Biology Reference
In-Depth Information
Another approach by the Stoffel laboratory utilized chemically
engineered single-stranded RNAs, known as antagomirs, that allow
for effi cient and specifi c silencing of various miRNAs within a cell
or a model organism [
24
]. These synthetic analogues consisted of
a cholesterol support fused to single-stranded RNA that was per-
fectly complementary to a specifi c miRNA. Gratifyingly, intrave-
nous administration of an antagomir signifi cantly decreased the
level of its corresponding miRNA in various mouse tissues. Since
many miRNAs are confi ned to certain types of tissues, such as the
liver-specifi c miRNA-122, antagomirs may be therapeutically use-
ful in selectively silencing endogenous miRNAs.
To this end, various antagomirs were synthesized, including
the miR-122-specifi c antagomir-122. Though partially and fully
modifi ed phosphorothioate RNA strands were also studied, the
cholesterol-conjugated antagomirs were found to be the most
effective and specifi c in silencing miRNA. Administration of
antagomir-122 in mice resulted in miR-122 degradation products
as well as signifi cantly decreased levels of miR-122, indicating that
antagomir-122 silences miR-122 through degradation.
This pharmacological silencing further elucidated the role of
miR-122 in the regulation of many mRNAs that contributed to the
liver phenotype. Antagomir-122-treated mice displayed specifi c
phenotypic changes, such as decreased plasma cholesterol levels,
linking miR-122 to the regulation of cholesterol biosynthesis. Gene
analysis from antagomir-treated mice identifi ed recognition motifs
within genes that interacted with miR-122, leading to upregulation
or downregulation of multiple genes. The recognition motif, or
miR-122 nucleus sequence, was the most over-represented hex-
amer found in a bioinformatics analysis. When the miR-122 nucleus
was incorporated into a luciferase reporter system, co-transfection
with miR-122 led to a signifi cantly repressed reporter when com-
pared to control miRNA transfections. Thus binding at the miR-
122 nucleus directly represses mRNA expression.
Antagomirs have been proven to provide a powerful means to
assess gene regulation in vivo. Silencing of select endogenous
miRNAs or a combination of miRNAs within mice leads to a better
understanding of the function and roles of various miRNAs.
Indeed, the long-lasting activity of antagomirs coupled with broad
biodistribution and low toxicity makes them ideal candidates for
therapies aimed at diseases linked to miRNA misregulation.
Though antagomirs provide an effi cient method to target
miRNAs, they require extensive sequence complementarity, and
the requisite pairing of one oligonucleotide per mature miRNA
often obviates high doses in order to obtain an in vivo effect.
Comparatively, an over-expressed miRNA decoy target can similarly
silence its cognate miRNA by acting as a competitive inhibitor [
62
].
This decoy may also be able to interact with specifi c sequences that
are shared by members of the same miRNA family. Ebert et al.
