|Advances in genetics and molecular biology have improved our
knowledge of the inner functions of cells, the basic building blocks of
the body. Behaviors of each human cell are controlled by its collection
of genetic information, which is included in about 25,000 genes in
humans. These genes and their products, RNAs and proteins, instruct
the cell what to do and when to grow and divide. According to the
central dogma of molecular biology, RNAs, transcribed from genes
on the chromosomal DNA, are in charge of transferring genetic
information and carry out DNA instructions by producing proteins
that guide each aspect of cell activities. However, this central dogma
is getting challenged by the recent findings that a family of small nonprotein-
coding RNAs, typically ~22 nucleotides in length, namely
microRNAs (miRNAs) are able to inhibit protein-coding genes by
interfering with the stability and function of mRNAs. Accumulating
studies have highlighted critical roles of miRNAs as central posttranscriptional
repressors of gene expression .
|miRNAs – A Family of Small Non-Coding RNAs
|MiRNAs are small endogenous noncoding RNAs that regulate gene
expression by repressing translation or promoting the degradation of
their target mRNAs. It has become an emerging concept that miRNAs
could interact with cellular networks to exert their function. They are
involved in almost every biological process, including development,
differentiation, cell-cycle control, apoptosis, and cancer. MiRNAs
regulate gene expression by binding to the 3′ untranslated region
(UTR) of their target mRNAs and mediating mRNA degradation
or translational inhibition. In the human genome, approximately
transcripts of 30% to 50% of genes are estimated to be targeted by
|MiRNAs are produced through sequential endonucleolytic cleavage
mediated by two evolutionarily conserved RNase III enzymes, Drosha
and Dicer . They can be transcribed from two different pathways.
Approximately half of miRNAs (intergenic miRNAs) are derived from
non-coding RNA transcripts, whereas intronic or exonic miRNAs are
located within protein-coding genes and share a common promoter
with their host genes. The majority of miRNAs is transcribed by RNA
Pol II as primary miRNAs (pri-miRNAs). Following transcription, the
pri-miRNA is recognized and processed by the Drosha-DGCR8 microprocessor
in the nucleus. This first cleavage step generates an approximately
70-nucleotide hairpin structure called precursor miRNA (premiRNA),
containing 25 to 30 base pair stems and relatively small loops
with 3′ overhangs. Next, the pre-miRNA is exported from the nucleus
through an interaction with exportin-5, a RanGTP-binding nuclear
transporter. The pre-miRNA is then further processed by Dicer, generating
a 20 to 25 nucleotide double-stranded mature miRNA consisting
of a functional guide strand and passenger strand. Dicer associates with
Ago2 and TRBP/PACT to form the RNA-induced silencing complex
(RISC), loading complex (RLC), allowing the tight coupling of Dicer
cleavage to the incorporation of miRNA into the RISC complex .
While the passenger strand is degraded, the mature miRNA is loaded
into the RNA-induced silencing complex (RISC) where it directs Ago2
to target mRNAs and repress protein expression.
|miRNAs as Tumor Suppressors and Oncogenesm
|When mammalian cells exhibit aberrant activities such as abnormal
growth and loss of apoptosis, they become precancerous and eventually
result in cancer initiation. A number of studies have shown that miRNA
regulates cell growth and apoptosis . As an example, miR-15 and
miR-16 induce apoptosis by targeting antiapoptotic gene BCL2 mRNA,
a crucial player in many types of human cancers, including leukemias,
lymphomas, and carcinomas . Utilizing many molecular techniques,
which include quantitative PCR, miRNA microarray, altered expression
of miRNAs, and RNAome deep sequencing, many miRNAs have been
identified as key players in human cancers, including lung, breast, brain,
liver, colon cancer, and leukemia. Proof-of-principle studies validate a
number of miRNAs as oncogenes or tumor suppressors. More than
50% of miRNA genes are located in cancer-associated genomic regions
or in fragile sites, suggesting that miRNAs may play a more important
role in tumorigenesis than previously thought . Overexpressed
miRNAs in cancers, such as mir-17-92, may function as oncogenes
and promote tumor progression by inhibiting tumor suppressor genes
that control cell differentiation or apoptosis . Silenced miRNAs
in cancers may function as tumor suppressor genes and may inhibit
cancers by downregulating oncogenes. miRNA expression profiles have
become useful biomarkers for cancer classification and diagnostics. It is
predicted that miRNA-based therapy will be a powerful tool for cancer
prevention and treatment.
|The initial evidence demonstrating the involvement of miRNAs
in human cancers came from a pioneering study on miR-15a/miR-
16-1 in human chronic lymphocytic leukemia (CLL). Calin et al. first
showed that these two miRNAs are located on a chromosomal region
that is frequently deleted in 50% of B-cell CLL cases . Further studies
identified anti-apoptotic gene BCL2 as one of the primary target of
miR-15a/miR-16-1. Meanwhile, the Slack group identified oncogene
RAS as specific targets for miRNAs in the let-7 family . In silico
prediction was first used to identify genes with 3′ UTRs (untranslated
regions) containing let-7 complementary sites. The top candidate
targets are the nematode RAS gene and the human KRAS, HRAS, and
NRAS genes. Supporting this finding is the fact that genomic regions
commonly deleted in lung cancer (where RAS is a cancer-initiating
oncogene) contain several human let-7 genes. RNA expression profiles
revealed specific downregulation of let-7 expression and concomitant
overexpression of RAS in samples of lung, as compared with normal
adjacent tissue. In addition to tumor suppressor miRNAs, miRNAs
with oncogenic properties can negatively regulate tumor suppressor proteins. One of the oncogenic miRNAs (oncomiRs) is miR-21. miR-
21 was reported to be aberrantly overexpressed in breast tumors,
glioblastoma and pancreatic cancer . In fact miR-21 targets the
tumor suppressor’s phosphatase and tensin homolog (PTEN) and
programmed cell death 4 (PCDC4). In the past decade, more than a
dozen of miRNAs have been shown to play important roles in tumor
initiation, progression and metastasis. More recently, miRNAs have
been reported to impact response to cancer drugs in chemotherapy
and targeted therapies. For examples, overexpression of miR-221 and
miR-222 is responsible for resistance to anti-estrogen therapies [12,13].
Inhibition of miR-21 and miR-200b increases sensitivity to gemcitabine
, a nucleoside analog used in various carcinomas: non-small cell
lung cancer, pancreatic cancer, bladder cancer and breast cancer.
|miRNAs in Cancer Prognosis and Treatment
|Deregulated miRNA expression in human cancer is due to chromosomal
abnormalities (amplification, deletion and translocation), mutations,
polymorphisms (SNPs), deficient miRNA biogenesis machinery
and epigenetic changes of miRNA genes as well . Genome-wide
profiling showed that miRNA expression signatures allowed various
types of cancer to be discriminated with high accuracy and the origin
of poorly differentiated tumors to be verified. In one study, Rosenfeld
et al. established a classifier based on 48 miRNAs from miRNA microarray
analysis of 253 samples [16,17]. Two-thirds of samples were
classified with high confidence, with accuracy >90%. Classification accuracy
reached 100% for most tissue classes, including 131 metastatic
samples. This finding demonstrates the effectiveness of miRNAs as
biomarkers for identifying the tissue of origin for cancers of unknown
primary origin. Recent progress in developing miRNA biomarkers allows
us to detect miRNAs in blood and other human fluids. It has been
shown that miRNAs circulate in a form of microvescicles called exosomes.
Therefore, they are extremely stable and resistant to degradation.
Weber et al. determined miRNA expression in 12 different types
of body fluids (amniotic fluid, breast milk, bronchial lavage, cerebrospinal
fluid, colostrum, peritoneal fluid, plasma, pleural fluid, saliva,
seminal fluid, tears and urine) collected from healthy individuals, and
showed that the highest concentrations of miRNAs were found in tears
. The ability to detect miRNAs in body fluids has generated great
interest in their clinical potential as cancer biomarkers. Many studies
have demonstrated that miRNAs can indeed be successfully employed
both as cancer diagnostic and prognostic biomarkers both in solid and
in hematological malignancies.
|The ability to target multiple genes or biological processes makes
miRNAs one of the most promising agents for cancer therapy. Initial
evidence of the feasibility and efficacy of a miRNA-based therapy
came from preclinical models aimed to understand the biological
roles of a specific miRNA. As examples, delivery of miR-15a and miR-
16 into human prostate cancer cells induces apoptosis and inhibits
tumor growth in vivo in a xenograft model . Inhibiting miR-21
by antisense oligonucleotides exerts an apoptotic response in vitro in
different types of cancer cells and also reduces tumor development
and metastasis in vivo . Despite the fact that basic knowledge of
miRNA biology is still providing new insights, the relative ease by
which miRNA can be manipulated pharmacologically appears to
provide an intriguing opportunity for the treatment of disease. To date,
there are several tools available to selectively target miRNA pathways.
One method is to increase miRNA levels by delivering chemically
synthesize miRNA mimics in vivo. miRNA mimics are synthetic RNA
duplexes designed to mimic the endogenous functions of miRNA with
chemical modifications for stability and cellular uptake. Synthetic miR- 34a was recently shown to inhibit lung tumor growth in a mouse model
[20,21]. To target oncogenic miRNAs, the most widely used approach
is to generate antimiRs that are modified antisense oligonucleotides
harboring the full or partial complementary reverse sequence of a
mature miRNA of interest. Stability, specificity and binding affinity
are the key requirements for an antimiR to achieve its efficacy in vivo.
Two types of chemical modifications have been used for this purpose,
including 2′-O-methyl-group modification and locked nucleic acid
|Similar to other therapeutic oligonucleotides, the main challenge is
the successful delivery of the therapeutic miRNAs to the target tissue
without compromising the integrity of the miRNA. Development of
clinical miRNA formulations often involves a complete evaluation
of existing technologies to identify those that are amenable to the
miRNA and its chemistry. Criteria in the evaluation process include
sufficient delivery to induce a therapeutic effect in tumor models
and a significant safety margin at therapeutic levels. Several delivery
methods have proven effective in delivering therapeutic miRNAs to
tumor tissues in vivo. These include viral vector-based systems and
nanoparticle-based systems. In recent years, a variety of nanoparticles
have been designed and developed. For example, systemic delivery of
a miR-16 mimic inhibited metastasis of PC-3M prostate cancer cells
intra-cordially injected 4 days before treatment . The therapeutic
delivery was facilitated using atelocollagen, a cationic polymer that
associates with RNA through electrostatic interactions and forms
particles in the nanometer diameter range. Because atelocollagen is a
natural product, these nanoparticles are highly biocompatible and are
able to achieve tumor-specific delivery via enhanced permeability and
retention. Another development of miRNAs in cancer therapeutics is
miR-34a. Therapeutic delivery of a miR-34a mimic using a neutral lipid
emulsion, either by direct injections into the tumor or by systemic tail
vein injections, prevented the outgrowth of viable subcutaneous lung
tumor xenografts . Importantly, miR-34a mimics formulated in
the neutral lipid emulsion failed to induce elevated levels of neither
cytokines nor liver and kidney enzymes in serum, suggesting that
tumor inhibition was a specific effect of the mimic and that treatment
was well tolerated. While recent progresses provide the experimental
bases for the utilization of miRNAs as both targets and tools in cancer
therapy, two major issues remain be addressed, which are development
of genetically engineered animal models to study cancer-related
miRNAs at various steps of tumorigenesis, and further improvement
of miRNA mimics and antagomiRs delivery under clinical contexts.
|The past decade has witnessed an explosion of research focused
on miRNAs. Accumulating evidence reveals that miRNAs play key
roles in regulating tumor initiation, progression and metastasis.
While cancer is a very complex disease, we are still at the verge of
understanding miRNAs in various types of cancer. Nevertheless, our
current knowledge of miRNAs has nourished the emergence of a wide
spectrum of alternative translational applications that involve miRNAs
and their associated molecules and pathways, including tumor
classification, diagnosis, prognosis and prediction of overall survival for
cancer patients, and administration of effective therapeutic targeting
using miRNA mimics and antagomiRs. Combinations of miRNAs with
current cancer therapeutics are believed to be highly effective towards
improving the well-being of cancer patients.
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