Luciferase Assay System
TECHNICAL NOTE:

High-throughput microRNA Target Screening:miR-122 Case Study
Patrick Collins, Michael Rose, Shelley Force Aldred, Nathan Trinklein

Introduction
MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role innumerous biological processes such as cellular signaling (1), cell differentiation, growth, development, andapoptosis (2). Mutations and improper regulation of miRNAs have been linked to a variety of physiologicaldisorders such as cancer and heart disease (3,4). In animals, miRNAs are usually complementary to one
or more a sites in the 3’UTRs of specific genes. Although current computational predictions of miRNA-UTR
interactions provide important guidance for experimental analysis of miRNAs, little functional data exists on
which to train prediction algorithms. Genome-wide transcript analysis can identify candidate target transcripts
but cannot measure both the changes in a transcript’s stability or translational efficiency attributable tomiRNAs. We have created a genome-wide library of human 3’UTR-luciferase reporter constructs to enable
researchers to screen thousands of potential miRNA targets in a high throughput fashion. Using this strategy,
we sought to identify new targets of miR-122, an important regulator of cholesterol and fatty-acid metabolism
in liver that has been suggested as a therapeutic target for metabolic disease (5).

Materials and Methods
SwitchGear Genomics has systematically identified human 3’ untranslated regions (3’UTRs) and createda genome-wide collection of 3’UTR regions cloned into our optimized luciferase reporter vector system
containing SwitchGear’s RenSP reporter cassette (GoClone) as a componenet of the LightSwitch Luciferase
Assay System. The RenSP reporter contains a PEST protein degradation sequence so that the destabilized
luciferase protein has a half-life of approximately 1 hour compared to the ~3 hour half-life of the native
luciferase protein, the CAT reporter protein half-life of ~50 hours, and GFP half-life of 25 hours. This modified
form of luciferase greatly facilitates detailed kinetic studies, especially those focusing on repression, whichmight otherwise be obscured by reporter protein accumulation. Co-transfection controls were originallydesigned to remove variability due to transfection efficiency alone (1). Such controls were not designed nor are
they adequate for comparing between different conditions or cell lines.

Cell culture
HT-1080 cells were cultivated in accordance with ATCC recommendations. The day before transfection, cells
were seeded in 96-well plates so that they would be 80-100% confluent at the time of transfection.

Preparation of 3’UTR Reporter GoClone Constructs and transfection
Using the SwitchGear Genomics recommended protocol(see Figure 1), we co-transfected HT-1080 cells with 100ngof individual 3’UTR-luciferase GoClone reporter plasmids
and either 20nM miR-122 mimic (Thermo Scientific, miRIDIAN
Mimic C-300591-05) or a non-targeting mimic control(miRIDIAN microRNA mimic negative control CN-001000-01).
All transfections were performed using the DharmaFECT Duo
Transfection Reagent (Thermo Scientific, T-2010).
Figure 1: Experimental design for miR-122 functional screen

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Luciferase Assay System

Measurement of luciferase activity and control measures
100uL of Luciferase reagent was added 24 hours after transfection to each well and the plate was incubatedfor 30 minutes in the dark before being read on a LmaxII-384 plate luminometer (Molecular Devices).
Knockdown of activity (interaction of the miRNA with the 3’UTR) for each construct was quantified as theratio of the luciferase signal observed with the miR122 mimic over the non-targeting control (luminescence= specific miRNA/non-targeting control). SwitchGear’s housekeeping, random, and vector-only GoClone
constructs were used in this assay to control for non-UTR specific treatment effects.

Results
After conducting a co-transfection experiment in HT1080
cells, we calculated the log2 ratio of luminescence observedfor each tested UTR reporter in the presence of the miR-122
mimic over the luminescence when transfected with the nontargeting control (Figure 2). Luminescence for 58/142 (40.8%)of the predicted targets was significantly different in the mimicco-transfection compared to the non-targeting control (P<0.05,t-test). Furthermore, of those 3’UTRs with significantly alteredluminescence, 25/58 (43.1%) were repressed 2-fold or moreby the miR-122 mimic. Thus, not only did this screen identify
numerous novel targets of miR-122, but we also found a numberof targets with more potent interactions with miR122 than werepreviously observed.

Figure 2: Summary of results from functional screen ofpredicted miR-122 targets in HT-1080 cells

In addition to performing a screen of putative targets using the 3’UTR GoClone
reporter collection, we also performed a number follow-up studies to provide
context for the initial results. We tested a number of the highly-repressed targets
from our screen at miRNA concentrations ranging from 0.0625 to 100nM. The tested
3’UTR reporters responded to miR-122 mimic in a dosedependent fashion, with
EC50 at or below 1.5 nM (Figure 3). We also tested for the specificity of the miR-122
knockdown by selectively mutating 2-3 bases in the seed recognition sequence ofthe 3’UTR reporter. In 5/6 cases, mutating the miR-122 seed recognition sequence
resulted in significantly decreased knockdown of luminescence in the presenceof the miR-122 mimic (Figure 4). The remaining 3’UTR mutant reporter exhibiteddecreased knockdown, but the change was not statistically significant. Finally,
we performed quantitative real-time PCR against endogenous 3’UTRs both in thepresence of the miR-122 mimic and the non-targeting control to test for knockdownof the endogenous message. Upon plotting the log2 ratio of mimic/non-targetingof both endogenous message and 3’UTR reporter luminescence we observed an
overall correlation of R=0.78 (N=14) (Figure 5).
Figure 3: Knock-down of 3’UTR luciferasechanges in a dose-dependent mannerwww.activemotif.com

Luciferase Assay System

Figure 4: Mutagenesis of miRNA seed recognition
sequence disrupts miRNA repression

Figure 5: 3’UTR luciferase activity is highly correlatedwith endogenous transcript levels

Summary
Using a number of complementary approaches, we have demonstrated that the results from the 3’UTR highthroughput reporter screen were dose-dependent, specific, and reproducible. Computational predictions
and transcript-based expression analysis alone cannot measure the functional roles of miRNAs, and our 3’UTR
reporter screen clearly demonstrates the ability to measure actual miRNA function. Luciferase assays provide
another advantage by measuring translational efficiency in addition to changes in message stability. In ourscreen, four of the 3’UTRs exhibited more repression by luminescence than by RT-PCR, highlighting transcriptsthat may be subject to translational repression. Our genome-wide library of human 3’UTR-luciferase reporter
constructs enables researchers to screen thousands of potential miRNA targets and understand the roles ofmiRNAs in a single experiment.

References
1. Cui Q, Yu Z, Purisima EO, Wang E (2006) Principles of microRNA regulation of a human cellular signaling
network. Mol Syst Biol 2: 46.
2. Esquela-Kerscher A, Slack FJ (2006) Oncomirs - microRNAs with a role in cancer. Nat Rev Cancer 6: 259–269.
3. Calin GA, Croce CM (2006) MicroRNA Cancer Connection:The Beginning of a New Tale. Cancer Research66, 7390-7394.
4. Chen JF, Murchison EP, Tang R, et al. (February 2008). Targeted deletion of Dicer in the heart leads to dilated
cardiomyopathy and heart failure. Proc. Natl. Acad. Sci. U.S.A. 105 (6): 2111-2116.

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