Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Molecular therapy in the microRNA era


MicroRNAs (miRNAs) consist of a growing class of non-coding RNAs (ncRNAs) that negatively regulate the expression of genes involved in development, differentiation, proliferation, apoptosis and other important cellular processes. miRNAs are usually 18–25 nt long and are each able to regulate several mRNAs by mechanisms such as incomplete base pairing and Post-Transcriptional Gene Silencing (PTGS). A growing number of reports have shown that aberrant miRNA expression is a common feature of human diseases including cancer, which has sparked interest in targeting these regulators of gene expression as a means of ameliorating these diseases. Here, we review important aspects of miRNA function in normal and pathological states and discuss new modalities of epigenetic intervention strategies that could be used to amend defects in miRNA/mRNA interactions.


Non-coding RNAs (ncRNAs) are genes that are able to function as RNA transcripts (reviewed in Eddy1 and Costa2). microRNAs (miRNAs) are part of the group of ncRNAs that can block mRNA translation and affect mRNA stability (reviewed in Ambros3 and Kim and Nam4). miRNAs are generally 18–25 nt long and were first described in the early 1990s in the worm Caenorhabditis elegans as regulators of development and differentiation.5, 6 Since then, several miRNAs have been identified in animals, plants and viruses. In the human genome, recent estimates point to at least thousands of miRNAs.7 So far, more than 462 different miRNAs have been described in humans.8, 9 miRNA genes are usually transcribed by RNA polymerase II into longer transcripts, referred to as primary transcripts or pri-miRNAs,10 and then processed into pre-miRNAs.11 Several important steps of miRNA biogenesis have been recently identified (reviewed in Bartel12), although the exact mechanisms by which specific miRNAs act still remains largely unclear. One therapeutically relevant concept is that one miRNA can downregulate multiple target proteins by interacting with different target mRNAs (‘one hit-multiple targets’ concept).13 There have been several reports implicating miRNAs in post-transcriptional regulation of proteins with diverse roles, from cell proliferation and differentiation to fat metabolism (reviewed in Filipowicz et al.14). Recently, miRNA deregulated expression has been extensively described in a variety of diseases, especially cancer (reviewed in Hwang and Mendell15). Some lines of evidence have already shown that up or downregulation of miRNAs correlates with many human cancers indicating that miRNAs can function as classical tumor suppressors or oncogenes.15, 16

The aim of this article is to review important aspects of miRNA biogenesis and function, and to introduce therapeutic concepts that could be used to ‘correct’ abnormalities in miRNA/mRNA expression associated with disease. We have named this new therapeutic opportunity a new modality of ‘epigenetic therapy’ and it might be applicable to multigenic diseases caused by deregulated expression of miRNAs.

miRNAs in normal and pathological states

It is already clear that miRNAs can negatively regulate their mRNA targets in two different ways depending on the degree of base pair complementarity. In the first case, miRNAs that bind with perfect – or nearly perfect – complementarity to protein-coding mRNA sequences are able to induce the RNA-mediated interference (RNAi) pathway. mRNA transcripts are then cleaved by endoribonucleases in the RNA Induced-Silencing Complex (RISC), which results in the irreversible degradation of target mRNAs. This mechanism of miRNA-mediated gene silencing is generally found in plants.17 On the other hand, animal miRNAs can use a second mechanism of gene regulation termed translational repression that does not result in the degradation of their mRNA targets.18, 19, 20 These miRNAs act by binding imperfectly within the 3′ untranslated regions (UTRs) of their mRNA targets with concomitant repression of their translation.18, 19, 20 miRNAs that use this mechanism are able to reduce the protein levels of their target genes, but the mRNA levels of these genes are not affected per se.18, 19, 20 The degradation pathway and the translational repression pathway both result in Post-Transcriptional Gene Silencing or PTGS. Bioinformatic analysis can be effective in the identification of miRNA/mRNA interactions. Although not always accurate, putative miRNA ‘seeds’ of no more that 7 nt which are conserved between animal miRNAs and the 3′ UTRs of their mRNA targets frequently predict miRNA/mRNA interactions.21, 22 Even though a number of questions remain to be answered regarding miRNA biogenesis and function, the decrease in target protein translation is a well established feature of miRNA function.

Several reports have implicated miRNAs in important aspects of differentiation and development in a cell type and tissue-specific manner. For example, miRNAs have been recently implicated in orchestrating epithelium differentiation in the formation of layers of skin.23 Additionally, Schratt et al.24 showed that a miRNA named miR-134 is a brain-specific ncRNA that regulates CNS development in mice, contributing to synaptic plasticity, development and maturation. These short RNA molecules are also expressed in specific stages of mammalian embryonic development, being able to control expression of genes implicated in tissue differentiation. For example, two miRNAs have been associated with skeletal muscle gene expression in a small circuitry. Interestingly, miR-1 was found to be able to promote myogenesis by targeting the transcript for histone deacetylase 4 (HDAC4), which represses transcription of muscle genes, and miR-133 was able to enhance myoblast proliferation by repressing the serum response factor (SRF), which promotes transcription of muscle genes.25 Another example is miR-181 which is able to specifically regulate homeobox proteins implicated in myoblast differentiation.26 These and other observations indicate that miRNAs are strongly involved in differentiation and development, two characteristics that are deregulated in several diseases.

In that regard, recent evidence has shown that miRNA expression can correlate with various human cancers and indicates that deregulated miRNAs can function as classical tumor suppressors and oncogenes.15, 27 miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in cancer diagnosis and therapy. For example, miRNAs such as miR-15a, miR-16 and let-7 have been found to be downregulated in different types of cancer, suggesting that they can act as a tumor suppressor genes.15 Interestingly, one of the mRNA targets for miR-15a and miR-16 is the transcript encoded by the anti-apoptotic gene BCL-228 and one of the mRNA targets for the miRNA let-7 is the mRNA transcript of the oncogene RAS.29 On the other hand, miRNAs such as the polycistron miR 17–92 (specifically miR-17-5p and miR-20a),30 miR-21,31 miR-155,32 miR-372 and miR-37333 have been implicated as oncogenes in different types of cancer. Targets for some of these miRNAs have also been described. The transcription factor E2F1 is one of the targets for miRNAs of the polycistron miR 17-92;34 the tumor suppressor LATS2 is one of the targets of miR-372 and miR-37333 and the angiotensin II type 1 receptor (hAT1R) is a target of miR-155.35 Several groups have also described the deregulated expression of miRNAs in cancer by microarray analysis. For example, Lu et al.,36 Calin et al.37 and Volinia et al.38 have recently shown that miRNAs expression profiling can be used to accurately distinguish between different types of cancers and even between different subtypes of tumors from the same cancer type, thereby revealing a higher sensitivity than mRNA fingerprinting. In order to evaluate whether overexpressed miRNAs can act as classical oncogenes, a transgenic mice overexpressing miR-155 was generated.39 Mice transgenic for miR-155 developed a lymphoproliferative disease resembling the human cancer, strongly suggesting that miR-155 is directly involved in the initiation and/or progression of cancer. In this study, the difference in mRNA expression for the transgenic mice and normal control mice was evaluated by microarrays, showing that several genes were up and downregulated in the transgenic mice. This is a strong indication that miR-155 can – directly and/or indirectly – regulate various protein-coding target genes.39

All the accumulating evidence indicates that normal miRNA expression is very important for proper development and differentiation in a tissue and cell-type specific manner. It is also becoming clear that miRNA deregulated expression is a common feature of several types of cancer and that this characteristic could be used as a prognostic marker for tumor diagnosis and treatment. As cancer is a multigenic disease, we also expect that similar defects in miRNA expression will be found in many other complex diseases in the years to come. Therefore, we believe that the therapeutic concepts discussed below could be applied in a general context to a wide variety of human diseases.

‘One hit – multiple targets’ – a new therapeutic concept

Since deregulated expression of miRNAs has been described in many types of cancer and might be a common feature of other multigenic diseases, we suggest that the ‘one hit-multiple target’ biological concept could be used to treat diseases. We postulate that if the primary molecular defect of the disease is in miRNAs or in the miRNA pathway and, as a consequence, the expression of the protein-coding mRNA targets is deregulated, one could intervene by ‘normalizing’ or ‘correcting’ the miRNA expression. This could result in recovery of the normal phenotype of the cells from a disease state to a normal state or even induce tumor cell death by apoptosis. As shown in Figure 1, therapeutic interventions for miRNAs that are deregulated in diseases such as cancer could be used to ‘correct’ the miRNA expression levels and, consequently, ‘normalize’ the expression of their numerous mRNA targets in cells, some of which may be encoded by oncogenes and tumor suppressor genes in this example. In addition, various diseases showing deregulated mRNA expression patterns, but relatively normal miRNA profiles, might also be normalized using the ‘one hit – multiple targets’ concept. Correcting defects in mRNA expression levels by using natural or synthetic miRNA regulators may prove to be of clinical relevance, since many of the potential miRNA targets are protein-coding mRNAs that are implicated in the disease state.

Figure 1

Schematic representation of miRNA expression ‘correction’ to recover normal phenotypes in multigenic diseases such as cancer by a new modality of ‘epigenetic therapy’. A growing number of reports have already shown that miRNAs are up or downregulated in cancer functioning as classical tumor suppressors and oncogenes (red arrows). Different therapeutic interventions can be applied to recover the normal expression of these miRNAs with an indirect ‘normalization’ of the expression of their mRNA targets (blue arrows). miRNAs, microRNAs; ONCOmiRNAs, oncogenic miRNAs; TS miRNAs, tumor suppressor miRNAs; TSGs, classical tumor suppressor genes.

Gene expression analysis has accumulated data regarding deregulated mRNA levels in a variety of diseases (reviewed in Mirnics and Pevsner40 Mocellin et al.,41Chung et al.,42). Only recently miRNA screens in healthy and affected tissues have started to reveal the role of miRNAs in different diseases and aberrant miRNA expression has been discovered in many affected tissues (Table 1). Various methods have been described for the screening of miRNA expression patterns. These include miRNA microarray techniques,43, 44, 45, 46, 47, 48, 49, 50, 51, 52 FACSorting using fluorescent nanoparticle miRNA probes,36 quantification using locked nucleic acids,53, 54 and qRT-PCR.55, 56, 57, 58, 59, 60, 61 As indicated in Table 1, altered miRNA expression has been linked to a variety of diseases, including cancer,7, 15, 16, 27, 28, 29, 30, 31, 32, 33, 38, 39, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 Tourette's syndrome and other diseases of the CNS,73, 74 metabolic diseases75, 76, 77, 78 and viral diseases.79, 80, 81, 82 Clearly, one of the most important questions remaining is whether the miRNAs are differentially expressed as a consequence of the pathologic cell state, or whether the particular disease is a direct cause of the deregulated expression of miRNAs. In any case, as pointed out above, ‘normalization’ of the miRNA expression pattern might result in restoration of the deregulated post-transcriptional control and therefore could have a therapeutic effect.

Table 1 miRNAs implicated in disease

The wide use of RNAi methodologies employing siRNAs or shRNAs to silence single target genes has contributed to recent advances in molecular biology (reviewed in Moffat and Sabatini83, Behlke84). Currently, siRNAs are being tested in experimental therapy for a variety of diseases and a few are starting to enter clinical trials (reviewed in Vidal et al.85). The use of siRNAs with perfect pairing to the target mRNA has also revealed a drawback of the RNAi approach, the frequent observations of off-target effects – or the RNAi-mediated silencing of genes other than the target gene.86, 87 With the miRNA mechanism in mind it is likely that many of the off-target effects are due to imperfect binding of the particular siRNA to non-target mRNAs.88 Also other mechanisms have recently been described to play a role, such as elicitation of the interferon response by high levels of siRNAs and the clogging of export of mRNAs from the nucleus by overloading with shRNAs.89, 90, 91, 92 Nonetheless, RNAi has proven its usefulness as a tool with good success in knocking down of particular single genes.93 However, as various microarray screens indicate, in unhealthy tissues there can be an imbalanced gene expression pattern involving many genes (reviewed in Chung et al.,42 and Hoheisel94). Therefore, it might be helpful to shift our focus towards the targeting of multiple genes using the miRNA mechanism of multiple-target regulation, which could be approached in two different ways. The first would be to restore normal cellular miRNA expression levels by either up or down-regulation of specific endogenous miRNAs. The second approach would be to design artificial miRNAs that have the potential to silence multiple deregulated target mRNAs in an RNAi-like fashion. Various strategies might be used and are discussed below.

Modifying endogenous miRNA expression levels

Inhibition of specific endogenous miRNAs has been achieved by the administration of synthetic anti-sense oligonucleotides that are complementary to the mature endogenous miRNAs. These anti-miRNA oligonucleotides (AMOs) were shown to specifically inactivate endogenous target miRNAs, although quite inefficiently.95, 96, 97 Improved AMO versions are now available containing at the 5′ end a 2′-O-methyl group97, 98, 99, 100 or 2′-O-methoxyethyl groups.76, 101 In addition, efficient locked-nucleic-acid antisense oligonucleotides (LNAs) have been designed.102, 103 Probes consisting of DNA/LNA oligonucleotides mixtures were described to form strong duplexes with complementary RNA in cells. These LNAs have proven to be useful in miRNA profiling by in situ hybridization,104, 105 and their locked feature may also result in a delayed clearance after systemic administration.102 In addition, there are antagomirs, which are AMOs conjugated to cholesterol.77 They have been described to efficiently inhibit miRNA activity in various organs when injected into mice, and may have therapeutic applicability in vivo.75

Overexpression of endogenous miRNAs can be achieved via expression systems that use viral or liposomal delivery.33, 106, 107 An efficient approach might be to express the miRNA or siRNA as hairpins using expression vectors containing polymerase III promoters such as H1108 or U6109 promoters. miRNAs can also be expressed from vectors containing polymerase II promoters, that is CMV controlled expression vectors.106, 107, 110 In this latter case, it was described that miRNA-mediated silencing was more efficient when expressing the miRNA in a pri-miRNA form, thus including miRNA flanking sequences and the hairpin structure, as was shown for miR-30107 and miR-155.106 Interestingly, also for the single-target RNAi approach, flanking pri-miRNA sequences improved shRNA-mediated silencing. Stegmeier et al.107 showed that miRNAs can be expressed from the 3′ UTR of a reporter gene, suggesting that mRNAs and miRNAs can functionally act from a single transcript. In addition, Chung et al.,106 recently showed that miRNAs can be expressed from an intron of a reporter gene, thereby allowing coupled expression of the miRNA and a reporter protein from the same transcript. They also demonstrated for their miR-155 based vector that multiple miRNAs can be expressed in a single polycistronic transcript to increase the silencing effect or to overexpress different miRNAs at the same time. Obviously, the use of polymerase II expression vectors opens the door for the use of tissue-specific promoters and inducible expression systems as well. Altogether, a number of ways are available to up or downregulate endogenous miRNAs in a specific manner.

Designing artificial miRNAs for down-regulation of multiple mRNAs

Besides the up or downregulation of miRNAs, multiple target regulation might be achieved by the design of artificial miRNAs with the ability to bind to similar target sites in different mRNAs. Currently computer programs are available that can be used for the design of shRNAs or siRNAs directed against single target sequences (i.e.111, 112, 113). For synthetic multi-target miRNAs, the miRNA-binding target sequences should obviously share high sequence homology between the different mRNAs to be downregulated. This may not always be possible without the allowance of multiple miRNA/mRNA mismatches and possible decreased efficiency of gene silencing. Computer program-assisted sequence predictions in combination with empirical efforts may result in the development of artificial miRNAs for the downregulation of multiple target mRNAs. Alternatively, different single-target siRNAs or shRNAs can be expressed at the same time, resulting in the simultaneous regulation of multiple mRNAs. Looking ahead, siRNA, miRNAs, and AMO's can even be applied in concert to specifically regulate a set of mRNAs that are deregulated in diseases.

Therapeutic implications

So far, miRNAs have been implicated in multigenic diseases and up or down-regulation of these molecules should serve to counterbalance, or correct, certain deregulated post-transcriptional processes (Table 1). miRNA downregulation has been achieved by using 2-O-methyl oligonucleotides,97, 98, 99, 100 AMOs containing 2-O-methoxyethyl groups,76, 101 cholesterol-conjugated AMOs,75, 77 and DNA/LNA oligonucleotides.103 Also, upregulation of miRNAs has been found to have an effect on abnormal cells by modulating particular mRNA levels (Table 1). As described by Plasterk,114 in general, there are four ways that deregulation of miRNA/mRNA interactions may cause disease. Mutations can occur in the DNA encoding the miRNA and/or deregulated expression of the corresponding RNA, resulting in (1) loss-of-function or (2) gain-of-function, exemplified by tumor-suppressor miRNAs and oncogenic miRNAs, respectively (Figure 1). Also, (3) the miRNA target sites may be corrupted due to mutation of the particular target gene, resulting in partial or complete loss of miRNA-mediated inhibition of translation. In addition, (4) mutations in the target gene may result in new, or improved, miRNA binding sites resulting in aberrant gene silencing, as was recently shown by Clop et al.115 to play a possible role in the phenotypic variation of sheep, and further exemplified by the Tourette's syndrome mutation described by Abelson et al.74 Thus, different types of defects may lead to deregulated miRNA/mRNA interactions. Currently, miRNA and mRNA, as well as genome and protein screens can be used to analyze differential expression patterns in different tissues to elucidate deregulated miRNA/mRNA interactions. On the basis of this knowledge, molecular therapy might be applied via different schemes (see also Figure 1). In the case of downregulated miRNAs (1), selective upregulation might be achieved using viral vector or liposomal delivery of miRNAs, thereby normalizing the mRNA expression levels in a ‘one hit-multiple target’ manner. For upregulated miRNAs (2), selective AMOs might normalize the particular miRNA expression level in the affected cells. In this way, target gene expression levels might be restored. In the case of mRNA mutations corrupting a particular miRNA/mRNA-binding site (3), single-target RNAi might be applicable, resulting in the specific downregulation of the mutated mRNA. Alternatively, specific inhibitors may be developed that neutralize the particular overexpressed protein product. The acquisition of a new miRNA-binding site in a particular mutated mRNA (4) might result in suppression of that mRNA. Abrogation of that mutant miRNA/mRNA interaction through downregulation of the particular miRNA may not be useful, since this might also result in the loss of suppression of all the other mRNA targets of that particular miRNA. Therefore, reintroduction of the wild-type mRNA using gene transfer methods might be a possible treatment option as an alternative for the more straightforward continuous administration of the particular protein product or other suitable substitute.

Conclusions and future directions

miRNAs have emerged as important players in the regulation of gene expression and their deregulation is a common feature of a variety of diseases, especially cancer. Currently, many efforts are focused on studying miRNA expression patterns, as well as miRNA target validation. The total number of identified miRNAs keeps increasing, revealing the tremendous complexity of these transcripts in eukaryotic cellular networks. Clearly, identification of miRNA/mRNA interactions may lead to new strategies of intervention in human diseases. With appropriate computational tools, artificial miRNAs can be constructed which may have the ability to target several mRNA targets at the same time, thereby providing the option to down-regulate whole sets of mRNAs. The complexity of gene expression in eukaryotic cells serves to challenge the control of large sets of deregulated mRNAs at the same time. However, the emergence of new technologies for high-throughput expression profiling of miRNAs and mRNAs, as well as proteins, in combination with loss-of-function and gain-of-function screens, should aid in applying ‘epigenetic multi-target’ intervention strategies in the treatment of complex multigenic diseases.



anti-miRNA oligonucleotides




Angiotensin II Type 1 receptor


Histone deacetylase 4


Locked nucleic acid anti-sense oligonucleotides




non-coding RNAs


precursor miRNAs


primary miRNAs


Post-Trancriptional Gene Silencing


RNA Induced Silencing Complex


RNA interference


Serum Response Factor


short hairpin RNAs


small interfering RNAs


Untranslated Region


  1. 1

    Eddy SR . Non-coding RNA genes and the modern RNA world. Nat Rev Genet 2001; 2: 919–929.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. 2

    Costa FF . Non-coding RNAs: new players in eukaryotic biology. Gene 2005; 357: 83–94.

    CAS  PubMed  Google Scholar 

  3. 3

    Ambros V . The functions of animal microRNAs. Nature 2004; 431: 350–355.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Kim VN, Nam JW . Genomics of microRNA. Trends Genet 2006; 22: 165–173.

    CAS  PubMed  Google Scholar 

  5. 5

    Wightman B, Ha I, Ruvkun G . Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell 1993; 75: 855–862.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6

    Lee RC, Feinbaum RL, Ambros V . The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993; 75: 843–854.

    CAS  Article  Google Scholar 

  7. 7

    Cummins JM, He Y, Leary RJ, Pagliarini R, Diaz Jr LA, Sjoblom T et al. The colorectal microRNAome. Proc Natl Acad Sci USA 2006; 103: 3687–3692.

    CAS  PubMed  Google Scholar 

  8. 8

    Hsu PW, Huang HD, Hsu SD, Lin LZ, Tsou AP, Tseng CP et al. miRNAMap: genomic maps of microRNA genes and their target genes in mammalian genomes. Nucleic Acids Res 2006; 34: D135–D139.

    CAS  PubMed  Google Scholar 

  9. 9

  10. 10

    Denli AM, Tops BB, Plasterk RH, Ketting RF, Hannon GJ . Processing of primary microRNAs by the Microprocessor complex. Nature 2004; 432: 231–235.

    CAS  Google Scholar 

  11. 11

    Cai X, Hagedorn CH, Cullen BR . Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs. RNA 2004; 10: 1957–1966.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Bartel DP . MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004; 116: 281–297.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 2005; 433: 769–773.

    CAS  Google Scholar 

  14. 14

    Filipowicz W, Jaskiewicz L, Kolb FA, Pillai RS . Post-transcriptional gene silencing by siRNAs and miRNAs. Curr Opin Struct Biol 2005; 15: 331–341.

    CAS  Google Scholar 

  15. 15

    Hwang HW, Mendell JT . MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 2006; 94: 776–780.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Esquela-Kerscher A, Slack FJ . Oncomirs – microRNAs with a role in cancer. Nat Rev Cancer 2006; 6: 259–269.

    CAS  Google Scholar 

  17. 17

    Tang G, Reinhart BJ, Bartel DP, Zamore PD . A biochemical framework for RNA silencing in plants. Genes Dev 2003; 17: 49–63.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Doench JG, Petersen CP, Sharp PA . siRNAs can function as miRNAs. Genes Dev 2003; 17: 438–442.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Zeng Y, Yi R, Cullen BR . MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc Natl Acad Sci USA 2003; 100: 9779–9784.

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Petersen CP, Bordeleau ME, Pelletier J, Sharp PA . Short RNAs repress translation after initiation in mammalian cells. Mol Cell 2006; 21: 533–542.

    CAS  Google Scholar 

  21. 21

    Rajewsky N . microRNA target predictions in animals. Nat Genet 2006; 38 (Suppl 1): S8–S13.

    CAS  PubMed  Google Scholar 

  22. 22

    Lewis BP, Burge CB, Bartel DP . Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 2005; 120: 15–20.

    CAS  Google Scholar 

  23. 23

    Yi R, O'Carroll D, Pasolli HA, Zhang Z, Dietrich FS, Tarakhovsky A et al. Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nat Genet 2006; 38: 356–362.

    CAS  PubMed  Google Scholar 

  24. 24

    Schratt GM, Tuebing F, Nigh EA, Kane CG, Sabatini ME, Kiebler M et al. A brain-specific microRNA regulates dendritic spine development. Nature 2006; 439: 283–289.

    CAS  PubMed  Google Scholar 

  25. 25

    Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE, Hammond SM et al. The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 2006; 38: 228–233.

    CAS  PubMed  Google Scholar 

  26. 26

    Naguibneva I, Ameyar-Zazoua M, Polesskaya A, Ait-Si-Ali S, Groisman R, Souidi M et al. The microRNA miR-181 targets the homeobox protein Hox-A11 during mammalian myoblast differentiation. Nat Cell Biol 2006; 8: 278–284.

    CAS  PubMed  Google Scholar 

  27. 27

    Chen CZ . MicroRNAs as oncogenes and tumor suppressors. N Engl J Med 2005; 353: 1768–1771.

    CAS  PubMed  Google Scholar 

  28. 28

    Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci USA 2005; 102: 13944–13949.

    CAS  PubMed  Google Scholar 

  29. 29

    Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A et al. RAS is regulated by the let-7 microRNA family. Cell 2005; 120: 635–647.

    CAS  Google Scholar 

  30. 30

    Hayashita Y, Osada H, Tatematsu Y, Yamada H, Yanagisawa K, Tomida S et al. A polycistronic microRNA cluster, miR-17-92, is overexpressed in human lung cancers and enhances cell proliferation. Cancer Res 2005; 65: 9628–9632.

    CAS  Google Scholar 

  31. 31

    Chan JA, Krichevsky AM, Kosik KS . MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 2005; 65: 6029–6033.

    CAS  Google Scholar 

  32. 32

    Eis PS, Tam W, Sun L, Chadburn A, Li Z, Gomez MF et al. Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci USA 2005; 102: 3627–3632.

    CAS  PubMed  Google Scholar 

  33. 33

    Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R et al. A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 2006; 124: 1169–1181.

    CAS  Google Scholar 

  34. 34

    O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Medell JT . c-Myc-regulated microRNAs modulate E2F1 expression. Nature 2005; 435: 839–843.

    CAS  Google Scholar 

  35. 35

    Martin MM, Lee EJ, Buckenberger JA, Schmittgen TD, Elton TS . Microrna-155 regulates human angiotensin II type 1 receptor expression in fibroblasts. J Biol Chem 2006; 281: 18277–18284.

    CAS  PubMed  Google Scholar 

  36. 36

    Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D et al. MicroRNA expression profiles classify human cancers. Nature 2005; 435: 834–838.

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE et al. A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 2005; 353: 1793–1801.

    CAS  PubMed  Google Scholar 

  38. 38

    Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Petrocca F et al. A microRNA expression signature of human solid tumors defines cancer gene targets. Proc Natl Acad Sci USA 2006; 103: 2257–2261.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39

    Costinean S, Zanesi N, Pekarsky Y, Tili E, Volinia S, Heerema N et al. Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci USA 2006; 103: 7024–7029.

    CAS  PubMed  Google Scholar 

  40. 40

    Mirnics K, Pevsner J . Progress in the use of microarray technology to study the neurobiology of disease. Nat Neurosci 2004; 7: 434–439.

    CAS  PubMed  Google Scholar 

  41. 41

    Mocellin S, Provenzano M, Rossi CR, Pilati P, Nitti D, Lise M . DNA array-based gene profiling: from surgical specimen to the molecular portrait of cancer. Ann Surg. 2005; 241: 16–26.

    PubMed  PubMed Central  Google Scholar 

  42. 42

    Chung CH, Bernard PS, Perou CM . Molecular portraits and the family tree of cancer. Nat Genet. 2002; 32: 533–540.

    CAS  PubMed  Google Scholar 

  43. 43

    Miska EA, Alvarez-Saavedra E, Townsend M, Yoshii A, Sestan N, Rakic P et al. Microarray analysis of microRNA expression in the developing mammalian brain. Genome Biol 2004; 5: R68.

    PubMed  PubMed Central  Google Scholar 

  44. 44

    Krichevsky AM, King KS, Donahue CP, Khrapko K, Kosik KS . A microRNA array reveals extensive regulation of microRNAs during brain development. RNA 2003; 9: 1274–1281.

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Shingara J, Keiger K, Shelton J, Laosinchai-Wolf W, Powers P, Conrad R et al. An optimized isolation and labeling platform for accurate microRNA expression profiling. RNA 2005; 11: 1461–1470.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Nelson PT, Baldwin DA, Scearce LM, Oberholtzer JC, Tobias JW, Mourelatos Z . Microarray-based, high-throughput gene expression profiling of microRNAs. Nat Methods. 2004; 1: 155–161.

    CAS  PubMed  Google Scholar 

  47. 47

    Thomson JM, Parker J, Perou CM, Hammond SM . A custom microarray platform for analysis of microRNA gene expression. Nat Methods 2004; 1: 47–53.

    CAS  PubMed  Google Scholar 

  48. 48

    Baskerville S, Bartel DP . Microarray profiling of microRNAs reveals frequent coexpression with neighboring miRNAs and host genes. RNA 2005; 11: 241–247.

    CAS  PubMed  PubMed Central  Google Scholar 

  49. 49

    Liang RQ, Li W, Li Y, Tan CY, Li JX, Jin YX et al. An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe. Nucleic Acids Res 2005; 33: e17.

    PubMed  PubMed Central  Google Scholar 

  50. 50

    Barad O, Meiri E, Avniel A, Aharonov R, Barzilai A, Bentwich Z et al. MicroRNA expression detected by oligonucleotide microarrays: system establishment and expression profiling in human tissues. Genome Res 2004; 14: 2486–2494.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Babak T, Zhang W, Morris Q, Blencowe BJ, Hughes TR . Probing microRNAs with microarrays: tissue specificity and functional inference. RNA 2004; 10: 1813–1819.

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    Liu CG, Calin GA, Meloon B, Gamliel N, Sevignani C, Ferracin M et al. An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues. Proc Natl Acad Sci USA 2004; 101: 9740–9744.

    CAS  PubMed  Google Scholar 

  53. 53

    Neely LA, Patel S, Garver J, Gallo M, Hackett M, McLaughlin S et al. A single-molecule method for the quantitation of microRNA gene expression. Nat Methods 2006; 3: 41–46.

    CAS  PubMed  Google Scholar 

  54. 54

    Castoldi M, Schmidt S, Benes V, Noerholm M, Kulozik AE, Hentze MW et al. A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA). RNA 2006; 12: 913–920.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Mattie MD, Benz CC, Bowers J, Sensinger K, Wong L, Scott GK et al. Optimized high-throughput microRNA expression profiling provides novel biomarker assessment of clinical prostate and breast cancer biopsies. Mol Cancer 2006; 5: 24.

    PubMed  PubMed Central  Google Scholar 

  56. 56

    Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT et al. Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005; 33: e179.

    PubMed  PubMed Central  Google Scholar 

  57. 57

    Schmittgen TD, Jiang J, Liu Q, Yang L . A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res 2004; 32: e43.

    PubMed  PubMed Central  Google Scholar 

  58. 58

    Tang F, Hajkova P, Barton SC, Lao K, Surani MA . MicroRNA expression profiling of single whole embryonic stem cells. Nucleic Acids Res 2006; 34: e9.

    PubMed  PubMed Central  Google Scholar 

  59. 59

    Raymond CK, Roberts BS, Garrett-Engele P, Lim LP, Johnson JM . Simple, quantitative primer-extension PCR assay for direct monitoring of microRNAs and short-interfering RNAs. Rna 2005; 11: 1737–1744.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Jiang J, Lee EJ, Gusev Y, Schmittgen TD . Real-time expression profiling of microRNA precursors in human cancer cell lines. Nucleic Acids Res 2005; 33: 5394–5403.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. 61

    Lao K, Xu NL, Yeung V, Chen C, Livak KJ, Straus NA . Multiplexing RT-PCR for the detection of multiple miRNA species in small samples. Biochem Biophys Res Commun 2006; 343: 85–89.

    CAS  PubMed  Google Scholar 

  62. 62

    Slack FJ, Weidhaas JB . MicroRNAs as a potential magic bullet in cancer. Future Oncol 2006; 2: 73–82.

    CAS  PubMed  Google Scholar 

  63. 63

    Yanaihara N, Caplen N, Bowman E, Seike M, Kumamoto K, Yi M et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006; 9: 189–198.

    CAS  PubMed  Google Scholar 

  64. 64

    Borkhardt A, Fuchs U, Tuschl T . MicroRNA in chronic lymphocytic leukemia. N Engl J Med 2006; 354: 524–525; author reply 524–525.

    CAS  PubMed  Google Scholar 

  65. 65

    Ruvkun G . Clarifications on miRNA and cancer. Science 2006; 311: 36–37.

    CAS  PubMed  Google Scholar 

  66. 66

    Hammond SM . MicroRNAs as oncogenes. Curr Opin Genet Dev 2006; 16: 4–9.

    CAS  PubMed  Google Scholar 

  67. 67

    Murakami Y, Yasuda T, Saigo K, Urashima T, Toyoda H, Okanoue T et al. Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene 2006; 25: 2537–2545.

    CAS  PubMed  Google Scholar 

  68. 68

    Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al. Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2002; 99: 15524–15529.

    CAS  PubMed  PubMed Central  Google Scholar 

  69. 69

    Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, Yendamuri S et al. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci USA 2004; 101: 2999–3004.

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 70

    Kluiver J, Poppema S, de Jong D, Blokzijl T, Harms G, Jacobs S et al. BIC and miR-155 are highly expressed in Hodgkin, primary mediastinal and diffuse large B cell lymphomas. J Pathol 2005; 207: 243–249.

    CAS  PubMed  Google Scholar 

  71. 71

    He L, Thomson JM, Hemann MT, Hernando-Monge E, Mu D, Goodson S et al. A microRNA polycistron as a potential human oncogene. Nature 2005; 435: 828–833.

    CAS  PubMed  PubMed Central  Google Scholar 

  72. 72

    Takamizawa J, Konishi H, Yanagisawa K, Tomida S, Osada H, Endoh H et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 2004; 64: 3753–3756.

    CAS  Google Scholar 

  73. 73

    Cao X, Yeo G, Muotri AR, Kuwabara T, Gage FH . Noncoding RNAs in the mammalian central nervous system. Annu Rev Neurosci 2006; 29: 77–103.

    CAS  PubMed  Google Scholar 

  74. 74

    Abelson JF, Kwan KY, O'Roak BJ, Baek DY, Stillman AA, Morgan TM et al. Sequence variants in SLITRK1 are associated with Tourette's syndrome. Science 2005; 310: 317–320.

    CAS  Google Scholar 

  75. 75

    Esau C, Davis S, Murray SF, Yu XX, Pandey SK, Pear M et al. miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting. Cell Metab 2006; 3: 87–98.

    CAS  PubMed  Google Scholar 

  76. 76

    Esau C, Kang X, Peralta E, Hanson E, Marcusson EG, Ravichandran LV et al. MicroRNA-143 regulates adipocyte differentiation. J Biol Chem 2004; 279: 52361–52365.

    CAS  Google Scholar 

  77. 77

    Krutzfeldt J, Rajewsky N, Braich R, Rajeev KG, Tuschl T, Manoharan M et al. Silencing of microRNAs in vivo with ‘antagomirs’. Nature 2005; 438: 685–689.

    PubMed  Google Scholar 

  78. 78

    Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE et al. A pancreatic islet-specific microRNA regulates insulin secretion. Nature 2004; 432: 226–230.

    CAS  Google Scholar 

  79. 79

    Gupta A, Gartner JJ, Sethupathy P, Hatzigeorgiou AG, Fraser NW . Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript. Nature 2006; 442: 82–85.

    CAS  PubMed  Google Scholar 

  80. 80

    Lecellier CH, Dunoyer P, Arar K, Lehmann-Che J, Eyquem S, Himber C et al. A cellular microRNA mediates antiviral defense in human cells. Science 2005; 308: 557–560.

    CAS  Google Scholar 

  81. 81

    Jopling CL, Yi M, Lancaster AM, Lemon SM, Sarnow P . Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 2005; 309: 1577–1581.

    CAS  PubMed  PubMed Central  Google Scholar 

  82. 82

    Sullivan CS, Grundhoff AT, Tevethia S, Pipas JM, Ganem D . SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 2005; 435: 682–686.

    CAS  Google Scholar 

  83. 83

    Moffat J, Sabatini DM . Building mammalian signalling pathways with RNAi screens. Nat Rev Mol Cell Biol 2006; 7: 177–187.

    CAS  PubMed  Google Scholar 

  84. 84

    Behlke MA . Progress towards in vivo use of siRNAs. Mol Ther 2006; 13: 644–670.

    CAS  PubMed  Google Scholar 

  85. 85

    Vidal L, Blagden S, Attard G, de Bono J . Making sense of antisense. Eur J Cancer 2005; 41: 2812–2818.

    CAS  PubMed  Google Scholar 

  86. 86

    Jackson AL, Linsley PS . Noise amidst the silence: off-target effects of siRNAs? Trends Genet 2004; 20: 521–524.

    CAS  Google Scholar 

  87. 87

    Jackson AL, Linsley PS . Expression profiling reveals off-target gene regulation by RNAi. Nat Biotechnol 2003; 21: 635–637.

    CAS  Google Scholar 

  88. 88

    Jackson AL, Burchard J, Schelter J, Chau BN, Cleary M, Lim L et al. Widespread siRNA ‘off-target’ transcript silencing mediated by seed region sequence complementarity. RNA 2006; 12: 1179–1187.

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 89

    Birmingham A, Anderson EM, Reynolds A, Ilsley-Tyree D, Leake D, Federov Y et al. 3′ UTR seed matches, but not overall identity, are associated with RNAi off-targets. Nat Methods 2006; 3: 199–204.

    CAS  PubMed  Google Scholar 

  90. 90

    Fedorov Y, Anderson EM, Birmingham A, Reynolds A, Karpilow J, Robinson K et al. Off-target effects by siRNA can induce toxic phenotype. RNA 2006; 12: 1188–1196.

    CAS  PubMed  PubMed Central  Google Scholar 

  91. 91

    Lin X, Ruan X, Anderson MG, McDowell JA, Kroeger PE, Fesik SW et al. siRNA-mediated off-target gene silencing triggered by a 7 nt complementation. Nucleic Acids Res 2005; 33: 4527–4535.

    CAS  PubMed  PubMed Central  Google Scholar 

  92. 92

    Grimm D, Streetz KL, Jopling CL, Storm TA, Pandey K, Davis CR et al. Fatality in mice due to oversaturation of cellular microRNA/short hairpin RNA pathways. Nature 2006; 441: 537–541.

    CAS  Google Scholar 

  93. 93

    Mello CC, Conte Jr D . Revealing the world of RNA interference. Nature 2004; 431: 338–342.

    CAS  PubMed  PubMed Central  Google Scholar 

  94. 94

    Hoheisel JD . Microarray technology: beyond transcript profiling and genotype analysis. Nat Rev Genet 2006; 7: 200–210.

    CAS  Google Scholar 

  95. 95

    Meister G, Landthaler M, Dorsett Y, Tuschl T . Sequence-specific inhibition of microRNA- and siRNA-induced RNA silencing. RNA 2004; 10: 544–550.

    CAS  PubMed  PubMed Central  Google Scholar 

  96. 96

    Davis S, Lollo B, Freier S, Esau C . Improved targeting of miRNA with antisense oligonucleotides. Nucleic Acids Res 2006; 34: 2294–2304.

    CAS  PubMed  PubMed Central  Google Scholar 

  97. 97

    Meister G, Tuschl T . Mechanisms of gene silencing by double-stranded RNA. Nature 2004; 431: 343–349.

    CAS  PubMed  PubMed Central  Google Scholar 

  98. 98

    Hutvagner G, Simard MJ, Mello CC, Zamore PD . Sequence-specific inhibition of small RNA function. PLoS Biol 2004; 2: E98.

    PubMed  PubMed Central  Google Scholar 

  99. 99

    Lee YS, Kim HK, Chung S, Kim KS, Dutta A . Depletion of human micro-RNA miR-125b reveals that it is critical for the proliferation of differentiated cells but not for the down-regulation of putative targets during differentiation. J Biol Chem 2005; 280: 16635–16641.

    CAS  Google Scholar 

  100. 100

    Cheng AM, Byrom MW, Shelton J, Ford LP . Antisense inhibition of human miRNAs and indications for an involvement of miRNA in cell growth and apoptosis. Nucleic Acids Res 2005; 33: 1290–1297.

    CAS  PubMed  PubMed Central  Google Scholar 

  101. 101

    Baker BF, Lot SS, Condon TP, Cheng-Flournoy S, Lesnik EA, Sasmor HM et al. 2′-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells. J Biol Chem 1997; 272: 11994–12000.

    CAS  Google Scholar 

  102. 102

    Vester B, Wengel J . LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 2004; 43: 13233–13241.

    CAS  Google Scholar 

  103. 103

    Orom UA, Kauppinen S, Lund AH . LNA-modified oligonucleotides mediate specific inhibition of microRNA function. Gene 2006; 372: 137–141.

    CAS  PubMed  Google Scholar 

  104. 104

    Wienholds E, Kloosterman WP, Miska E, Alvarez-Saavedra E, Berezikov E, de Bruijn E et al. MicroRNA expression in zebrafish embryonic development. Science 2005; 309: 310–311.

    CAS  Google Scholar 

  105. 105

    Kloosterman WP, Wienholds E, de Bruijn E, Kauppinen S, Plasterk RH . In situ detection of miRNAs in animal embryos using LNA-modified oligonucleotide probes. Nat Methods 2006; 3: 27–29.

    CAS  PubMed  Google Scholar 

  106. 106

    Chung KH, Hart CC, Al-Bassam S, Avery A, Taylor J, Patel PD et al. Polycistronic RNA polymerase II expression vectors for RNA interference based on BIC/miR-155. Nucleic Acids Res 2006; 34: e53.

    PubMed  PubMed Central  Google Scholar 

  107. 107

    Stegmeier F, Hu G, Rickles RJ, Hannon GJ, Elledge SJ . A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells. Proc Natl Acad Sci USA 2005; 102: 13212–13217.

    CAS  Google Scholar 

  108. 108

    Brummelkamp TR, Bernards R, Agami R . A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550–553.

    CAS  PubMed  PubMed Central  Google Scholar 

  109. 109

    Miyagishi M, Taira K . U6 promoter-driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nat Biotechnol 2002; 20: 497–500.

    CAS  PubMed  PubMed Central  Google Scholar 

  110. 110

    Xia H, Mao Q, Paulson HL, Davidson BL . siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20: 1006–1010.

    CAS  PubMed  PubMed Central  Google Scholar 

  111. 111

  112. 112

  113. 113

  114. 114

    Plasterk RH . Micro RNAs in animal development. Cell 2006; 124: 877–881.

    CAS  PubMed  Google Scholar 

  115. 115

    Clop A, Marcq F, Takeda H, Pirottin D, Tordoir X, Bibe B et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet 2006.

Download references


We thank Dr Xandra O Breakefield, Dr Casey A Maguire, Dr Anna M Krichevsky and Dr Newton V Verbisck for critically reading this manuscript and for helpful suggestions. We also thank Suzanne McDavitt for helping with the manuscript format.

Author information



Corresponding authors

Correspondence to T Wurdinger or F F Costa.

Additional information

Duality of Interest

None declared.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Wurdinger, T., Costa, F. Molecular therapy in the microRNA era. Pharmacogenomics J 7, 297–304 (2007).

Download citation


  • non-coding RNAs
  • miRNAs
  • mRNA targets
  • multigenic diseases
  • cancer
  • ‘epigenetic’ therapy

Further reading


Quick links