Biology Reference
In-Depth Information
HRP
Horseradish peroxidase
RISC
RNA-induced silencing complex
1
Introduction
MicroRNAs (miRNAs, miRs) are a family of small (19-25
nucleotides in length) noncoding RNAs that regulate gene expres-
sion by sequence-selective targeting of mRNAs [
1
], leading to a
translational repression or mRNA degradation, depending on the
degree of complementarity between miRNAs and the target
sequences [
2
]. Since a single miRNA can target several mRNAs and
a single mRNA may contain several signals for miRNA recognition,
it is calculated that at least 10-40 % of human mRNAs are targets of
microRNAs [
1
]. In general, a low expression of a given miRNA is
expected to be potentially linked with an accumulation of targets
mRNAs; conversely, a high expression of miRNAs is expected to be
the cause of a low expression of the target mRNAs [
2
,
3
].
Since the involvement of microRNAs in human pathologies is
a fi rmly established fact, the pharmacological modulation of their
activity appears to be a very appealing issue in the development of
new types of drugs (miRNA therapeutics). One of the most inter-
esting issues is the possible development of miRNA therapeutics
for development of anti-cancer molecules [
4
]. In this respect pep-
tide nucleic acid (PNA)-based molecules are appealing [
5
].
In PNAs the pseudo-peptide backbone is composed of
N
-
(2-aminoethyl)glycine units [
6
]. PNAs are resistant to both nucle-
ases and proteases [
7
,
8
] and, more importantly, hybridize with
high affi nity to complementary sequences of single-stranded RNA
and DNA, forming Watson-Crick double helices [
1
]. For these
reasons, PNAs were found to be excellent candidates for antisense
and antigéne therapies [
9
-
11
]. The major limit in the use of PNA
for alteration of gene expression is the low uptake by eukaryotic
cells [
12
]. In order to solve this drawback, several approaches have
been considered, including the delivery of PNA analogues with
liposomes and microspheres [
7
,
13
,
14
]. One of the possible strat-
egy is to link PNAs to polyarginine (R) tails, based on the observa-
tion that this cell-membrane penetrating oligopeptides are able to
facilitate uptake of conjugated molecules [
15
]. Peptide-PNA con-
jugates have been shown to be effi ciently incorporated in cells by
gymnosis, i.e., without the need of transfecting agents, showing
high uptake effi ciency [
16
].
The aim of this chapter is to describe methods for determining
the activity of PNAs designed to target oncomiRNAs, using as
model system miR-221 and its target p27
Kip1
mRNA [
17
-
19
]. The
effects of PNAs targeting miR-221 are here presented discussing
data obtained using as cellular model system the human breast can-
cer cell line MDA-MB-231, in which miR-221 is up-regulated and
p27
Kip1
down-regulated [
20
].
