Review

RESEARCH PAPER New Biotechnology �Volume 27, Number 6 �December 2010

Strategies to identify microRNA targets:New advancesHongtao Jin1, Wenbin Tuo2, Hai Lian1, Quan Liu1, Xing-Quan Zhu3 and Hongwei Gao1

1 Institute of Military Veterinary, Academy of Military Medical Sciences, Key Laboratory of Jilin Province for Zoonosis Prevention and Control, 1068 Qinglong Road,Changchun 130062, Jilin Province, People’s Republic of China2Animal Parasitic Diseases Laboratory, Animal and Natural Resources Institute, United States Department of Agriculture, Beltsville, MD 20705, USA3 State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, CAAS, Lanzhou 730046, Gansu Province,People’s Republic of China

MicroRNAs (miRNAs) are small regulatory RNA molecules functioning to modulate gene expression at

the post-transcriptional level, and playing an important role in many developmental and physiological

processes. Ten thousand miRNAs have been discovered in various organisms. Although considerable

progress has been made in computational methodology to identify miRNA targets, most predicted

miRNA targets may be false positive. Due to the lack of effective tools to identify miRNA targets, the study

of miRNAs is seriously retarded. In recent years, some molecular cloning strategies of miRNA targets have

been developed, including RT-PCR using miRNAs as endogenous primers, labeled miRNA pull-down

assay (LAMP) and RNA ligase-mediated amplification of cDNA end (RLM-RACE). The identified miRNA

targets should be further validated via effects of miRNA alteration on the target protein levels and

bioactivity. This review summarizes advances in strategies to identify miRNA targets and methods by

which miRNA targets are validated.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

Bioinformatics prediction of miRNA targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

Molecular cloning of miRNA targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

Labeled miRNA pull-down assay (LAMP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

RNA ligase-mediated amplification of cDNA end (RLM-RACE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

Other methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736

Functional validation of miRNA targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736

Over-expression of miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736

Loss-of-function of miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737

Corresponding authors: Liu, Q. (liuquan1973@hotmail.com), Gao, H. (ccltdz@sohu.com)

734 www.elsevier.com/locate/nbt 1871-6784/$ - see front matter � 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2010.09.006

New Biotechnology �Volume 27, Number 6 �December 2010 REVIEW

Review

IntroductionMicroRNAs (miRNAs) are short non-coding RNAs that play impor-

tant roles in gene regulation during physiological or disease-asso-

ciated processes by specifically suppressing the translation of

target genes by binding to the mRNAs. MiRNAs are transcribed

by RNA polymerase II or III in larger precursors as primary miRNAs

(pri-miRNAs) containing characteristic stem-loop structures that

are processed in the nucleus by a complex of the RNase III enzyme

Drosha [1,2]. Drosha products are about 65-nt long hairpins (pre-

miRNAs), which are exported into the cytoplasm by Exportin-5

and Ran-GTP, where they are cleaved by the RNase III enzyme

Dicer to release the mature miRNAs that are RNA duplexes of about

22-nt in length [3,4]. One strand of the duplex (miR strand) is

selectively loaded onto an argonaute protein (Ago), the RNA-

induced silencing complex (RISC) is formed and can now bind

to, and repress target mRNAs containing sites of partially com-

plementary to the miRNA [5,6]. These miRNAs perform a variety of

significant functions for cell such as growth, metabolism, devel-

opment, and cell differentiation [7,8].

The first miRNAs found in Caenorhabditis elegans are lin-4 and

let-7, which control the developmental timing by regulating

translation of their respective targets [9,10]. miR-1 and miR-133

modulate muscle gene expression and sarcomeric actin organiza-

tion in zebrafish [11]. In mice, miR-21 plays a role in tumor

growth, invasion and metastasis by targeting at multiple tumor/

metastasis suppressor genes, including programmed cell death 4

(PDCD4) and maspin [12,13]. miR-1 regulates cardiomyocyte

apoptosis by post-transcriptional repression of Bcl-2 [14]. miR-

124a regulates human IkappaBzeta, which is involved in the fine-

tuning of NF-kappaB-mediated gene expression [15]. In herpes-

virus, including human cytomegalovirus (HCMV), Kaposi’s sar-

coma-associated herpesvirus (KSHV), and Epstein-Barr virus (EBV),

miRNAs function to target at the stress-induced immune ligand

MICB to escape recognition by natural killer cells [16]. Only a few

examples are mentioned here, and there are many more miRNA

functions, which cannot be described in this paper.

Up to now, an estimated 9539 mature miRNAs have been found

in animals, plants and viruses (http://microrna.sanger.ac.uk/

sequences/), and miRNAs are estimated to account for about 1–

2% of the known eukaryotic genes, and regulate at least 30% of

genes in animals, which has not been experimentally proven yet

[17].

Computational analysis suggests that a single miRNA could

regulate more than 200 mRNAs and that a single mRNA may be

regulated by multiple miRNAs [18]. However, only a few miRNAs

targets have been identified by biological methods.

Bioinformatics prediction of miRNA targetsSome computational programs based on sequence alignment,

including TargetScanS, PicTar, and miRanda, have been developed

to predict miRNA targets [19–21]. However, these methods have

their inherent limitations. Firstly, computational approaches have

been successful in plants, due to the perfect complementarity

between miRNAs and mRNA targets. In animals, however, the

miRNA/mRNA is usually less complementary, making it inaccu-

rate to predict target sites. Secondly, conventional sequence align-

ment usually requires longer sequences, and the short sequence

makes it difficult to rank and score targets. Therefore, the com-

monly used sequence analysis tools appear to be relatively less

useful to predict miRNA target [22]. Thirdly, it would not seem to

be realistic for a single miRNA to regulate hundreds of targets, and

effective regulation of translation from transcripts may require

miRNAs and their targets to be located in the same cellular

compartments. Hence, most of these theoretical targets may be

false [23]. Finally, 30-UTR data are very limited, making it difficult

to predict miRNA targets.

Molecular cloning of miRNA targetsRT-PCRMiRNA can be used as a primer for direct synthesis of the target

cDNA because it is completely or partially complementary to the

30-end of its target mRNA, and form a temporarily stable complex,

which can initiate cDNA synthesis in cases of weak complemen-

tarity between the 30-end of the miRNA and the target mRNA. The

resultant cDNA, with miRNA on its 50-end, can be easily cloned

and sequenced [24,25].

The method contains two rounds of reverse transcription. The

first is performed on an mRNA template using miRNAs as the

endogenous cytoplasmic primer, which can extend miRNA and

overcome the problem of low complementary binding of miRNAs

to their targets. The second is performed on purified hybrid 30-

cDNA–miRNA-50 molecules (primeRNA), which can anneal to

target mRNA in a highly gene-specific manner, and increase the

length of cDNA for detection [24].

Labeled miRNA pull-down assay (LAMP)LAMP is a simple, direct and cost-effective approach to identify

miRNA targets in vitro. The precursor miRNA (pre-miRNA) is

synthesized in vitro, labeled with digoxigenin (DIG), and mixed

with cell extracts. The endogenous Dicer in cell extract processes

the pre-miRNA and produces mature miRNA in vitro. The DIG-

labeled miRNA then binds to its target gene(s) in cell extract,

presumably through the endogenous RNA-induced silencing com-

plex (RISC). The DIG-miRNA–target mRNA complex is precipi-

tated by anti-DIG antiserum. The isolated miRNA-specific mRNA is

reverse transcribed, and the total cDNAs are amplified, subcloned

and sequenced [26].

Compared to the conventional bioinformatics approach, the

LAMP assay has its advantages. Firstly, there is no need to have a

complete and genome-wide database of 30-UTR. Secondly, the

cDNA array is not necessarily required for LAMP analysis, which

is particularly important to study certain organism without cDNA

array. Finally, compared with the conventional microarray ana-

lysis, the LAMP assay is relatively simple to perform and specific for

isolation of miRNA targets from cell extracts [26]. However, DIG

label may influence Dicer processing or strand selection, and there

are some non-specific amplifications using LAMP to identify target

genes of miRNAs, which may result from random ligation of the

primers used in RT-PCR, or non-specific binding between target

mRNA and RISC.

RNA ligase-mediated amplification of cDNA end (RLM-RACE)This strategy is designed to isolate the pre-miRNA cleavage pro-

ducts from biological samples. Poly(A)+ RNA is ligated to the RNA

adapter and reverse transcribed using an oligo(dT) primer. Synth-

esis of the second strand of cDNA is directed from an oligonucleo-

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tide primer complementary to the RNA adapter containing the

sequence for the bacteriophage T7 promoter. Synthesized double-

stranded cDNA is subjected to in vitro transcription with T7 RNA

polymerase, allowing linear amplification of cDNA containing the

ligated adapter. In vitro transcribed RNA is used as a template for

the synthesis of single-stranded cDNA, and the resulting cDNA is

biotinylated and hybridized to a transcriptome microarray.

Since RNA adapter and/or T7-oligonucleotide may anneal non-

specifically to RNA and cDNA, increasing the background signal,

negative controls consisting of cDNA populations, in which the

RNA adapter is included, but not ligated to 50 cleaved transcripts,

are used in an attempt to identify background hybridization

[27,28].

This method identifies miRNA targets by T7-mediated amplifi-

cation and microarray hybridization. The T7 RNA polymerase

amplification allows enrichment of mRNA targets, increasing

the sensitivity for target detection. Subtraction of negative control

hybridization signals from those of the enriched cDNAs greatly

reduces non-specific signals as a result of binding to non-target

mRNAs, minimizing the number of false positives. In addition, the

use of biological replicates reduces variability and increases sta-

tistical confidence. One of the disadvantages of RLM-RACE strat-

egy is that it does not discriminate between miRNA and siRNA

cleavage products, since both RNA species contain ligation-com-

petent 50 monophosphates [29].

Other methodsThere are some techniques to purify microribonucleoprotein

(miRNP) complexes associated with miRNAs and mRNA targets.

For example, isolation of miRNA/mRNA complexes by immuno-

purification of Ago1-containing protein complexes using anti-

influenza protein hemagglutinin (HA) antibody in Drosophila

melanogaster expressing functional HA epitope tagged Ago1 [30],

purification of RISC by Ago2-coimmunoprecipitation approach

combined with microarray analysis of mRNAs [31], imunopreci-

pitation of Ago-bound mRNAs using specific monoclonal antibo-

dies against the Ago protein family [32], affinity purification of

AIN-1 and AIN-2, other RISC associated proteins using correspond-

ing antibody [33], ribonucleoprotein immunoprecipitation-gene

chip (RIP-Chip) using antibodies against wild-type human Ago2 in

Hodgkin lymphoma cell lines [34]. These approaches allow the

analysis of the complete transcriptome for miRNA targets, and

permit more direct identification of physiologically relevant

miRNA targets.

The cross-linking and immunoprecipitation assays (CLIP), a

method for identifying protein–RNA interaction sites in living

cells, have also been used for miRNA target identification recently

[35,36]. This method exploits UV-irradiation to covalently cross-

link RNA–protein complexes, allowing them to be stringently

purified using immunoprecipitation. RNA fragments resulted from

digested complexes can be sequenced by high throughput meth-

ods. However, CLIP is limited by the low efficiency of UV 254 nm

RNA–protein cross-linking, and the location of the crosslink is not

readily identifiable within the sequenced crosslinked fragments,

making it difficult to separate UV-crosslinked target RNA segments

from background non-crosslinked RNA fragments. So an improved

method, called photoactivatable-ribonucleoside-enhanced cross-

linking and immunoprecipitation (PAR-CLIP) has been developed,

736 www.elsevier.com/locate/nbt

in which 4-thiouridine is incorporated into transcripts of cultured

cells and the RNA-binding protein (RBP) binding sites can be

identified by scoring for thymidine to cytidine transitions in

the sequenced cDNA [37,38].

Functional validation of miRNA targetsIf an mRNA is a true target of a miRNA, change of miRNA should

have predictable effects on target protein. The typical approach to

validate the functional importance of a miRNA target is a transient

over-expression of a given miRNA in a cell, and the target protein

levels and bioactivity are determined [39,40].

Over-expression of miRNAOver-expressed miRNA can be performed by transfection with

chemically synthesized ds-RNA precursors in vitro, which may

imitate endogenous miRNAs. Due to their small size and directly

introduction into cells by transfection, they can be easier to deliver

and use in dosage response studies than vectors [41,42]. Long-term

studies request numerous miRNA, which can be produced by

miRNA expression plasmids and viral vectors. The design of

miRNA expression constructs is relatively simple. miRNAs are

transcribed by RNA polymerase II, and their primary transcripts

contain a cap and poly(A) tail. Introducing miRNA sequences

flanked by at least 40-nt from their precursor into DNA plasmids

is necessary to yield mature miRNAs [43]. Such constructs are

being widely used in cultured cells. In order to overcome the

low transfection efficiency of miRNA expression plasmids in pri-

mary cells, adenovirus-, adeno-associated virus-, and lentivirus-

delivered system have been developed [44–46].

The reporter system is usually be used to validate the functions

of miRNA, whose rationale is that binding of miRNA to its specific

mRNA will repress reporter protein expression and the altered

miRNA will increase or decrease reporter protein expression level.

The experimental approach is to clone the 30-UTR of the candidate

target gene immediately downstream of the reporter gene open

reading frame (ORF) sequence to generate the reporter construct.

The reporter construct and a miRNA of interest are then transiently

transfected into host cells. Thereafter, the reporter gene is detected

24–48 h after transfection [47,48].

Loss-of-function of miRNADue to possible mis-expression of mRNAs, which may result from

low expression of the endogenous miRNA or spatial differences

between the miRNA and its target, results of over-expression

should be further confirmed by loss-of-function experiments [49].

Gene knockout and antisense technologies are used to silence

miRNA function. Knockout of the miRNA processing gene Dicer1

can cause deficiency of all mature miRNAs, and knockout of

miRNA genes and mutation of miRNA target sites in protein-

encoding genes can interrupt miRNA-mediated gene regulation

[50,51]. However, conditional inactivation of Dicer cannot define

the exact role of a single miRNA. In such a case, it is necessary to

restore the expression of specific miRNAs.

Oligoribonucleotides complementary to miRNA, for example,

20-O-methyl-modified oligoribonucleotides and cholesterol-con-

jugated single-stranded RNAs (antagomirs), can inhibit miRNA

expression [52–55]. The 20-O-methyl modifications act as irrever-

sible, stoichiometric inhibitors of small RNA function. Antagomirs

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are stabilized with a phosphorothioate backbone with several

sulfates and 20-O-methyl modification. Antagomirs are also mod-

ified at the 20 carbon (at least some nucleotides). An alternative

strategy to target miRNAs in vivo has recently been reported using

antisense oligonucleotides (ASOs), which are unconjugated single-

stranded RNAs carrying phosphorothioate backbone and 20-O-

methoxyethyl modification [56,57].

Locked nucleic acids (LNAs) are a class of conformationally

restricted nucleotide analogs, whose incorporation in an oligo-

nucleotide increases the affinity of that oligonucleotide for

its complementary RNA or DNA target, and can be used to

modify oligoribonucleotides to silence miRNA expression

[58,59].

Recently, miRNA sponges have been used as miRNA inhibitors,

which are transcripts expressed from strong promoters (RNA poly-

merase II promoter), containing multiple, tandem miRNA binding

sites in its 30-UTR, allowing to soak up miRNA and relieve its targets

from binding of miRNA [60,61]. When vectors encoding these

sponges are transiently transfected into cultured cells, sponges

depress miRNA targets as strongly as chemically modified anti-

sense oligonucleotides. This new tool has several advantages, such

as the possibility to express the inhibitor in conjunction with

reporter genes, making it easy to sort sponge-treated cells. In

addition, the expression of sponges from stably integrated trans-

genes can be used to explore tissue-specific miRNA functions in

vivo [62].

SummaryMiRNAs are important regulators of cell growth, differentiation,

and apoptosis, and dysregulation of miRNA function may lead to

certain diseases. It is critically important to be able to identify and

validate miRNA targets. Due to high false predicted miRNA targets,

all kinds of molecular cloning of miRNA targets have been devel-

oped. However, the exact mechanism of miRNA-mediated repres-

sion is still debated and it is not known how miRNA-mediated

mRNA destabilization and translational repression, and some

genes may be repressed on the protein level without being affected

on the mRNA level. Many proteomic methods should be produced

to complement present DNA strategies in the future, and the wider

availability of experimentally validated miRNA targets and their

action mechanisms will certainly permit more reliable computa-

tional predictions. A high throughput strategy for miRNA func-

tional validation is also needed. We also expect that more and

more functional roles of disease-specific miRNAs will be elucidated

and novel therapeutic strategies be developed.

AcknowledgementsThis work was supported by grants from the National Key

Technology R&D Program in China (Grant No. 2010BAD04B01),

the National Natural Science Foundation of China (Grant No.

30972178), and ‘‘Gold Idea’’ foundation of Institute of Military

Veterinary, Academy of Military Medical Sciences (Grant No.

YCX0901).

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