Biology Reference
In-Depth Information
duplexes onto RISCs, resulting in enhanced RNAi activity.
Moreover, TRBP knockdown suggested that enoxacin activity is
TRBP dependent in vivo.
To better determine the action of enoxacin in vivo, researchers
utilized a similar shGFP/GFP reporter assay in transgenic mice.
Enoxacin was injected, and after incubation the mRNA levels of
GFP were determined. Similar to the in vitro studies, the addition
of enoxacin increased the knockdown of GFP by up to 60 %. The
use of small molecules such as enoxacin to regulate the RNAi path-
way may provide important information about the cellular effects
of miRNA biogenesis. This exciting result indicates that it is pos-
sible to also upregulate the effect of miRNAs in cells and that it is
possible to recapitulate cellular studies and the small molecule
effect in model organisms.
The Jeang laboratory reported another example of a
small-molecule modulator of miRNA maturation [
61
]. Using cells
transfected with a dual-luciferase reporter assay and an shRNA tar-
geting fi refl y luciferase, 530 small molecules were screened for the
ability to inhibit the knockdown of fi refl y luciferase relative to the
Renilla
control. Two compounds, polylysine and trypafl avine,
were found to reproducibly affect the luciferase ratios relative to a
non-treated control. After several elegant experiments to elucidate
the mechanism of action, polylysine was found to inhibit the for-
mation of Dicer-RNA complexes, effectively inhibiting the last
maturation step in the biogenesis pathway. Alternatively, trypafl a-
vine was found to block miRNA loading into the RISC complex by
inhibiting either TRBP/AGO2 or RHA/AGO2 associations. Even
more excitingly, the effi cacy of these compounds was examined in
a mouse model where cancerous cells over-expressing miR-93 and
miR-130 were implanted in a mouse leading to tumor formation.
Treatment with either compound resulted in a signifi cantly reduced
ability of the cells to promote tumor formation (Fig.
12
). This
result demonstrates that it is possible to have therapeutic com-
pounds that alter miRNA maturation in a nonspecifi c fashion.
While various other small molecule approaches to miRNA/siRNA
regulation have been reported, most focus on the modulation of
miRNA function and not necessarily maturation and thus will not
be discussed. Nonetheless, this represents an exciting and rapidly
developing fi eld for the development of therapeutic small-molecule
modulators of miRNAs.
4
Manipulation of miRNA Function in Living Cells with Oligonucleotides
Due to the importance of miRNAs in multiple disease pathologies,
various studies focus on developing loss-of-function models in
vivo. The most direct route to achieve these knockdowns is via the
use of complementary oligonucleotides which directly base pair to