Biology Reference
In-Depth Information
2.1 Small-Molecule
Regulators In Vivo
Regulation of miRNA levels via small-molecule modulators has
recently been demonstrated. The utilization of small-molecule
therapeutics has several signifi cant advantages over oligonucle-
otides, most notably substantially improved pharmacokinetic prop-
erties [
27
]. Additional advantages typically include more facile
synthetic production and easier cellular delivery/targeting [
28
].
Moreover, the ability to screen large compound libraries for their
ability to affect the entire miRNA biogenesis and regulatory path-
way represents a unique mechanism to potentially increase the
degree of target specifi city that has previously hindered other small
molecule approaches.
In order to screen small-molecule libraries for activity, a robust
assay is required that facilitates screening of cells in a high-
throughput fashion. Work in the Deiters and Huang labs has made
substantial progress towards this requirement [
40
]. Initial screen-
ing attempted to ascertain an inhibitor of miR-21 due to its
overexpression in a variety of cancers, including glioblastomas.
The original assay was developed using a stably transfected lucifer-
ase reporter system with a miRNA-binding site cloned into the 3
′
UTR of the luciferase gene. Various constructs (including a miR-
21-, miR-30-, or non-miRNA-binding site) were stably introduced
into HeLa cells due to their well-reported misregulation of miR-21.
In the absence of small molecules, cells harboring the miR-21-
binding site construct exhibited a signifi cant decrease in luciferase
expression relative to the other constructs due to the overexpres-
sion of miR-21 knocking down the luciferase expression. A similar
result could be achieved in the miR-30 line via the exogenous
introduction of miR-30; however, due to its minimal levels in
HeLa cells a signifi cant effect on luciferase levels was not observed
without this addition. Due to the ability of the assay to knock
down luciferase expression, inhibition of the miRNA pathway at
any point should lead to a notable decrease in the miRNA levels
and consequently increase the levels of luciferase, providing a posi-
tive readout for the assay (Fig.
2
). Thus, when cells were grown in
the presence of a small-molecule inhibitor, compounds could be
easily identifi ed as miRNA effectors. Screening ~1,000 compounds
resulted in the discovery of
1
which increased luciferase expression
approximately 500 % relative to a DMSO-treated control (Fig.
3
).
In order to identify the mechanism of action, the miRNA and pri-
miRNA levels were examined by qRT PCR, demonstrating
decreased miR-21 and pri-miR-21 levels. Interestingly,
1
had no
effect on the levels of a variety of other miRNAs, indicating a
degree of miRNA specifi city. Unfortunately, this compound did
not result in the apoptosis of a glioblastoma cell line, and its actual
mechanism of action has yet to be established.
While the previously described assay was useful for the identi-
fi cation of small-molecule miRNA inhibitors, several issues pre-
cluded its true utilization in a high-throughput screen, including
