A P P L I C AT I O N N O T E

Modulating
endogenous
microRNA targets
with miRIDIAN
microRNA Mimics
and Inhibitors:
miR-122 inhepatocarcinoma
cells (Huh-7).

Introduction
MicroRNAs (miRNAs) have been shown to regulate gene
expression through both translational attenuation andmessenger RNA (mRNA) degradation. These small non-coding
RNAs typically target multiple genes simultaneously, inducing
subtle but reproducible shifts in target gene expression.
Synthetic miRNA mimics and inhibitors designed for potency
and specificity can be extremely valuable tools for probing
the function of target genes, thus helping to reveal the roleof miRNAs in development, differentiation, and disease.
Major challenges for researchers investigating miRNA function
include the confirmation of subtle phenotypic changes
associated with miRNA over-expression and inhibition, as wellas identification of miRNA gene targets. The identificationof a miRNA of interest can be performed using microarray
expression profiling, by RT-qPCR, or with reporter constructs
that register the status or changes to a specific biologicalpathway and/or phenotype (for example, target gene
expression, cell growth or differentiation). Another emerging
approach is the identification of miRNAs by screening withsynthetic reagents that mimic or inhibit function to demonstrate

Authors
Barbara Robertson
Allison St. Amand
Annaleen Vermeulen
Revvity, Inc.

a specific phenotypic effect.
Endogenous miRNAs are transcribed as stem-loop
structures and subsequently processed by several enzymes,
including the RNA-induced silencing complex (RISC), intofunctional, mature miRNAs with one or two mature (targeting)strands. miRIDIAN™ miRNA Mimics are synthetic duplexes
representing mature miRNAs as recorded in miRBASE(www. mirbase.org/). Like the naturally occurring miRNAs,
the synthetic mimics are loaded into RISC which enables
regulation of mRNA primarily through partial complementarity
to target sites in the 3’ UTR of a particular transcript.

Modulating endogenous microRNA targets with miRIDIAN microRNA Mimics and Inhibitors: miR-122 in hepatocarcinoma cells (Huh-7).

miRIDIAN Mimics have chemical modifications that areincorporated into a given strand of the molecule to establish

A.

A.

strand bias and ensure that the mature (targeting) miRNA
strand is preferentially loaded into RISC. Thus, by introducing

HumanMouse
RatDogthese molecular mimics into a cell type of interest, theresearcher can enhance or supplement endogenous miRNA
activity representing a gain-of-function assay.
Conversely, the addition of miRNA inhibitors into abiological system results in a loss-of-function assay witha predicted decrease in endogenous miRNA function.
In general, inhibitors comprise a non-hydrolyzable,
single-strand reverse complement to the mature miRNA
strand. The mechanism of inhibition is likely mediated byirreversible binding of the inhibitor to mature miRNA thus

B.B.

1........10........20..........30.........40
GCGGAGGUGUUCCCAGGCUGCCCC--CAACACUCCAGG-CCCUG
CCAGAGCUGAACUAAGGCUGCUCCAUCAACACUCCAGGCCCCUG
CCAGAGCUGAUCUAAGGCUGCUCCAUCGACACUCCAGGCCCCUG
GCGGAGGUGUUCUAAGGCUGCCCCCUCAACACUCCAGC-CCCCA
miR-122seed region

ALDOA A 3' UTR
5'...UCCCAGGCUGCC--CCCAACACUCCA...3'||::| :|||||||||
3'UGUUUG-UGGUAACAGUGUGAGGU 5'miR-122

Figure 1: Three miRNA target prediction programs predict thesame site for miR-122 in the ALDOA 3’ UTR (PicTar, TargetScan,miRBASE). A. The site is in a highly conserved region of the 3’ UTRof several species. B. The predicted binding of the miR-122 (blue)
to the ALDOA target is through a 8mer seed match (red).

preventing binding of the mature miRNA to its endogenous
targets1. Inhibitor potency can be improved by either
enhancing the binding of the inhibitor to the mature miRNAthrough chemical modification and/or by incorporating
additional attributes, such as structure, into the molecule.

Materials and methods
Cell cultureThe newest generation of inhibitors, miRIDIAN miRNA Hairpin

Huh-7 cells were obtained from ATCC and cultured under

Inhibitors incorporate terminal stem-loop structures andrecommended media conditions. The cells were plated inchemical modifications to further enhance potency andserum-containing medium without antibiotics in 96-well plates

extend inhibitory activity . The combination of these design(10,000 cells/well) approximately 24 hours before transfection.

2elements in loss-of-function assays increases the likelihoodof observing otherwise subtle phenotypes often associatedwith miRNA activity.
To demonstrate the utility of miRIDIAN miRNA Mimics
and Hairpin Inhibitors in assays for miRNA function
in an endogenous system, we chose a well-studied
miRNA:target-pair, miR-122:Aldolase-A (ALDOA). The ALDOA
message has a conserved miR-122 target site in its 3’
UTR and is normally repressed in hepatocytes (Figure 1).

miRNA mimics and inhibitors
Targeting and negative control miRIDIAN miRNA Mimics
and Inhibitors were synthesized by our researchers. Threedifferent inhibitor designs were synthesized: (1) a fully 2’O-methylated inhibitor reverse complement to the mature
miRNA (RC), (2) a first generation inhibitor design and(3) the current miRIDIAN miRNA Hairpin Inhibitor containing
flanking hairpin structures.

Previous studies suggested that a primary function for miR-

Exogenous dual luciferase assay for detection of

122 was to maintain the adult liver phenotype by suppressing

miR-122 function

expression of “non-liver” genes like ALDOA. This model issupported by work in mice where inhibition of miR-122 led

The dual-luciferase plasmid, psiCHECK-2 vector, containing

to up-regulation of ALDOA, a decrease in hepatic fatty acidboth the synthetic Firefly Luciferase (Fluc) gene and theand cholesterol synthesis, and reduced plasma cholesterolsynthetic Renilla Luciferase (hRluc) gene, each with its own

levels3-5. Here we describe an application for miRIDIAN miRNApromoter and poly(A)-addition sites, was obtained fromMimics and Inhibitors that further reinforces a regulatory role

Promega (Cat #C8021). A fully complementary target site

for miR-122 in liver tissue identity (phenotype and function),for miR-122 miRNA was inserted between the XhoI–NotI

and more importantly highlights a practical utility for theserestriction sites in the multiple cloning region in the 3’

novel reagents.

UTR of the hRluc gene2. For luciferase assays, cells wereco-transfected with 100 ng/well of the reporter plasmid
and a range of concentrations for the mimic or the inhibitor(1, 6, 16, and 40 nM) using 0.3 μL/well DharmaFECT™ Duo.

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2Modulating endogenous microRNA targets with miRIDIAN microRNA Mimics and Inhibitors: miR-122 in hepatocarcinoma cells (Huh-7).

Cells were grown at 37 °C and harvested at multiple
time points after transfection for luciferase and viability
assays. Firefly and Renilla luciferase activities weremeasured using the Dual-Glo™ Luciferase Assay System(Promega, Cat #E2980)2.
Endogenous ALDOA mRNA knockdown assay
For ALDOA mRNA knockdown assays, cells were transfectedwith a concentration range of either the miR-122 miRIDIAN
miRNA Mimic or Inhibitor (1, 6, 16, and 40 nM). Cells were
cultured at 37 °C and harvested 1, 3, 5 and 7 days
post-transfection. mRNA knockdown was determinedusing the branched DNA assay QuantiGene™ Screen Kit(Panomics, Cat #QG-000-050)6.
Cell viability assay
For both luciferase and mRNA detection, cell viability
was determined on a duplicate plate using the Thermo
Scientific™ alamarBlue™. Cell viabilities for control andexperimentally treated cells were always within 20% of
untreated (or mock treated) cells.

Results
Exogenous dual luciferase assay with miR-122 mimic
and inhibitors
Previous studies demonstrated that miR-122 targets
ALDOA mRNA in the liver of animal models5. Microarray
analysis performed in-house (data not shown) revealed
that the heptocellular carcinoma derived cell line, Huh7, expresses moderate levels of miR-122 as well. For thisreason, we chose the Huh-7 cell line as a good in vitro system
for miRNA gain and loss-of-function experiments. To test
mimic and inhibitor function, respectively, molecules wereco-transfected with either a dual-luciferase reporter plasmidthat detects miR-122 expression (see Materials & Methods)
or an empty psiCHECK-2 reporter plasmid. The untreatedand negative controls confirmed that miR‑122 is expressedat moderate but readily detectable levels in Huh-7 cells(Figure 2). Cells transfected with the miR‑122 reporter had
decreased Renilla luciferase expression levels relative tocells transfected with the empty psiCHECK-2 reporter. WhenmiRIDIAN miR-122 Mimics were co-transfected with the miR122 reporter, expression of Renilla luciferase was furtherdecreased (> 3 fold). Conversely, co-transfection with miR-122
hairpin inhibitor resulted in an increased (> 8 fold) expressionof Renilla luciferase.
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Figure 2: psiCHECK-2 dual luciferase assay in Huh-7 cells.
Huh-7 were transfected with reporter plasmids alone orco-transfected with miRIDIAN products with DharmaFECT Duo(0.3 μg/well). Luciferase expression was measured 48 hours aftertransfection. Transfection efficiency was normalized to Firefly
luciferase expression. Firefly/Renilla luciferase ratios were further
normalized to psiCHECK-2 reporter alone.

Endogenous ALDOA assay
While reporter assays permit rapid assessment offunctionality, monitoring the effect of miRNA mimics orinhibitors towards a biologically relevant target provides
greater insight into their effectiveness on putative endogenousmiRNA-target pairs. To explore how well the miRIDIAN
miRNA Mimics and Inhibitors performed when monitoring an
endogenous target, we transfected each class of regulatory
molecules into Huh-7 cells at 1, 6, 16, and 40 nM per welland monitored mRNA expression of the endogenous target(ALDOA) over time. As expected, transfection of the miRIDIAN
miRNA Mimic resulted in further downregulation of ALDOA
mRNA levels (Figure 3). Addition of miRIDIAN miRNA Hairpin
Inhibitor molecules to the cells resulted in increased in ALDOA
mRNA levels (Figure 3). While both the RC and first generation
version of miRNA inhibitors resulted in increased ALDOA
mRNA level, the inhibition was less pronounced compared tohairpin inhibitors.
Of note, modulation of miR-122 function with miRIDIAN
miRNA Mimics and Inhibitors revealed that the optimal time
for detecting effects on the ALDOA target may differ forthe corresponding modulator. For example, in this study
down-regulation of ALDOA mRNA levels is most pronounced
at day 3 posttransfection whereas increased expression
in response to miR-122 miRIDIAN miRNA Inhibitor wasbest observed at day 5 and 7 (Figure 3). Whether this is aconsequence of these specific reagents or a general feature3Modulating endogenous microRNA targets with miRIDIAN microRNA Mimics and Inhibitors: miR-122 in hepatocarcinoma cells (Huh-7).

of this methodology requires further investigation. Until there
is a greater body of evidence, we would suggest a timecourse as part of assay optimization for miRIDIAN miRNA
Mimic and Inhibitor studies.

hundreds of genes by a single miRNA and the potential for
combinatorial regulation of a single gene. Furthermore,
miRNA-mediated regulation and the resulting mRNA
attenuation often result in subtle phenotypic effects which
are difficult to detect. Consequently, there are currently
only a few well-characterized direct targets of miRNAs.
Recently, identification of miRNA-target relationships reliedon labor intensive miRNA and gene expression profiling and/or surrogate reporter assays. Now with the availability of
reliable miRNA-based mimics and inhibitors, researchers
have the necessary tools to perturb specific miRNA activities
and assess the phenotypic consequences in gain- or loss-offunction assays.
Here we describe the specific application of miRIDIAN miRNA
Mimic and Inhibitors in an endogenous system for gain- andloss-of-function experiments. To demonstrate the power ofFigure 3: Modulation of ALDOA mRNA levels in Huh-7 cell line
using miRNA mimics and inhibitors. Dashed red line indicates
ALDOA expression level as a result of endogenous miR-122
regulation. ALDOA mRNA expression values > 1 indicates
inhibition of miR-122, while values < 1 indicates additional
down-regulation of ALDOA by miRNA. miRIDIAN Hairpin and firstgeneration inhibitor performs well to inhibit miR-122, while RC
inhibitor effect is slight.

these novel molecular tools, we selected a gene target for
which evidence pointed to regulation by a defined miRNA,miR-122. Delivery of the corresponding miR-122 miRIDIAN
miRNA mimic resulted in reduction of the endogenous target(ALDOA), while delivery of either of two inhibitor designs
resulted in an increase in ALDOA mRNA level. The more
recent inhibitor design includes terminal hairpin structures,and unlike the first generation of short RC inhibitors, exhibited

Summary
miRNAs are non-coding RNAs that mediate posttranscriptional gene regulation via a mechanism thatinvolves RISC-mediated binding to complementary
sequences in the 3’ UTR of target genes. While miRNAs areclearly involved in critical steps of animal development,
cellular differentiation and disease, target identification
remains challenging. Efforts to identify gene targets areconfounded by unique attributes of miRNA regulation which
include regulation mediated by partial complementarityof the miRNA: target pair, the potential for modulation of

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highly functional and long‑lasting activity. This work further
reinforces the concept that secondary structure likely
promotes sustained function of the inhibitor permitting
detection of a potentially subtle endogenous phenotype2.
Therefore, the combination of gain-of-function (mimic-induceddown regulation) and loss‑of function (inhibitor-inducedup regulation) experiments provided further support for
direct miR-122-ALDOA target relationships. In summary,
we have demonstrated that miRIDIAN miRNA Mimic andHairpin Inhibitors can serve as complementary moleculartools providing critical phenotypic information and rapid
confirmation of putative miRNA-target interactions.

4Modulating endogenous microRNA targets with miRIDIAN microRNA Mimics and Inhibitors: miR-122 in hepatocarcinoma cells (Huh-7).

References
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function. RNA 13(5), 723-730 (2007).
3. J. Krutzfeldt, N. Rajewsky, Silencing of microRNAs in vivo
with ‘antagomirs.’ Nature 438(7068), 685-689 (2005).
4. Esau, C., S. Davis, miR-122 regulation of lipid metabolism
revealed by in vivo antisense targeting. Cell Metab.
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5. J. Elmen, M. Lindow, LNA-mediated microRNA silencing in
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7. J. Elmen, M. Lindow, Antagonism of microRNA-122 in miceby systemically administered LNA-antimiR leads toup-regulation of a large set of predicted target mRNAs inthe liver. Nucleic Acids Res. 36(4), 1153-1162 (2008).

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