for known miRNAs. It pro-
vides an integrated web interface to analyze miRNA
sequence data and to predict gene targets. The miRBase
database has three main functions: 1) the miRBase
Registry acts as an independent arbiter of miRNA gene
nomenclature, assigning names prior to publication of
novel miRNA sequences; 2) the miRBase Sequences
provides known miRNA sequence data, references, and
links to other resources; and 3) the miRBase Targets is
used for the prediction of miRNA target genes.
The deepBase (http://deepbase.sysu.edu.cn/) is a com-
prehensive web-based database for annotating and dis-
covering small and long non-coding RNAs, ncRNAs
(miRNAs, siRNAs, piRNAs, etc.), from high-throughput
deep sequencing data. In the current version of deepBase,
NGS data from 185 small RNA libraries from diverse
tissues and cell lines of seven organisms (human, mouse,
chicken, Ciona intestinalis, Drosophila melanogaster,
Caenhorhabditis elegans, and Arabidopsis thaliana) have
been curated. Its integrative, interactive, and versatile
web graphical interface facilitates analyzing and visua-
lizing NGS data on the internet.
The microRNA.org (http://www.microrna.org/ microrna/
home.do) is a database for experimentally-observed
miRNA expression patterns and predicted miRNA targets
and target downregulation scores. Through its graphical
interface, the microRNA.org web resource provides
several functions: 1) exploring genes that are potentially
regulated by a particular miRNA; 2) searching for the set
of miRNAs that potentially regulate a particular gene
cooperatively; and 3) comparing miRNA expression
profiles in different tissues. The strategy for miRNA
target prediction is to treat miRNAs as adaptors of genes
in the 3’-UTR region by using near-prefect, base-pairing
in a small region in the 5’ end (positions 2-8) of the
miRNA. The most valuable information of this web
resource is the experimental data that can be used to
verify miRNA regulation.
The miRGen 2.0 (http://diana.cslab.ece.ntua.gr/ mirgen/)
is a database of miRNA genomic information and regu-
lation. It contains 812 human miRNA coding transcripts
and 386 mouse miRNA coding transcripts as well as
expression profiles of 548 human and 451 mouse
miRNAs and over 172 human and 68 mouse small RNA
libraries derived from cell lines and tissues. It is imple-
mented in a MySQL relational database management
system. Its interface allows users to search for miRNAs
and transcription factors of interest.
The miRNAMap (http://mirnamap.mbc.nctu.edu.tw/)
provides genomic maps of miRNAs and their target
genes in mammalian genomes. Experimentally-verified
miRNAs and experimentally-verified miRNA target
genes in human, mouse, rat, and other metazoan geno-
mes are collected in this database. Target genes of
miRNAs are predicted using three bioinformatics tools:
miRanda [50], TargetScan [51], and RNAhybrid [52]
independently. To reduce false positives for predicted
target genes, three strategies were used: 1) a predicted
target site is one that is predicted by at least two of the
three bioinformatics tools; 2) the target site must be
within an accessible region of a gene; and 3) the target
gene has to have multiple target sites. Its interface is
well designed to facilitate access to the data and analyz-
ing data as well as visualizing the data and associated
analysis results.
PMRD (http://bioinformatics.cau.edu.cn/PMRD/) is a
plant miRNA database. This database contains 8433
miRNAs from 121 plant species and possible target
genes for each miRNA with a predicted interaction site.
The data in PMRD include available plant miRNA data
deposited in the public database, the ones curated from
literature, and miRNA profiling data generated in-house.
4.3.2. miRNA NGS Data Analysis Tools
There are several web servers and standalone programs
for analysis of miRNA expression profiling and novel
miRNA discovery from NGS data. The web servers
include miRanalyzer [53,54] and miRCat [55]. Examples
of standalone analysis tools are miRDeep [56] and
miRExpress [57].
miRanalyzer (http://web.bioinformatics.cicbiogune.es/
microRNA/) is a web server for identifying and analy-
zing miRNA in deep-sequencing data. After inputting
NGS data (a list of unique reads and their copy numbers)
into the web server tool, users can conduct data analysis
in three steps: 1) detect all known miRNA sequences
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annotated in miRBase; 2) match against other libraries of
transcribed sequences; and 3) predict new miRNAs. It
accepts two different input file formats: 1) a tab-se-
parated file with a row representing a short read se-
quence and its count and 2) a multi-FASTA format file
with the copy number of the unique short reads (read
count) as the description in the header. To detect the ex-
pression levels of known miRNAs—a major objective of
many miRNAs studies—miRanalyzer aligns the short reads
to the known miRNA sequences using the miRBase
repository [4] which offers mature (the mature sequences
of known miRNAs), mature-star (the sequence which pairs
with the mature miRNA in the pre-miRNA secondary
structure), and precursor miRNA sequences (sequence of
the hairpin). Since a group of miRNAs that can be ali-
gned with the same read normally belong to the same
family, miRanalyzer reports these ambiguous matches, sta-
ting all miRNAs where alignments were found. After
known miRNAs are detected, their corresponding target
genes (the genes predicted to be regulated by the detec-
ted miRNA) are predicted and precalculated ontological
analyses are given. For the remaining reads that are not
aligned to the known miRNAs, miRanalyzer maps them
to databases of transcribed sequences as miRNA, non-
coding RNA, and (retro)-transposons. The mapping step
is “perfect”, meaning that no mismatch is allowed in the
mapping. In the last step—the most important task of
miRNA NGS data analysis—detecting previously-un-
reported miRNAs is conducted. A machine-learning ap-
proach based on the random forest method [58] is used to
detect new miRNAs in miRanalyzer.
The miRCat (http://srna-tools.cmp.uea.ac.uk/) is a
miRNA NGS data analysis tool that was developed for
identification of mature miRNAs and their precursors. It
takes a FASTA format file of small RNA reads as input
and then maps them to a reference sequence database
using PatMaN [59]. Analysis results from miRCat are
output in three files: 1) a comma-separated text file with
the details for predicted miRNA candidates; 2) the
RNAfold output for candidate precursors; and 3) a
FASTA format file of predicted mature miRNA se-
quences. The miRCat program has been tested on several
high-throughput plant sRNA datasets and showed high
sensitivity and specificity.
The miRDeep package (http://www.mdc-berlin.de/en/
research/research_teams/systems_biology_of_gene_regu
latory_elements/projects/miRDeep/) is a stand-alone tool
for identifying known and novel miRNAs from NGS
data. The miRDeep program employs a probabilistic
model of miRNA biogenesis to score the compatibility
of the nucleotide position and frequency of sequenced
RNA reads with the secondary structure of the miRNA
precursor. The false positive rate and the sensitivity of its
predictions are statistically controlled in miRDeep. There-
fore, not only known and novel miRNAs can be detected
from deep-sequencing data by using miRDeep, but also
the quality of the detection results can be estimated. The
key function of miRDeep is the detection of miRNAs by
analyzing how sequenced RNAs are compatible with the
way miRNA precursors are processed in the cell. It
should be noted is that miRDeep is designed to detect
miRNAs without cross-species comparisons.
The miRExpress (http://miRExpress.mbc.nctu.edu. tw)
software package is written in the C++ programming
language and can be executed on 32- or 64-bit Linux
machines. It was developed to extract miRNA expres-
sion profiles from sequencing reads obtained by NGS
technology. The data analysis pipeline of miRExpress
consists of three steps. In the first step, the identical
short reads are merged into a unique read with “a count
of reads.” Each unique short read is also checked to
deter- mine whether it contains a full or a partial adaptor
sequence. In the second step, each unique short read is
aligned with the sequences of known mature miRNAs in
miRBase [4]. In the third step, expression levels of
miRNAs are measured by computing the sum of read
counts for each miRNA according to the alignment
criteria (e.g., the length of the read equals the length of
the miRNA sequence and the identity of the alignment is
100%). The cutoff of alignment identity can be set by
users based on their requirements when using miR-
Express.
5. APPLICATIONS OF NGS IN DRUG
DEVELOPMENT
Drug development is a complex and lengthy process
(Figure 3) that starts from pharmaceutical target identi-
fication and validation followed by lead identification
and optimization (usually termed as drug discovery). The
lead compounds are subsequently subjected to precli-
nical tests and clinical trials of increasing levels of com-
plexity (usually termed as drug development). Following
the completion of all three phases of clinical trials, a
pharmaceutical company analyzes all of the data and
files a new drug application with the FDA. If the data on
the new drug successfully demonstrate both safety and
effectiveness in careful review, the FDA approves the
new drug to be distributed on the market. For some
drugs, the FDA requires additional trials (commonly
called Phase IV) to evaluate long-term effects. This
whole process (including discovery and development)
for a new drug takes about ten to five years and costs
around $1 - $2 billion [60,61].
In a relatively short time, high-throughput sequencing
of small RNAs have provided a great potential for the
profiling of known and novel small RNAs. Given the
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Figure 3. Drug development process.
ability of miRNAs to target multiple genes and play a
major role in most biological processes, miRNAs have
received intensive research interest both as biomarkers
and as therapeutic agent targets [9,15,23,62] for drug
development.
Some miRNAs are involved in the regulation of
tumorigenesis and are tumor tissue-specific. Therefore,
these miRNAs are potential biomarkers for diagnosis
and prognosis of cancers and could serve as phar-
maceutical targets for development of anti-cancer drugs
[16]. For example, twenty-two miRNAs were up-regu-
lated and thirteen were down-regulated in gastric cancer
[63]. In that study, miR-125b, miR-199a, and miR-100
were also found to be involved in the progression of
gastric cancer. Another example is the let-7 miRNA. It
regulates the RAS oncogene. Expression of let-7 miRNA
in some human lung tumors causes increased expression
of the RAS oncogene and may contribute to tumori-
genesis [24]. Expression levels of miRNAs have been
shown to play a controlling role in tumor growth rates,
suggesting possible new strategies for therapeutic treat-
ment [64]. The serum levels of miR-141 (a miRNA
expressed in prostate cancer) differ significantly between
prostate cancer patients and healthy controls [65].
Some miRNAs are particularly abundant in the nervous
system and regulate processes such as neurogenesis,
synapse development, and plasticity in the brain, con-
trolling the expression of hundreds of genes involved in
neuroplasticity and synapses. For example, it was ob-
served that the expression level of miR-16 causes ada-
ptive changes in production of the serotonin trans- porter,
miR-133b regulates the production of tyrosine hy-
droxylase and the dopamine transporter, and miR-212
affects production of striatal brain-derived neurotrophic
factor and synaptic plasticity upon cocaine [14,62]. These
miRNAs could be potential drug targets for more ef-
ficient therapies.
A recent study found that the miRNA (miR-101) target
sites within Alzheimer’s amyloid-β precursor protein
(APP) 3’-untranslated region (3’-UTR) and down-regu-
lates APP levels in human cell cultures. It is differen-
tially expressed and involved in the regulatory network
of Alzheimer’s disease (AD). The results suggest that
miR-101 could be a potential target for AD therapeutics
[66].
Recently, NGS has been applied to identify miRNA
biomarkers for diagnosis and prognosis of disease. For
example, the expression levels of the oncogenic miRNAs
of the miR17-92 cluster and the miR-181 family deter-
mined by using SOLiD NGS were higher in five un-
favorable neuroblastomas. In contrast, the expression
levels of the tumor suppressive miRNAs of miR-542-5p
and miR-628 were much higher in five favorable neu-
roblastomas compared to the five unfavorable neuro-
blastomas in which the expressions of these two miRNAs
were virtually absent [14].
Theoretically, inhibition of a particular miRNA in-
volved in a disease can block the expression of a thera-
peutic target protein and administration of a miRNA
mimetic can boost the endogenous miRNA population
repressing a detrimental gene. Therefore, miRNA inhi-
bitors and some miRNA mimetics can serve as thera-
peutic candidates for various diseases such as cancer,
cardiovascular disease, neurological disorders, and viral
infection. A lot of pharmaceutical companies are invest-
ed in the development of drugs that target miRNAs.
Currently, a number of new drug products targeting
miRNAs are in pre-clinical studies and in clinical trials.
Ta b l e 4 summarizes some of these. A detailed example
of the utilization of miRNAs in drug development will
be reviewed.
A liver specific miRNA, miR-122, regulates a host of
messenger RNAs in the liver, many of which encode
proteins involved in lipid and cholesterol metabolism. It
is abundant in healthy individuals. Replication of
hepatitis C virus (HCV) is dependent on miR-122
expression [67]. When normal liver cells are infected by
HCV, miR-122 binds to the two target sites located in the
5’-end of the HCV genome, increasing infectious virus
production [68] as depicted in Figure 4 (box A). There-
fore, if a drug can be designed to specifically recognize
and bind to miR-122, the replication process of HCV
will be effectively blocked as shown in Figure 4 (box B).
Miravirsen (SPC-3649), developed by Santaris Pharma
(http://www.santaris.com/), is such an inhibitor of
miR-122 and is expected to provide a very high barrier to
the generation of viral resistance. Pre-clinical studies
showed a potent, dose-dependent, and long lasting
inhibition of miR-122 in mice, cynomologus monkeys,
and green African monkeys as indicated by decreases in
cholesterol levels. A phase II clinical study in patients
infected with HCV started in September 2010 and cur-
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J. Liu et al. / J. Biomedical Science and Engineering 4 (2011) 666-676
673
Table 4. Examples of miRNAs in drug development.
Generic Name Target Indication Status Company
Miravirsen miR-122 Hepatitis C virus Phase IIA Santaris Pharma
Unspecified miR-21 Fibrosis Clinical Regulus Therapeutics
Unspecified miR-21 Cancer Clinical Regulus Therapeutics
Unspecified mi-R122 Hepatitis C virus Clinical Regulus Therapeutics
Unspecified mi-155 Inflammation Pre-clinical Regulus Therapeutics
Unspecified miR-33a Metabolic diseases Pre-clinical Regulus Therapeutics
MGN-9103 miR-208/499 Chronic Heart Failure Pre-clinical Miragen Therapeutics
MGN-1374 miR-15/195 Post-MI Remodeling Pre-clinical Miragen Therapeutics
MGN-4893 miR-451 Polycythemia Vera Pre-clinical Miragen Therapeutics
MGN-4420 miR-29 Cardiac fibrosis Lead optimization Miragen Therapeutics
Unspecified Let-7 Lung cancer Pre-clinical Mirna Therapeutics
Unspecified miR-34 Prostate cancer Pre-clinical Mirna Therapeutics
TCDD miR-191 Hepatocellular carcinoma Pre-clinical Rosetta Genomics
Unspecified miR-34a Liver cancer Pre-clinical Rosetta Genomics
rently SPC-3649 is in a multiple ascending dose study in
healthy volunteers. SPC-3649 is the first drug targetting
miRNA to enter human clinical trials.
The roles of miRNAs in diseases have been very well
established over the last few years. Although there is still
much to be learned concerning the mechanism of
miRNAs in biological processes, scientists have been
able to apply their knowledge to use miRNAs as bio-
markers for diagnosis and prognosis of diseases and as
potential pharmaceutical targets for drug development.
Within the last few years, many studies on miRNAs
have moved into animal models with highly encouraging
results for drug development. With the progress of NGS
technology, expression levels of known miRNAs will be
more precisely and rapidly detected and more and more
novel miRNAs will be discovered as biomarkers for
diagnosis and prognosis of diseases and as potential tar-
gets for drug development. Thus, we look forward to a
brighter future for utilizing miRNA expression profiling
in the drug development process.
6. FUTURE PERSPECTIVE
Overall, manipulation of miRNA functions through the
activation or the silencing of miRNAs could become a
promising therapeutic tool and novel strategy for disease
treatment. NGS technologies have facilitated an enor-
mous potential for miRNA detection and gene regulation
research for drug development. miRNA expression pro-
filing plays an important role in the identification of new
therapeutic strategies, clinical diagnostics, and persona-
lized medicine [23,69,70].
7. ACKNOWLEDGEMENTS
This publication was made possible by NIH Grant # P20 RR-16460
from the IDeA Networks of Biomedical Research Excellence (INBRE)
Program of the National Center for Research Resources. The views
presented in this article are those of the authors and do not necessarily
reflect those of the US Food and Drug Administration. No official
endorsement is intended nor should be inferred.
(a)
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