MINI REVIEW

MicroRNA and cancer: Current status and prospective

Wei Wu1*, Miao Sun1, Gang-Ming Zou2 and Jianjun Chen1*

1Department of Medicine, University of Chicago, Chicago, IL2Herman B Wells Center for Pediatrics Research, Indiana University School of Medicine, Indianapolis, IN

Gene expression in normal cells is highly regulated by complexgene regulatory networks. Disruption of these networks may leadto cancer. Recent studies have revealed the existence of an abun-dant class of small nonprotein-coding regulatory RNAs, known asmicroRNAs (miRNAs). MiRNAs may regulate diverse biologicalprocesses including development, cell proliferation, differentiationand apoptosis, through suppressing the expression of their targetgenes. Posttranscriptional silencing of target genes by miRNAsoccurs either by cleavage of homologous target messenger RNAs(mRNAs), or by inhibition of target protein synthesis. Computa-tional predictions indicate that 1 miRNA may target on hundredsof genes, and suggest that over 50% of human protein-codinggenes might be regulated by miRNAs. MiRNAs are receiving in-creased attention in cancer genomic research. We are beginningto understand that miRNAs may act as oncogenes and/or tumorsuppressor genes within the molecular architecture of gene regula-tory networks, thereby contributing to the development of cancer.MiRNAs may provide useful diagnostic and prognostic markersfor cancer diagnosis and treatment, as well as serving as potentialtherapeutic targets or tools.' 2006 Wiley-Liss, Inc.

Key words: microRNAs; targets; oncomir; tumor suppressor gene;apoptosis; antagomir; cancer

Cancer is a complex genetic disease in which oncogene amplica-tion and/or tumor suppressor gene mutation leads to step-wisederegulation of cell proliferation and apoptosis. Evidence is emerg-ing that particular microRNAs (miRNAs) may play a role in humancancer pathogenesis. MiRNAs are endogenous �22 nucleotide (nt)noncoding RNAs (ncRNAs), which can play important regulatoryroles in animals and plants by pairing to the messenger RNAs(mRNAs) of target genes and specifying mRNA cleavage orrepression of protein synthesis.1–4 Recent evidence indicates thatmiRNAs exhibit important regulatory roles in development, cellproliferation, cell survival, and apoptosis, and thus play a centralrole in gene regulation in health and disease.5–7 The miRNAs thatwere first discovered, lin-48,9 and let-7,10,11 were identified geneti-cally in Caenorhabditis elegans (C. elegans); it became clear thatmiRNAs represent novel means of regulating developmental tim-ing in C. elegans. Subsequently, hundreds of nonprotein-codingmiRNAs were identified across species, showing that they arehighly conserved.6,12–14

In practice, when a small RNA is discovered by cDNA cloning,typically, it can be classified as a miRNA if it meets 2 of the follow-ing 3 criteria: (i) its expression should be confirmed by Northernblotting, RT-PCR or RNase protection assay, and so on (i.e. expres-sion criterion); (ii) the sequence should be present in 1 arm of thehairpin precursor, which lacks large internal loops or bulges (i.e.structure criterion); (iii) the sequences should be phylogeneticallyconserved (i.e. conservation criterion).15 If a small RNA is found bymethods other than cDNA cloning, its expression must be demon-strated along with the precursor structure and conservation.15 Todate, the public miRNA database, MiRBase (http://microrna.san-ger.ac.uk; release 8.1), has collected 462 human and 340 mousemiRNA sequences. Both cloning and bioinformatic approacheshave shown that the human genome contains a much larger numberof miRNAs than previously appreciated.16 It has been suggestedthat the total number might be above 800 or even 1,000.5 MiRNA

genes can be located in the introns and/or exons of protein-codinggenes or in the intergenic regions between protein-coding genes.They can form polycistronic clusters or exist individually.1,13,17

Biochemical approaches have provided a basic understandingof the molecular details of miRNA biogenesis.15 As shown inFigure 1, a primary transcript RNA (pri-miRNA) transcribed from amiRNA gene by RNA polymerase II is first processed into a stem-loop structure of about 70–100 nt (precursor miRNA; pre-miRNA)by a double-strand (ds)-RNA-specific ribonuclease, Drosha, with thehelp of its binding partner DGCR8.18 These pre-miRNAs are trans-ported into the cytoplasm via an Exportin-5-RanGTP dependentmechanism.19–21 In the cytoplasm, they are digested by a second,dsRNA-specific ribonuclease called Dicer with the help of TRBPand AGO2.22–24 The released 17–25 nt mature miRNA is bound bya complex called miRNA-associated RNA-induced silencing com-plex (miRISC). Two mechanisms for miRNA action are currentlyknown: mRNA cleavage and translational repression of mRNAwithout RNA cleavage. In animals, the complex-bound, single-stranded miRNAs binds specific target mRNAs through the sequencethat is significantly, though not completely complementary to the tar-get mRNAs.25 By a mechanism that is not fully characterized, thebound mRNA remains untranslated, resulting in reduced expressionof the corresponding genes. A single miRNA could regulate multiple tar-get genes, while a single gene could be targeted by multiple miRNAs,suggesting that the miRNAome and mRNAome interaction is acomplicated network. For more details, see reviews.1,6,15,17,26–28

Several excellent review papers focusing on the link betweenmiRNAs and cancer, e.g., Refs. 7, 26, 27 and 29–32 have beenpublished in recent years. Particularly, Esquela-Kerscher andSlack’s review26 is a very elegant and comprehensive one on thistopic, in which the connection of miRNAs and cancer has beendiscussed in depth. In the present review, we are trying to describea more general and up-to-date picture regarding the association ofmiRNAs with cancer, as well as miRNA regulation, signalingpathways and target prediction and validation. Some recentadvancements in this field that have not been included in the pre-vious reviews are also discussed here.

Deregulation of miRNA expression in cancer

Rapidly accumulating evidence has revealed that miRNAs areassociated with cancer because of deregulation.7,26,29,30,33 Genome-wide studies demonstrated that miRNA genes are frequently located

Wei Wu’s current address is: Division of Cancer Biology, Departmentof Medicine, Northwestern University, Chicago, IL 60201, USA.*Correspondence to: Division of Cancer Biology, Department of Med-

icine, Northwestern University, ENH research Institute, 1001 UniversityPlace, Evanston, IL 60201, USA. Fax:11-224-364-7402.E-mail: wei-wu-0@northwestern.edu or Department of Medicine, Univer-sity of Chicago, 5841 S. Maryland Avenue, MC2115, Chicago, IL 60637,USA. Fax:11-773-702-3002. E-mail: jchen@medicine.bsd.uchicago.edu

Grant sponsors: G. Harold and Leila Y. Mathers Charitable Foundation;Cancer Research Foundation Young Investigator Award.

Received 28 March 2006; Accepted after revision 29 September 2006DOI 10.1002/ijc.22454Published online 12 December 2006 inWiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 120, 953–960 (2006)' 2006 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

at cancer-associated genomic regions or in fragile sites, as well as inthe minimal regions of loss of heterozygosity, in the minimalregions of amplifications or in the common breakpoint regions. Thissuggests that miRNA might be a new class of genes involved inhuman tumorigenesis. Downregulation of miRNAs is observed fre-quently in cancer samples. For example, miR-15 and miR-16 at13q14 were deleted and/or downregulated in about 68% of B-cellchronic lymphocytic leukemia (CLL) cases.34,35 MiR-143 and miR-145 consistently display reduced steady-state levels of the maturemiRNAs in colon cancer36 and in lung cancer37 compared to thelevels in normal tissues. Three research groups37–39 subsequentlyreported the reduced expression of the miRNA let-7 in human lungcancers relative to normal tissues, which indicates a poor prognosis.Lu et al.40 found that the absolute expression levels of many miR-NAs were reduced significantly in tumors. Thus, the frequentlyobserved downregulation of miRNAs in cancer tissues relative tonormal tissues suggests that many miRNAs may function as tumorsuppressor genes. Because the abrogation of differentiation is a hall-mark of cancers, the low-level expression of such miRNAs may berelated to the state of poor differentiation of cancer cells. If so, theexpression of these miRNAs would be upregulated when the cancercells are induced to differentiation. Indeed, Sempere et al. showedthat 19 miRNAs existing in brain tissue (including lin-4 and let-7orthologs) were downregulated in both human and mouse embry-onal carcinoma cells, and their expression is upregulated duringneuronal differentiation induced by retinoic acids.41 Similarly, Luet al. treated the myeloid leukemia cell line HL-60 with all-trans ret-inoic acid for 5 days, and miRNA profiling revealed the induction

of 59 miRNAs being coincident with differentiation.40 Therefore, ageneral downregulation of miRNAs in tumors and an upregulationof these same miRNAs during differentiation, indicate that miRNAexpression reflects the state of cellular differentiation. Thus, Luet al.40 speculated that abnormalities in miRNA expression mightsimilarly contribute to the generation or maintenance of ‘‘cancerstem cells’’, which was proposed to be responsible for cancerousgrowth in both leukemias and solid tumours.42 In addition, restoringthe expression of such miRNAs in cancer cells probably could pro-mote differentiation and induce malignant cells into a benign or nor-mal state.

On the other hand, upregulation of miRNAs is also observed fre-quently. For example, over-expression of precursor miR-155/BICRNA was reported in children with Burkitt lymphoma,43 human B-cell lymphomas44 and Hodgkin lymphoma.45 Further, Yanaiharaet al. found that high expression of miR-155 has a significantlyworse prognostic impact on patients with lung adenocarcinoma asan independent risk factor, and therefore could serve as a marker forsurvival.37 Although miR-155 is overexpressed in several types ofhuman cancers, its biological function remains uncertain. However,a previous study has shown that BIC (host gene of miR-155) isimplicated as a collaborator with c-myc in an avian lymphomamodel system.46 A polycistronic miRNA cluster, mir-17-92(including 7 miRNAs: miR-17-5p, miR-17-3p, miR-18a, miR-19a,miR-20a, miR-19b-1 and mir-92-1), residing in the C13orf25 genelocus on chromosome 13, was upregulated in 65% of B-cell lym-phoma samples.47,48 The increased expression of these miRNAs cor-relates with the presence of the 13q31 amplicon.47 The high-level

FIGURE 1 – Diagram of microRNA biogenesis. MicroRNA (miRNA) genes are transcribed from genomic DNA to produce the pri-miRNA.Pre-miRNA products (�70 nt) are generated by Drosha/DGCR8/Pasha complex, also known as microprocessor complex. The pre-miRNA isexported from the nucleus to the cytoplasm by Exportin 5/Ran-GTP. In the cytoplasm, a second RNasIII enzyme, Dicer, together with its part-ner, such as the trans-activator RNA-binding protein (TRBP), produces miRNA duplexes (�22 nt). Mature miRNA is produced, and guarded bythe effector complex, called miRISC to target mRNA. MiRNAs regulate the target genes by translational inhibition or mRNA degradation.

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amplification of 13q31 has been observed in hematological andother solid neoplasms,48,49 including diffuse large B-cell lym-phoma,50 primary cutaneous B-cell lymphoma,51 follicular lym-phoma,52 mantel cell lymphoma,53 nasal-type natural killer/T-celllymphoma,54 glioma,49 nonsmall cell lung cancer,49 bladder can-cer,49 squamous-cell carcinoma of the head and neck,49 peripheralnerve sheath tumor,55 malignant fibrous histiocytoma,56 alveolarrhabdomyosarcoma57 and liposarcoma.58 The oncogenic functionof the mir-17-92 cluster is further validated by a number of piecesof evidence as follows. MiR-19a and miR-92-1 are overexpressedin cells from patients with B-cell CLL.35 MiR-17-5p is overex-pressed in breast, colon, lung, pancreas and prostate tumors, whilemiR-20a is overexpressed in colon, pancreas and prostate tumors.59

MiR-17-3p is overexpressed in lung adenocarcinoma and is relatedto the patient’s survival.37 MiRNAs from the mir-17-92 cluster areoverexpressed in lung cancers and enhance cell proliferation.60

Moreover, introduction of the expression construct of the mir-17-92 cluster, but not the putative open reading frame of C13orf25,could enhance lung cancer cell growth.60 Most importantly, to testthe hypothesis directly that increased expression of this cluster con-tributes to cancer formation, He et al.47 overexpressed the mir-17-19b-1 cluster (the vertebrate-specific portion of the mir-17-92 clus-ter) using a mouse model of human B-cell lymphoma. Hematopoi-etic stem cells (HSCs) derived from fetal livers of c-myc transgenicmice generate B-cell lymphomas by 4–6 months of age when trans-planted into lethally irradiated recipients.61 Irradiated animals thatreceived HSCs overexpressing both Myc and the mir-17-19b-1cluster developed malignant lymphomas faster (�51 days) thanthose animals that received HSCs expressing Myc alone, or in com-bination with either 96 unrelated, single miRNAs or any individualmiRNA of the mir-17-19b-1 cluster (3–6 months).47 More recently,Volinia et al.59 identified a large number of overexpressed miR-NAs, including miR-17-5p, miR-20a, miR-21, miR-92, miR-106aand miR-155, in solid tumor samples. Therefore, overexpression ofmiRNAs appears to play an important role in tumorigenesis, withthe miRNAs functioning directly or indirectly as oncomiRNAs.The aberrant expression of miRNAs in tumor cells appears to bestrongly associated with cancer development.

A current challenge is to reveal the mechanism of regulation ofmiRNA gene expression itself, i.e. why and how the miRNAs arederegulated. It is very important for us to understand further, themechanism of the development of miRNA-associated cancer. Insome cases, the cause of miRNA deregulation is clear. For exam-ple, in the case of the mir-17-92 cluster, it is now very clear thatthe increase expression of these miRNAs correlates with the pres-ence of the 13q31 amplicon.47,48 Similarly, the downregulation ofmiR-15 and miR-16 at 13q14 observed in about 68% of B-cellCLL cases is largely due to a deletion at 13q14.34,35 However, inmost of cases, there is no clue as to why and how the miRNAs arederegulated. Given that miRNAs, like protein-coding genes, aretranscribed by RNA polymerase II,62 and that many miRNAs arelocated within the introns of protein coding genes,63 the regulationof miRNA gene expression is probably similar to the regulation ofprotein-coding gene expression. For example, CpG-island methyl-ation and/or histone deacetylation might be a possible mechanismfor downregulation of miRNAs,64 although it might not be a com-mon mechanism in the regulation of miRNAs.37,65 In addition,some miRNAs probably are regulated along with their host genes,e.g., the mir-17-92 cluster is upregulated along with the host geneC13orf25, because of locus amplification.47,48 Furthermore, theexpression of miRNAs could be regulated through regulatingmicroprocessors of the miRNAs, for example, by regulating theexpression of Dicer.27,66

Computational prediction of targets of miRNAs

Another challenge is to accurately identify targets that are regu-lated by miRNAs.26 In animals, miRNAs confer inhibitory regula-tion of the target-gene translation level through binding in the 30UTRs. This was first evidenced by lin-4 targeting of the lin-14/lin-

28 genes,8,9 and let-7 targeting of the lin-41 gene11 in C. elegans.Recently, 4 bioinformatics approaches have been developed forpredicting miRNA targets in humans.3,67–70 Most bioinformaticalgorithms use an ‘‘miRNA seed’’ that encompasses the first 2–8bases of the mature miRNA sequence to search for complementar-ity to sequences in the 30 UTRs of all expressed genes.71 Thesestudies have revealed that a single miRNA might bind to hundredsof gene targets which can be diverse in their function, and that 1target gene might be regulated by several different miRNAs.26

Briefly, Lewis et al.3,67 developed a sophisticated algorithm, calledTargetScan, and its improved version, TargetScanS. TargetScan3

searches the UTRs for segments of perfect Watson-Crick comple-mentarity to base 2–8 of the miRNA (numbered from the 50 end),calculates a folding free energy G to each miRNA-target site inter-action using RNAeval, and then assigns a Z score to each UTR.TargetScanS67 is an improved algorithm that requires a shorterseed (6 nt), which is preceded by an adenosine and is located in ashort ‘‘island’’ of conservation in at least 4 mammalian genomes(i.e., human, mouse, rat and dog). It simply relies on perfect Wat-son-Crick seed matches that are conserved in the UTR regions ofwhole genome alignments, and does not rely on free-energy calcu-lation.67 John et al.70 developed an algorithm named miRanda,which optimizes sequence complementarity using position-specificrules and relies on strict requirements of interspecies conservation.Kiriakidou et al.69 developed an algorithm called DIANA-MicroT,which is trained to identify miRNA targets having a single bindingsite. This algorithm takes an approach that is different from thoseof the other algorithms described earlier71: (i) It focuses on single-binding-site targets and (ii) it seeks binding sites that have a typicalcentral bulge and require 30-binding, beyond the obligatory 50-seed.Krek et al.68 developed an advanced algorithm called PicTar,which is trained to identify both binding sites targeted by a singlemiRNA, and those that are coregulated by several miRNAs in acoordinated manner. It employs pair-wise alignment for accuratefiltering for binding sites that are conserved across 8 vertebrates,and takes into account clustering and coexpression of miRNAs,ontologic information, as well as free energy of RNA:RNAduplexes by using RNA hybrid.72

Recently, we collected all the putative miRNA-target pairs pre-dicted by each algorithm to set up a complete list of all the pre-dicted human miRNA-target pairs, because there is no evidence asyet to show that one is much better than the others. Briefly, wecollected 21,293 unique miRNA-target pairs predicted by TargetS-canS,67 46,155 unique miRNA-target pairs predicted by PicTar,68

54,578 unique miRNA-target pairs predicted by miRanda70 and185 unique miRNA-target pairs predicted by DIANA-MicroT.69

After combination, we obtained 101,031 predicted unique miRNA-target pairs. Of the 101,031 pairs, 0.01%(12), 2.8% (2,817), 15.4%(15,510) and 81.8% (82,692) were predicted by 4, 3, 2 and a singlealgorithm, respectively (Sun M, Chen J. unpublished). Notably,most of the pairs (81.8%) were predicted by only a single algo-rithm. Although the false positive rate of each of the 4 algorithmswas estimated to be 20–30%,71 it is unlikely that most of the82,692 pairs are false-positive ones. Instead, it is possible that eachalgorithm may only predict a part of the whole miRNA-target pairs.For example, although the KIT gene was predicted only by miR-anda as a target of miR-221, it has been biologically validated to bea real target.73,74 Thus, the combination of these 4 predictions mightprovide a much more comprehensive list of putative miRNA-targetpairs than do any single prediction.

Current methodologies of miRNA target validation

Because of the potential high false-positive rate of computa-tional prediction of miRNA targets, validating target predictions isvery important. MiRNA target prediction algorithms may be infor-matically validated by evaluation of an algorithm’s success in cor-rectly identifying known miRNA targets (i.e. targets that have al-ready been validated biologically, and scoring them highly), orthey may be validated by comparing the number of postulated

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binding sites that an algorithm finds for a real miRNA withthat found for a control group of artificially generated ‘‘fictitiousmiRNAs’’. Nonetheless, all of these strategies merely provide indi-rect validation, and they have several limitations.71 However,whereas the ultimate validation of predicted miRNA targets is bio-logical validation, at present there is no a high-throughput method forbiologically validating miRNA targets.71 The conventional biologicalvalidation methodologies are still extremely labor-intensive and donot allow high-throughput target validation. The commonly used val-idation methodologies include luciferase reporter assays,3,39,68,69,75

mutation studies,69,75 gene-silencing techniques,39,75,76 rescueassays77 and classic genetic studies.8,39,77 Overall, only about 30animal miRNA targets have been validated to date with use ofthese various techniques.71

A new mechanism of miRNA-mediated gene regulation inanimals and a potential high-throughput method foridentifying miRNA-target pairs

In a classic model of miRNA function in animals,1,2 miRNAsthat form imperfect duplexes with their targets inhibit proteinexpression without affecting mRNA levels (as illustrated in Fig. 1).However, recent findings indicate that miRNAs that share only par-tial complementarity with their targets can also induce mRNA deg-radation.74,78–83 For example, in C. elegans, regulation by the let-7miRNA results in degradation of its lin-41 target mRNA. Further-more, lin-14 and lin-28 mRNA levels significantly decrease inresponse to lin-4 miRNA expression.81 In addition, transfection ofexogenous miRNA duplexes into HeLa cells can cause moderatedownregulation of hundreds of mRNAs, many of which contain therecognition motif of the overexpressed miRNA in their 30 UTR.80Moreover, in vivo knockdown of a liver-specific miRNA (miR-122) has shown that hundreds of mRNAs, many of them seeminglyto be direct targets of this miRNA, were moderately upregulated.78

Taken together, these studies suggest that mRNAs containing par-tial miRNA complementary sites can be targeted for degradationin vivo, and that miRNA-dependent regulation of mRNA stabilitymay be more common than previously appreciated.82

Thus, as shown by some pilot studies,74,82,83 the predicted miRNA-target pairs could be roughly validated on a large scale by compari-son of expression patterns between miRNAs and the predicted tar-gets through conducting microarray analysis on both miRNAs andprotein-coding genes. However, the aims of the pilot studies arenot to set up a high-throughput method for biological validation ofthe predicted miRNA-target pairs, and the specificity, robustnessand reproducibility of the approaches have not been systematicallytested and evaluated. In fact, because only a few miRNA targetshave been biologically validated to be affected by miRNAs at themRNA level,78,80,81 it is unclear whether the majority of miRNAtargets can be significantly degraded at the mRNA level by miR-NAs in animals. In addition, although it was reported that the folddecrease of lin-14 mRNA is consistent with that in LIN-14 proteinlevels mediated by lin-4 miRNA,81 it was suggested that thechanges in target mRNAs are usually weak.82 It is unclear to whatextent the mRNA-level changes of the miRNA targets can reflectthe changes at their protein level. Thus, a large-scale protein-levelanalysis technique, such as a protein array, probably should also beintegrated in the potential high-throughput technique for miRNA-target-pair validation.

MiRNA signaling pathways in cancer development

As described earlier, thousands of target genes of human miR-NAs have been predicted using bioinformatic tools. Interestingly,predominant miRNA targets are transcription factor or kinases.3

Researchers are currently attempting to dissect the target genes ofmiRNAs and their signaling pathways involved in cancer. SeveralmiRNA signaling pathways have been carefully studied (Fig. 2).As an example, Johnson et al. showed that the 30 UTR of thehuman RAS gene contains multiple let-7 complimentary binding

sites, allowing let-7 to regulate RAS expression. Furthermore, theoverexpression of let-7 in human cancer cell lines results in adecreased level of RAS compared with untreated cells. Conversely,knockdown of let-7 in human cancer cell lines, in which let-7 isnormally expressed at a high level, leads to a remarkable increasein RAS protein expression, providing a mechanism for let-7 nega-tively regulating RAS. Consistent with the observations in vitro,expression of let-7 is lower in lung tumors than in normal lung tis-sue, while that of RAS protein is significantly higher in lungtumors.39 Introduction of let-7 isoforms (let-7a and let-7f) into alung cancer cell line A549 resulted in a 78.6% reduction in thenumber of colonies,38 indicating that growth inhibition may be thebiological significance of let-7, presumably through negatively reg-ulating RAS signaling pathways. A few additional genes have alsobeen predicted to be a potential target for let-7, such as LIM kinase2,3 which belongs to a gene family having a role in the regulationof cell shape and motility and possibly in metastasis as well.

As aforementioned, the mir-17-92 cluster is overexpressed inmultiple malignancies. To reveal the mechanisms of the mir-17-92oncogenic effect directly, He et al.47 utilized a mouse B-cell lym-phoma model and demonstrated that enforced expression of themir-17-92 cluster gene acted with c-myc expression to acceleratetumor development in this model. Tumors derived from haemato-poietic stem cells expressing a subset of the mir-17-92 cluster and c-myc possesses less apoptosis than c-myc-induced lymphomas, whichunderwent extensive apoptosis. Because of its oncogenic function,the authors nominate this miRNA cluster as the first candidate non-coding oncogene, ‘‘oncomiR-1.’’47 Interestingly, O’Donnell et al.75

independently found that c-Myc binds directly to the miR-17-92locus and activates expression of a cluster of 6 miRNAs. One ofthe transcriptional targets of c-Myc, E2F1 is negatively regulatedby 2 miRNAs in this cluster, miR-17-5p and miR-20a. This studyexpands the known classes of transcripts within the MYC targetgene network, and uncovers a mechanism through which MYCsimultaneously activates E2F1 transcription and limits its transla-tion by the miR-17-92 cluster of miRNAs, allowing a tight controlof proliferative signal. Collectively, these findings suggest that themiR-17-92 cluster acts as an oncogene at one set of conditions,while as a tumor suppressor at another.

In most malignant tumors, the PI3K/Akt signaling pathway isactivated and results in an aggressive proliferation. The tumor sup-pressor gene, phosphatase and tensin homolog deleted on chromo-some ten (PTEN), inhibits PI3K function. Loss of PTEN expres-sion is associated with PI3K/Akt signaling activation in many

FIGURE 2 – Schematic representation of miRNA-mediated signalingpathways. MiRNAs functioning as either tumor suppressors (Red) oroncogenes (green) participate in the cell survival and apoptosis path-way, by which may contribute to cancer initiation and development.

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types of tumors.84 MiR-19a, b has been demonstrated to bind the30 UTR of PTEN mRNA in vitro.3 In lymphomas, overexpressionof miR-19 is correlated with underexpression of PTEN protein,35

suggesting that overexpression of mir-19 in tumor cells could bean alternative mechanism by which the PI3K/Akt signaling path-way is activated.

Other studies have linked miRNAs to the regulation of apopto-sis. Chan et al.85 revealed a strong over-expression of miR-21 inhuman glioblastoma tumor tissues and cell lines. Knockdown ofmiR-21 in cultured glioblastoma cells triggers activation of cas-pases and leads to increased apoptotic cell death, suggesting thataberrantly expressed miR-21 may contribute to the malignant phe-notype by blocking expression of critical apoptosis-related genes.It appears that miR-21 has a broad function in tumor progression,because miR-21 is also upregulated in breast cancer86 and lungcancer.37 Cimmino et al.87 demonstrated that expression of miR-15a and miR-16-1 is inversely correlated to expression of BCL-2in CLL and that both miRNAs negatively regulate BCL-2 proteinat a posttranscriptional level; BCL-2 repression by these miRNAsinduces apoptopsis in a leukemic cell lines. Cheng et al.88

employed a library of miRNA inhibitors to screen for miRNAinvolved in cell growth and apoptosis, and observed that miRNA-mediated regulation has a complexity of cellular outcomes andthat miRNAs can be mediators of regulation of cell growth and ap-optosis pathways.

KIT is an important tyrosine kinase receptor in cell differentia-tion and growth. Inhibiting the KIT signaling via miRNAs maycontribute to uncontrolled cell growth in certain type of cells.73

He et al.74 reported that miR-221, -222 and -146 are upregulatedin papillary thyroid carcinoma and are associated with dramaticdecrease of expression of KIT at both transcript and protein levels.The authors further demonstrated that point mutations occurredfrequently in the KIT loci (5 out of 10 samples) within the compli-mentary binding regions for these miRNAs, and therefore dis-rupted the miRNA–mRNA target interactions in these tumors.

Volinia et al.89 observed that miR-106a overexpression in coloncarcinoma inversely correlates to RB1 protein expression, and thatdownregulation of miR-20a in breast cancer is associated to highexpression of TGFBR2 protein, while overexpression of miR-20ain some lung cancer samples is associated with downregulation ofTGFBR2 protein. These results suggest that miR-106a and miR-20a might negatively regulate RB1 and TGF b signaling, respec-tively, which was confirmed by a luciferase assay in vitro.

More recently, to perform genetic screens for novel functions ofmiRNAs, Voorhoeve et al.90 developed a library (miR-Lib) ofvectors expressing the majority of cloned human miRNAs andmade a corresponding microarray (miR-Array) containing allmiR-Lib inserts. In a screen for miRNAs that cooperate with onco-genes in cellular transformation, the authors identified miR-372and miR-373 as potential oncogenes in testicular germ cell tumors.Each of them permits proliferation and tumorigenesis of primaryhuman cells that harbor both oncogenic RAS and active wild-typep53. These miRNAs neutralize p53-mediated CDK inhibition, pos-sibly through direct inhibition of the expression of the large tumorsuppressor homolog 2 (LATS2), a serine-threonine kinase whosedeletion accelerates cellular proliferation and tumorigenic develop-ment. In all, the authors provided compelling evidence that thesemiRNAs are potential novel oncogenes participating in the devel-opment of human testicular germ cell tumors by numbing the p53pathway, thus allowing tumorigenic growth in the presence ofwild-type p53.

Taken together, the deregulation of miRNAs participates inactivation of cell proliferation or inactivation of apoptotic signal-ing pathway in conjunction with other genetic changes leading tocancer pathogenesis.91 Further studies would reveal that miRNAsfunction as key regulators of many more cancer-related genes.Besides the fact that miRNAs inhibit the target genes directly,Gregory and Shiekhattar27 provided evidence that defect of micro-processor of miRNA is one alterative mechanism through which

the expression of miRNA can be altered and miRNA perturbationis linked to cancer. Reduced expression of Dicer is associated withpoor prognosis in lung cancer patients.66

MicroRNA expression profiles for cancer diagnosis

Emerging data indicate that miRNAs play an important role inregulating gene activation in cancer initiation and progression.Researchers are now using the miRNA-expression signatures toclassify cancers and to define miRNA markers that might predictfavorable prognosis.26 Various methods have been employed forthis purpose. Northern blotting was used in early profiling studies,and is still widely used, because it can convincingly and simulta-neously detect both the precursor and mature miRNAs. It providesdata about the Dicer processing step, which may be regulated.92

However, Northern blotting is not a high-throughput and sensitivemethod. Thus, over the past years, real-time RT-PCR and microar-ray methods have been adapted to detect the expression of miR-NAs. High-throughput real-time RT-PCR methods have beendeveloped to screen the expression of precursor and/or maturemiRNAs using either SYBR Green dye or TaqMan chemistry(probe) (e.g., Refs. 93–95). To date, largely, there are 4 platformsof microRNA-arrays including dotted cDNA arrays,96 oligonu-cleotide arrays,97–99 bead-based flow cytometric miRNA expres-sion profiling40 and ‘‘locked nucleic acid’’ (LAN)-modified oligo-nucleotide arrays100,101 (for reviews, see Refs. 92 and 102). Thesetechnologies have been utilized in screening miRNA expressionpattern in hematopoietic malignancies and solid tumors.17 Forexample, using Northern blotting, Chen et al.103 demonstrated thatmiR-181, miR-223 and miR-142s were differentially or preferen-tially expressed in hematopoietic tissues. Using a high-throughputTaqman real-time RT-PCR method, Jiang et al.94 screened 222miRNA precursors in 32 commonly used cell lines of lung, breast,head and neck, colorectal, prostate, pancreatic and hematopoieticcancers. Using oligonucleotide microchips,99 Croce’s group hasrevealed distinct signatures of miRNAs in human breast cancer,86

in human megakaryocytopoiesis104 and in B cell CLL.35,105 Theyobserved that a unique miRNA signature is associated with prog-nosis factors and disease progression in B cell CLL.105 Using asimilar oligonucleotide microchip, Yanaihara et al.37 analyzed themiRNA expression in 104 pairs of primary lung cancers and corre-sponding noncancerous lung tissues, and found that the expressionof 43 miRNAs was statistically differentially displayed in cancertissues; their further analysis revealed that the high expression ofmiR-155 and the low expression of let-7a-2 are correlated withpoor survival. In addition to studies on individual type of tumors,a large-scale microRNAome analysis was performed on 540 sam-ples including lung, breast, stomach, prostate, colon and pancre-atic tumors with oligonucleotide microchips; cancer-associatedmiRNAs were identified as miRNA signatures, including somewell characterized ones such as miR-17-5p, miR-20a, miR-21,miR-92, miR-106a and miR-155 in solid tumors.59 Lu et al.40

used a new, bead-based flow cytometric miRNA expression profil-ing method to conduct a systematic expression analysis of 217mammalian miRNAs on 334 samples, including multiple humancancers. They observed a general downregulation of miRNAs intumors when compared with that in normal tissues. Furthermore,they successfully classified poorly differentiated tumors usingmiRNA expression profiles.

Taken together, miRNA expression profiles can be utilized todefine the expression pattern of diverse cancers, to predict theprognosis, and to monitor response to therapy or toxicity. It isnoteworthy that small RNAs can be measured easily from the for-malin-fixed tissue specimens used routinely in hospital pathologylaboratories. Thus, potential miRNA-based diagnostics could fitsimply into the standard hospital workflow.98 To improve thespecificity and sensitivity of miRNA microarrays, Neely et al.100

recently developed a new approach, LAN-modified oligonucleo-tide array, in which they designed 2 probes for each miRNA (eachprobe is half of the length of the mature miRNA) and labeled with

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different fluorophores. With such an approach, the authors wereable to quantitate miRNA expression from as little as 50 ng totalRNA with a high specificity.100 In addition, Kloosterman et al.101

designed LNA oligonucleotides and applied them for in situhybridization to monitor the spatial and temporal expression ofmiRNAs in vivo. Such technique improvement and renovationwill make it more promising to use miRNA expression profiles forcancer diagnosis, for defining a treatment strategy for patients, forprediction of the prognosis, and for monitoring treatment re-sponse.

Targeting miRNA regulation with antisenseoligonucleotides: a potential therapeutic intervention

To investigate the potential causative roles of deregulatedmiRNA processes in human disease, synthetic, chemically-modi-fied antisense oligonucleotides targeting to miRNAs or protein-coding genes have been widely used. They have been proven to bea powerful research tool, and may eventually become a new thera-peutic tool.106–108 On the one hand, if a disease phenotype isrelated to the overexpression of miRNAs, antisense oligonucleo-tides that are complementary to either the mature miRNAs or theirprecursors can be designed to specifically inhibit the activity ofthe miRNAs. There are 3 types of modifications for anti-miRNAoligonucleotides (AMO)106: (i) 20-O-Methyl (OMe) AMOs, (ii) 20-O-Methoxyethyl (MOE) AMOs and (iii) Locked nucleic acid(LNA) AMOs. Various AMOs have been implemented in experi-mental studies. For example, Cheng et al.88 applied a library ofMe-AMOs to screen for miRNAs that are involved in cell growthand apoptosis. Esau et al.109 inhibited miR-122 in mice with aMOE-AMO to uncover the role of the liver-specific miR-122 inthe adult liver and demonstrated that miR-122 is a key regulator ofcholesterol and fatty-acid metabolism in the adult liver. Chanet al.85 successfully applied 20-O-methyl- and DNA/LNA-mixedoligonucleotides to specifically knockdown miR-21 to investigatethe potential contribution of this miRNA in the regulation of apo-ptosis-associated genes in glioblastoma cell lines. On the otherhand, to enhance the function of miRNAs due to a deletion or aloss of function mutation, a therapeutic approach could entail ex-ogenous delivery of corrective synthetic miRNAs in the form of(siRNA-like) double strand oligoRNAs.106 One example showsthat enforced expression of let-7 in the lung adenocarcinoma cellline A549 inhibited lung cancer cell growth in vitro.38 This holdsa promise that let-7 may be useful in treatment of lung cancer orenhancing the current treatments. Synthesized miRNA-14U wasused to inhibit the overexpressed HER-2 protein in ovarian cancercell line SKOV3.110

To develop a pharmacological approach for silencing miRNAsin vivo, Krutzfeldt et al.78 designed chemically modified, cholesterol-conjugated single-stranded RNA analogues (termed ‘‘antagomirs’’),

which are complementary to miRNAs. Their study shows that thetermed antagomirs are a powerful tool to silence specific miRNAsin vivo.78 Therefore, we may expect that in the future, activation ofmiRNAs with ‘‘agomiR’’ or silencing of miRNAs with ‘‘anta-gomirs’’ could become a therapeutic strategy for cancer treatment.Nevertheless, there are still several difficulties to overcome beforeapplying such strategies in clinic. For example, as miRNAs mayhave dual functions (oncogene or tumor suppressor) in differentorgans or conditions, is it possible to design ‘‘agomiR’’ or ‘‘anta-gomirs’’ that target specific tissues or disease sites?108 In addition,the regulation and stability of the injected ‘‘agomiR’’ or ‘‘anta-gomirs’’ in vivo, the proper dosages for the optimal effect, and thepotential side effects, should be carefully examined and addressed.Moreover, because a single miRNA may regulate hundreds of differ-ent targets while a single target gene may be regulated by several dif-ferent miRNAs, the regulation network may be very complex. Thus,more cautions should be taken in considering clinical applications.

Conclusion

The microRNA field is rapidly developing, as shown by thefacts that the number of experimentally identified miRNAs andtheir targets is increasing, and that the functions and the signalingpathways of more and more miRNAs have been carefully studied.The recent findings suggest that miRNAs play an important role inregulation of gene activation by binding to the mRNAs of targetgenes, by switching the genes on or off, or by fine-tuning thegenes, to control cell viability, cell cycle, proliferation or apopto-sis. It was predicted that more than half of human genes might beregulated by miRNAs. Deregulation of miRNA expression is oftenassociated with cancer. These studies may change the landscapeof cancer genetics and uncover new mechanisms that contribute tocancer development. The future challenges are to learn how miR-NAs are regulated in human genome, and to identify more biologi-cal targets of miRNAs and the relevant signaling pathways. None-theless, activation of miRNAs with ‘‘agomiR’’ or silencing ofmiRNAs with ‘‘antagomirs’’ could probably become a therapeuticstrategy for cancer treatment in the future, although we are farfrom that point.

Acknowledgements

We thank Dr. Janet D. Rowley and Dr. Suzanne D. Conzen fortheir critical comments and valuable suggestions, and 3 anony-mous referees for their constructive comments. We also appreciatethe help of Ms. Mary Beth Neilly in manuscript revision. We apol-ogize to our colleagues whose outstanding contributions to thegrowing miRNA field were not cited as primary references becauseof the space constraints.

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