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.
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.
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.
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.
Angiotensin II Type 1 receptor
Histone deacetylase 4
Locked nucleic acid anti-sense oligonucleotides
Post-Trancriptional Gene Silencing
RNA Induced Silencing Complex
Serum Response Factor
short hairpin RNAs
small interfering RNAs
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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.
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Wurdinger, T., Costa, F. Molecular therapy in the microRNA era. Pharmacogenomics J 7, 297–304 (2007). https://doi.org/10.1038/sj.tpj.6500429
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