Biology Reference
In-Depth Information
immune disorders, and viral infections [
8
-
12
]. Small molecule
modifi ers of miRNA function [
13
-
15
] could serve as important
tools for elucidating the biological roles of miRNAs and as lead
structures for the development of potential new therapeutic agents.
In order to identify small molecule modifi ers of miRNAs, fi rst
functional assays for specifi c miRNAs must be developed. Here, we
report the development of an assay for miRNA miR-122 function
based on a luciferase reporter.
MiRNA miR-122 is involved in the regulation of lipid and
cholesterol metabolism and is the most abundant miRNA in the
liver [
16
]. Furthermore, miR-122 is down-regulated in hepatocel-
lular carcinoma (HCC) and targets the anti-apoptotic Bcl-2 family
member Bcl-w [
17
]. It was discovered that miR-122 is required by
the hepatitis C virus for replication and infectious virus production
through interaction with the viral genome [
18
]. The HCV genome
contains two miR-122 target sites in the 5
noncoding region of
the virus and miR-122 recognition results in the up-regulation of
viral RNA production [
19
]. Moreover, miR-122 may stimulate
HCV translation [
20
] through interactions with the HCV internal
ribosome entry site (IRES) and argonaute proteins [
21
]. It was
recently reported that knockdown of miR-122 with antisense
agents resulted in a decrease in HCV RNA replication in human
liver cells [
18
] and reduced HCV levels in chronically infected pri-
mates [
22
]. These promising results suggest that small molecule
inhibitors that target miR-122 could provide a new approach for
antiviral therapy. The development of a functional assay followed
by a high-throughput screen has the potential to deliver such
molecules.
A reporter assay for mature miR-122 function was constructed
based on the psiCHECK-2 (Promega) reporter plasmid [
15
]. The
psiCHECK-2 vector has a multi-cloning site at the 3
′
terminus of
the
Renilla
luciferase gene where a miRNA target sequence can be
inserted. Here, the complementary sequence of mature miR-122
was cloned between the
Pme
I and
Sgf
I restriction sites, generating
the reporter construct psiCHECK-miR122. After transfection of
the psiCHECK-miR122 construct into cells expressing endoge-
nous miR-122,
Renilla
luciferase expression is inhibited (Fig.
1a
).
In the presence of a miR-122 inhibitor, the
Renilla
luciferase
expression will be restored, leading to an increased luciferase signal
(Fig.
1b
). Utilizing a reporter assay that results in an increased lucif-
erase signal in the presence of an active miR-122 inhibitor rules out
false-positive hits due to compound toxicity, which can occur in an
assay based on a decrease in reporter signal [
23
]. The ability of the
reporter to detect endogenous miR-122 was confi rmed by tran-
siently transfecting the generated psiCHECK-miR122 construct
into Huh7 human hepatoma cells [
15
]. The assay was further vali-
dated by co-transfection of a miR-122 antagomir antisense agent as
a positive control. The empty psiCHECK-2 vector without the
′
