The principles discussed above have implications for the study of miRNAs in human disease. This is a burgeoning research topic, and we note here only a few recent examples.
The loss of individual miRNAs will derepress targets. At present, it is difficult to predict what consequences that will have for the organism. Systematic analysis of a nematode deletion collection that covers nearly all nematode miRNAs revealed that surprisingly few were obviously required for normal viability, fertility, morphology or behaviour86
. Undoubtedly, many of these mutants will eventually prove to be less fit than their wild-type counterparts in some respect. Taken the other way around, the fact that so many miRNA mutants seem to be viable may have substantial implications for disease. For example, mice carrying mutations in haematopoietic-system-expressed miRNAs such as miR-155 (REFS 87
), miR-150 (REF. 69
) or miR-223 (REF. 73
) are all viable and fertile, but they have abnormalities in immune cell numbers that can be of substantial consequence following immune challenge. Similarly, mice with mutations in cardiac miRNAs such as miR-1-2 (REF. 89
) or miR-208 (REF. 90
) are viable and fertile, but they exhibit profound stress-dependent defects in cardiac function and/or remodelling. Although some mammalian miRNA loci are essential (for example, the miR-17-92 cluster74
), it seems likely that loss of many individual human miRNA genes will prove to be compatible with life and reproduction but will underlie postnatal cardiac, immune, neurological or metabolic disorders.
As discussed, miRNAs might conceivably exert their major activities through handfuls of key targets or through the sum regulation of large batteries of targets with subtle individual regulation. In cases of the former type, the escape of individual targets from miRNA-mediated regulation may be of particular consequence. A long history of model-organism genetics indicates that very few genes are likely to cause a substantial gain-of-function phenotype simply by losing their 3′ UTRs. On the other hand, it is well worth knowing these few, as they are potentially attractive therapeutic targets. For example, HMGA2
encodes a chromatinassociated protein and contains seven let-7 sites in its 3′ UTR. The specific loss of these miRNA-binding sites strongly potentiated its ability to induce tumours91
. This correlates well with the observation that various malignant HMGA2
translocation alleles similarly delete the 3′ UTR and let-7 sites. Thus, HMGA2
may represent a type of miRNA genetic switch, especially in cancer cells. Generally speaking, a small number of targets maintain multiple conserved sites for a given miRNA, and many of these have proved to be of substantial genetic importance (BOX 3
Box 3 Frequent occurrence of multiple target sites in microRNA (miRNA) genetic switches
It is a challenge to discern individual predicted targets of particular consequence from large numbers of conserved predicted sites. However, it is striking that many of the animal miRNAs that have been identified through the discovery of loss-of-function alleles in forward genetic screens have genetic-switch functions that are substantially mediated by individual targets with multiple conserved sites. These include lin-4–lin-14
) and miR-279-nerfin
). In fact, these particular targets have the most conserved octomeric sites for the cognate miRNA, according to TargetScan21
) emerged from forward genetic screens as key miRNA targets that have multiple Brd-box miRNA sites or K-box miRNA sites8
, respectively, and similar trends apply to several miRNAs that have been analysed by reverse genetic screens, such as let-7 sister miRNAs: hbl-1 (REFS 41
) and miR-223–Dmef2c73
. This is not to say that individual targets with single binding sites are not important or cannot be genetic switches. A classic example is lin-28
, which mediates a genetic-switch function through a single lin-4 target site4
. On the other hand, the existence of target genes that maintain exceptional pairing to individual miRNAs may reflect regulatory linkages with biology that is worth investigating.
In the other direction, the overexpression of miRNAs could lead to the potentially detrimental over-repression of targets (). This is probably of broad consequence, as apparently both genetic-switch and fine-tuning targets are susceptible to strong repression by ectopic miRNAs, yielding target loss-of-function phenotypes32
. As miRNAs can operate through as little as seven nucleotides of complementarity, one must also consider the idea that repression through illegitimate sites created by mutations may have phenotypic consequence. For example, the muscular phenotype of the Texel sheep strain is due to a mutation in the myostatin 3′ UTR that creates a binding site for miR-1 and miR-206 — miRNAs that are highly expressed in skeletal muscle93
. As a key negative regulator of muscle mass, even slight decreases in myostatin activity yield muscle overgrowth.
A potentially broad connection to human disease was inferred from the location of many miRNA loci near fragile chromosomal sites94
. If such aberrations are causally due to miRNA dysfunction in the heterozygous state, it seems likely that many of them either remove repressive cis
-regulatory elements or bring into proximity novel transcriptional activation elements that deregulate miRNA expression. The appropriate transcriptional regulation of miRNAs is therefore of great importance. This is well illustrated by Myc, a transcription factor with both activator and repressor function. It was recently reported that there is widespread direct repression of miRNAs by Myc. This may be functionally linked to the oncogenic activity of Myc, as the enforced expression of Myc-repressed miRNAs could suppress Myc-induced tumours95
. Conversely, some miRNAs, including the six miRNAs that are encoded by the mir-17-92
Operon, are directly activated by Myc96
. This also has clinical consequences, as the mir-17-92
locus is highly amplified in various solid tumours and B cell lymphomas97
, and enforced expression of this miRNA Operon accelerates Myc-induced tumorigenesis98
. The cis
-regulatory control of few miRNAs is known in detail, but these observations indicate that it will be crucial to elucidate both the transcriptional activation and the transcriptional repression of miRNAs.