Biology Reference
In-Depth Information
Chapter 14
Molecular Methods for Validation of the Biological Activity
of Peptide Nucleic Acids Targeting MicroRNAs
Eleonora Brognara , Enrica Fabbri , Nicoletta Bianchi , Alessia Finotti ,
Roberto Corradini , and Roberto Gambari
Abstract
The involvement of microRNAs in human pathologies is a fi rmly established fact. Accordingly, 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 interesting issues is the possible development
of miRNA therapeutics for development of anti-cancer molecules. In this respect appealing molecules are
based on peptide nucleic acids (PNAs), displaying a pseudo-peptide backbone composed of
N
-
(2-aminoethyl)glycine units and found to be excellent candidates for antisense and antigéne therapies. The
major limit in the use of PNAs for alteration of gene expression is the low uptake by eukaryotic cells. The
aim of this chapter is to describe methods for determining the activity of PNAs designed to oncomiRNA
targets, using as model system miR-221 and its target p27
Kip1
mRNA. The effects of PNAs targeting miR-
221 are here presented discussing data obtained using as model system the human breast cancer cell line
MDA-MB-231, in which miR-221 is up-regulated and p27
Kip1
down-regulated.
Key words
PNA, MicroRNA, Cell assays, Anti-miRs, FACS analysis, Taqman assays
Abbreviation
AEEA
2-(2-aminoethoxy)ethoxyacetyl spacer
FACS
Fluorescence-activated cell sorter
FBS
Fetal bovine serum
Fl
Fluorescein
PBS
Phosphate-buffered saline
PNA
Peptide nucleic acid
3
-untranslated region
RT-qPCR Retro transcription-quantitative polymerase chain reaction
EDTA
′
UTR
3
′
Ethylenediaminetetraacetic acid
SDS
Sodium dodecyl sulfate
DTT
Dithiotreithol
TBS
Tris-buffered saline
