Biology Reference
In-Depth Information
Chapter 11
Cellular MicroRNA Sensors Based on Luciferase Reporters
Colleen M. Connelly and Alexander Deiters
Abstract
Recently, microRNAs (miRNAs) have been linked to a variety of human diseases including cancer and viral
infections. Small molecule modifi ers of miRNAs could represent new therapeutic agents and be used as
tools for elucidating the biological roles of miRNAs. In order to identify small molecule modifi ers of miR-
NAs, functional assays for specifi c miRNAs must be developed and optimized. Here, we report the con-
struction of a luciferase reporter assay for miRNA miR-122 function and the development of a stable
Huh7 cell line that can be used for high-throughput screening of small molecule miR-122 inhibitors. The
steps described here can be applied not only to Huh7 cells and miR-122 but also to virtually any cell line
and miRNA combination.
Key words
MicroRNA, Cell-based assay, Luciferase, High-throughput screen
1
Introduction
MicroRNAs (miRNAs), single-stranded RNAs of approximately
22 nucleotides, regulate gene expression in a sequence-specifi c
fashion by binding partially complementary sequences in the
3
-UTR) of target messenger RNAs
(mRNAs) [
1
]. MiRNAs are transcribed from the genome into
primary miRNAs (pri-miRNAs) and undergo several posttranscrip-
tional processing steps via a dedicated miRNA pathway to produce
mature miRNAs. MiRNAs down-regulate gene function by inhib-
iting translation, accelerating the degradation of the target mRNA,
or mediating deadenylation of the mRNA [
1
,
2
]. Almost 2,000
human miRNAs are currently listed in miRBase [
3
,
4
], and it is
estimated that they are involved in the control of more than 30 %
of all genes [
5
]. Biological processes regulated by miRNAs include
embryonal development, differentiation, apoptosis, and prolifera-
tion [
6
], and the targets of miRNAs range from signalling proteins
and transcription factors to RNA binding proteins [
7
]. Recently,
the aberrant expression of certain miRNAs has been linked to a
variety of human diseases, including cancer, cardiovascular diseases,
′
-untranslated region (3
′
