INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 4, ISSUE 12, DECEMBER 2015

ISSN 2277-8616

Micro RNA Mimics And Antagonists

Nabajit Das, Naveen Tripathi, Sukant Khurana
Abstract: Gene regulation is vital for life and it involves plethora of mechanisms, including microRNA (miRNA) based RNA inhibition. Messenger RNA
(mRNA) inhibition by miRNA requires less sequence specificity than inhibition by small inhibitory RNA (siRNA) and has different set of enzymes required
for processing. The regulation and richness of RNA inhibition and specifically miRNA action is just beginning to be studied, while much work has
undergone in the study of synthesis and processing of miRNA. More than 1800 unique human miRNAs have been computationally predicted and several
have been experimentally validated. Given their ability to act as sequence specific regulators of the transcriptome, miRNAs have potential in
therapeutics and diagnostics. We specifically focus on synthesis of current therapeutic applications of miRNA. We discuss the two different strategies in
miRNA treatment: mimics and antagonists, and bring forward the promises and perils of miRNA therapy in its journey from lab to medicine.
Index Terms: antagonists, clinical trial, diseases, drugs, therapeutics, gene, mimics, miRNA.

1 INTRODUCTION
GENE regulation is necessary to sustain the fine-tuned
orchestra of life and give unique identity and function to
different cells. Our bodies have many different cell types but
these cells have the same genome as they all came from the
same zygote. What makes cells different from each other is
how genes are regulated. Understanding differential gene
regulation can shed light on both the identities and functions of
cells, their differential sensitivities to different signals and
sturdiness of function in the face of vastly different
expressions[1-3].Different
regulatory
and
epigenetic
mechanisms partly enable different identities and functions of
cells. Micro Ribonucleic Acids (miRNAs) are small RNA
molecules that regulate genes, but are too tiny to code for
proteins. They work like Small interfering RNA (siRNA) but do
not require as high a degree of sequence complementarity;
and different sets of enzymes are involved in their processing
than siRNA[4].The first reported miRNA, lin-4 was first
discovered in Caenorhabditis elegans in 1993 by Victor
Ambros group and was found to be regulating timely
developmental events[5]. This discovery was followed by
several thousand microRNAs in almost all metazoan studied
for miRNA[6-8]. With implications for the treatment of cancer,
diabetes and brain disorders, miRNAs can play an important
role in the development of future therapeutics and diagnostics.

2 REVIEW
2.1 Status and unsolved questions on the genesis of
MicroRNAs
MicroRNA synthesis and processing has been described in
great detail in several other works[9-11], so here we would
only briefly touch upon the topic to provide the necessary
background for the understanding of mimics and
antagonists.

___________________________
Sukant Khurana is an assistant professor in the
department of biological sciences at Indian Institute of
Science Education and Research-Kolkata, PH-+918443954472. E-mail: [email protected]
Naveen Tripathi and Nabajit Das worked as visiting
student
in
DBS
at
IISER-Kolkata,
PH+919040474100.
E-mail:
[email protected],
[email protected]

MicroRNAs, the smallest of all small eukaryotic RNAs,

suppress messages of mRNA by RNA silencing.
Genesis of miRNAs begins in the nucleus, where RNA
polymerase II (Pol II) transcribes primary transcripts including
ones containing miRNA sequences. Such primary miRNA
transcripts (pri-miRNA) get processed by an enzyme Drosha,
which removes the tails of the primary miRNA, leaving a
shorter stem loop structure known as pre-miRNA. PremiRNA associates with an exportin complex (RAN GTP) and
enters the cytoplasm [12]. Once it reaches the cytoplasm,
pre-miRNA is released from exportin complex and associates
with DICER for further processing[13-16]. DICER cleaves the
loop structure resulting in an asymmetrical double stranded
RNA(miRNA) of 19-23 nucleotides[15, 17]. This double
stranded RNA associates with RNA induced silencing
complex (RISC complex)[15]. MiRNAs get unbound inside the
RISC complex and then guide the RISC to conserved
recognition sites of target mRNA[10]. Binding of RISC to the
target mRNA triggers the silencing. Between different
metazoans there is variability in the genesis of
microRNAs[18]. MicroRNAs are physically clustered and often
exist within protein coding and long non coding RNA
genes[19, 20]. For example, roughly 1/3 of microRNAs lie in
the introns of protein coding gene where the host gene drives
the expression of the microRNAs[21, 22]. Given the fact that
RISC complex holds on to the driver strand once a target
mRNA is degraded, the answer which is yet not clear is: what
is the fate of the RISC complex after the first round of target
mRNA degradation? One possibility is that the complex gets
ready for the next possible target mRNA match. If so, then
how many times is the cycle repeated inside the cytoplasm? It
would be interesting to find out the destiny of the passenger
strand, which leaves the RISC complex before the miRNA
held inside the RISC finds its target. There are reports that
the enzymes in the cytoplasm degrade the passenger
strand[23] but there is a small, as yet unexplored possibility
that the passenger strand might also find a complementarity
in the mRNA strand in the cytoplasm and block any other
mRNA processing. We also do not know if the single stranded
free passenger miRNA strand can again get bound to the free
ends of career strands in RISC complex, diminishing its
function.

2.2 Status of development in miRNA therapeutic

approaches
Inappropriate expression of miRNA leads to numerous
diseases in humans[24, 25], such as miR-195 contributing to
postnatal loss of cardiac regenerative capacity [26]; miR-1,
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miR-133a/b and miR-208 being dysregulated in human

myocardial infraction[27]; and miR-33 negatively regulating
cholesterol homeostasis[28]. These molecules need to be
systematically blocked in order to regain normal functionality
in the diseased condition. Many of them are in pre-clinical
testing and might end up being drugs in the near future.
Depending on the expression pattern in pathological
condition, there are two major kinds of disease therapies:
miRNA mimics[29, 30]and miRNA antagonists[31, 32]. Figure
1 summarizes the effect of miRNA mimics and antagonists on
the target gene protein expression. To better understand their
biological roles, we can use tools to increase or decrease
miRNA availability and function. MiRNA mimics are used to
restore loss of function of miRNAs in the diseased tissues.
Such mimics carry out processes in the same way as the
mature miRNAs in any cell. They are double stranded
miRNA-like non-natural RNA fragments designed to silence
genes. Mimics results in an increase in the number of RISC
complexes in the cytoplasm. Coupled with the RISC complex,
the mimic acts on the target mRNA to block its posttranscriptional activity. In this regard, miRNA can be used to
replace traditional gene therapy approaches. For example,
miR-34 is known to inhibit human pancreatic cancer tumorinitiating cells[33], down-regulation of which may lead to a
pathological conditions. A mimic of miR-34 is the first of its
kind to enter a phase I clinical trial after having been shown to
globally downregulate target mRNA. No side effect has been
reported so far [34]. Another example that gives a clearview of
the beneficiary role of miRNAs in human disease and why we
need mimics to restore their loss, is that loss of let-7 function
enhances lung tumour formation[35]. Mimicking the nature of
this candidate miRNA regains the appropriate functioning.
Let-7mimic is being tested in non-human primates[35, 36].
Successful development of a miRNA mimic requires the RNA
molecule to be capable of entering the RISC complex and
targeting the genes of interest. Oligonucleotide engineering
for miRNA mimics is conducted in a manner that would retain
sequence complementarity with target mRNAs. The original
function to act as a guide sequence within RISC is retained by
such mimics to cope with the loss of endogenous miRNA in
pathological conditions[37]. This is known as miRNA
replacement therapy[38].It helps regain identical miRNA
activities that are reduced or missing in the diseased state. In
addition to mimics, we posit that the transcribing DNA
sequence can also be inserted in the genome or introduced
as plasmids to induce a gain in the function of miRNAs in
various organisms. In humans, this approach would likely not
find much ground in the immediate future due to ethical
concerns but can be of use in economically important
organisms. In a diametrically opposite approach, use of
miRNA antagonists (anti-miR or antagomiR or antisense
oligonucleotides) is an effective strategy to block endogenous
miRNAs that acquire a gain in function in human disease.
MicroRNA antagonists are short, single stranded
oligonucleotide molecules that target the active miRNA in
question with partial or full complementarity, before the
endogenous miRNA reaches its target mRNA. This triggers a
break-down of the endogenous miRNA by forming a duplex
structure of the endogenous and exogenous miRNA. For
example, miRNA-122 is believed to promote the Hepatitis C
Virus (HCV) [39]. Systematic silencing of miR-122 with antimiR-122 locked nucleic acids (LNA) in non-human primates
with the Hepatitis C infection triggers a breakdown of

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endogenous miR-122, the causative agent in the diseased

phenotype[40, 41]. This candidate is ahead of all other antimiRNAs in the drug development pipeline currently being
tested in phase II of a clinical trial[42]. In order to determine
the right miRNA antagonist construct, a thorough in vitro and
in vivo examination is needed. Such an examination, with
various modifications, can reveal the function of individual
microRNAs. MiRNA antagonists are single-stranded
antisense oligonucleotides that are completely or partially
complementary to the target miRNA[43, 44]. The result of any
such exogenous oligonucleotide injection is endogenous
miRNA degradation [45]. The fate of degraded mRNA needs
to be systematically studied. Compared to siRNA, where
100% sequence complementarity is required for functioning,
miRNAs provide far more sturdy tools as even in much lower
complementarity; they are capable of RNA inhibition. This
feature of lower complementarity would also drive the price of
therapies down, and their effectiveness higher because one
does not have to design a personalized RNA inhibition
approach using miRNAs. A potential flip side of the coin might
be the additional testing required for potentially off-target
effects. Of the eight novel drug candidates listed in table1,
Miravirsen, a miR-122 blocker, is leading the table, after
having successfully passed phase I of a clinical trial and is
currently being tested in phase II. Miravirsen is a product of
SantarisPharma. Listed second in the table, MRX 34 is
designed to mimic the functions of the natural miR-34 and is
the first of its kind to enter Phase I of a clinical trial. MRX 34 is
a product of Mirna Therapeutics Inc. TargomiRs, a mimic of
miR-116 is in the clinical phase 1 which has a role in
malingnant pleural mesothelioma and non-small cell lung
cancer. Another potential drug candidate listedin the table1,
miR-Rxlet-7 is designed to mimic functions of natural let-7. It
is next in line to enter a Phase I clinical trial and is currently
being tested in non-human primates. The other advanced
potential anti-miRNA drug candidates miR-195, miR-208,
miR-33, and miR-155 are yet to enter clinical trials. We
gathered
information
on
clinical
trials
from
https://clinicaltrials.gov/.

2.3 Figures and Tables

Fig.1. Effect of microRNA, microRNA mimics, and

antagonists on protein expression: Hypothetical
expression levels of same protein for control, mimics
and antagonists to show the effect of miRNAs.

CONCLUSION

Both approaches of mimics and antagonists have started

showing promising results. We expect several more miRNA
based therapies to enter clinical trials in the coming years and
to make it to market soon. While progress in miRNA
therapeutics has been noticeable, for continued progress in
the field, unanswered questions relating to the biology of
miRNA will need to be answered. We present a few
outstanding unsolved issues here. Identification of the trigger
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factors that promote miRNA expression across species has

not begun in earnest yet. The regulatory network of miRNA,
feedback based on miRNA levels and activity remain
unexplored. Epigenetic regulation of inhibitory RNAs including
miRNA remains a mystery at present. Evolution of miRNA and
overall inhibitory RNA machinery also remain largely
unexplored. Detailed, structured function studies of Drosha
and Dicer are much needed. Even with a smaller sequence
than siRNA incomplementarity match, miRNA can regulate
target mRNAs. This raises the possibility of regulation of more
than one mRNA. This can be double edged sword, enabling
design of miRNA that works for larger population segments of
whole human race on one hand and a concern for potential
off-target on the other hand. Systematic analysis of both
on-target effects and off-target side-effects, if such sideeffects are indeed the case for miRNA therapy, would be
required for case by case basis. Additionally, routes of
administration, especially enteral and parenteral, remain to be
studied systematically for the degradation and half life of
these potential therapeutic agents. At present, the long-term
and short-term effects of molecular medicine in-vivo are still
not entirely clear; as the question of how many gene
transcripts can be regulated by one miRNA is an open one. In
summary, we expect several promises of miRNA therapeutics
to start bearing fruit and expect basic biology research on
miRNA to be filled with several surprises.

ACKNOWLEDGMENT
The funding for this work came from intramural funding to
Sukant Khurana from IISER-K.

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