Abstract

Introduction

MicroRNAs are small, non-coding RNAs of 18 to 24 nucleotides, discovered in 1993 in the nematode Caenorhabditis elegans. Lee et al. [2] found that a gene crucial for C. elegans post-embryonic development, lin-4, does not code for a protein, but rather is transcribed into a 22-nucleotide RNA molecule. This molecule could repress the expression of the lin-14 mRNA by directly interacting with its 3' untranslated region (UTR) [3]. While this was recognized as a new method of gene regulation, it was initially considered as an oddity peculiar to C. elegans. The discovery of let-7, another small RNA involved in developmental timing in C. elegans [4], and the finding that both its sequence and temporal expression pattern were largely conserved in a variety of organisms [5] immediately suggested these small RNAs might in fact play important and conserved roles in gene regulation, and the identification of hundreds of miRNAs in the worm, fly, and mammalian genomes followed [1].

To date, more than 300 miRNAs have been discovered in humans, and computational analyses predict that up to 1,000 miRNAs exist in the genome [6,7]. Since miRNAs can regulate more than one target, estimates indicate they may be able to regulate up to 30 percent of the protein-coding genes in the human genome [8], highlighting their importance as global regulators of gene expression.

miRNA Biogenesis and Method of Action

MiRNAs are transcribed by RNA Polymerase II into large precursor RNAs, often several kilobases in length, called pri-miRNAs. Notably, the majority of human miRNAs are transcribed from regions found within introns of either protein-coding or non-protein-coding transcripts, but a minority is found in isolated regions of the genome, within the exons of noncoding mRNA genes, or within the 3'UTRs of mRNA genes.

In the nucleus, these pri-miRNAs are capped and polyadenylated prior to being processed by Drosha, a member of the RNase III enzyme family, in conjunction with the double-stranded RNA-binding protein DGCR8/Pasha. This processing step produces segments 70 nucleotides in length, which fold into stem-loop structures known as pre-miRNAs [9]. These are exported from the nucleus in a GTP-dependent fashion by exportin 5 and are subject to an additional processing step by another RNase III enzyme, Dicer. This step releases a double-stranded RNA duplex, 22 nucleotides in length, which in turn is incorporated into the miRISC complex, in analogous fashion to that observed in RNA interference (RNAi). In this complex, the mature miRNA strand is retained, and the complex is now capable of regulating its target genes.

The identification of miRNA target transcripts remains one of the greatest challenges in the field today. MiRNAs can be organized into families based on sequence homology, which is particularly strong at the 5' end of the mature miRNA, suggesting this section of the mature transcript has been preserved through evolution and thus plays an important role in the process of target recognition. Indeed, studies have shown this 5' region, often called the "miRNA seed," to be crucial for both the stability of the mature miRNA and its incorporation into the miRISC complex [10-14]. Bioinformatic approaches have taken advantage of this "miRNA seed" to predict miRNA targets across the genome. It has been predicted that a single miRNA can bind over 200 different target transcripts, and, notably, these targets are highly diverse, from transcription factors to transporters [15-24].

There is nevertheless evidence supporting a role for the regions not encompassed within the "miRNA seed." Perhaps the clearest example of this is found in let-7, whose complete mature miRNA sequence is conserved across species [25], suggesting an important role for the 3' region of the miRNA in target transcript binding. Further insights into the miRNA-target recognition mechanism are needed, as they will make target prediction much more accurate and efficient.

A Potential Role for miRNAs in Cancer

Cancers of all types share a number of characteristics, such as the loss of cellular identity, an increased ability to grow and proliferate, and alterations in the systems controlling cell death. Studies performed in a variety of organisms have revealed that miRNAs have the ability to regulate these cellular processes, suggesting that they could be involved in cancer. For instance, lin-4 and let-7 control the timing of developmental events in C. elegans. Mutations in these miRNAs resulted in abnormalities in cell-cycle exit as well as in the execution of a terminal differentiation program, preventing cells from reaching their fully differentiated state [2].

In Drosophila, it has been found that over-expression of the Bantam miRNA causes excessive growth of wing and eye tissue by blocking the pro-apoptotic action of the gene hid [26,27]. It was also found that another Drosophila miRNA, miR-14, can act as a strong suppressor of apoptosis. Deletion of this miRNA caused increased expression of the apoptotic effector caspase, Drice, suggesting a direct regulation of this factor by miR-14 [28]. Likewise, the miR-2/6/11/13/308 family of Drosophila miRNAs has been reported to induce widespread apoptosis in embryos through the regulation of the proapoptotic factors hid, grim, reaper, and sickle, which act through the inhibition the caspase inhibitor Diap1 in response to a number of natural and toxic conditions [29].

Another aspect in which miRNAs appear to have a role is the proper differentiation of cells into different tissues. For example, miR-273 and the miRNA encoded by lys-6 have been shown to play important roles in the patterning of the C. elegans nervous system [30,31], and miR-430 has been implicated in brain development [3]. The importance of miRNA in development has been shown also in mammalian systems, examples of which are miR-181 in the differentiation of hematopoietic cells toward the B-cell lineage [33], miR-374 in pancreatic islet-cell development [34], miR-143 in adipocyte differentiation [35], miR-196 in limb patterning by SHH [36], and miR-1 in heart development [37].

miRNAs as Tumor Suppressors

miRNAs as Oncogenes

miRNA Signatures and Detection Methods

The multiple lines of evidence indicating that microRNAs are differentially expressed in normal and in tumor samples suggest that miRNAs could serve as useful tumor profiling tools. Indeed, profiling studies have shown that each cancer type possesses a distinct miRNA expression signature, and this signature can provide useful information about the malignancy. The adaptation of high-throughput technologies have facilitated the study of the expression of multiple miRNAs in a given sample, making it possible to profile substantial sample numbers with relative ease. Among the most important methods for miRNA profiling is the use of oligonucleotide miRNA microarray analysis, which allows the researcher to simultaneously determine the expression levels of hundreds of miRNAs in a given sample [65-67]. Multiple variations of this technique have been developed, underscoring its position as the central miRNA profiling tool [68-71][68-71]. Other important approaches to the problem are also available, such as quantitative PCR for precursor miRNAs [72] or mature miRNAs [73,74]; genome-wide approaches with serial analysis of gene expression (SAGE), such as miRAGE [75]; and bead-based flow cytrometric techniques [76].

Taking advantage of these tools, several groups have studied the miRNA expression of several types of cancer. Michael et al. [49] and Cummins et al. [75] have studied the expression profile of the known miRNAs in colorectal cancer samples. These studies independently found a general down-regulation of miRNA levels in tumor cells compared to normal colonic epithelium, while also observing a significant decrease in the levels of miR-143 and miR-145 beyond this general effect. Cummins et al. found a signature of 50 differentially expressed miRNAs in malignant vs. normal colonic epithelium, of which 32 miRNAs had reduced levels in tumor cells. Yanaihara et al. studied over 100 patient-matched pairs of primary malignant and normal adjacent lung tissue and found that the expression of 43 miRNAs was significantly different in the tumor tissues compared to the normal adjacent tissues. Of these miRNAs, 28 were down-regulated and 15 were up-regulated in the malignant tissue [46]. Similar studies have been carried out in CLL [77], breast cancer [50], glioblastoma [78], pancreatic cancer [79], hepatocellular carcinoma [80], and thyroid papillary carcinoma [58].

The ability to profile the miRNA expression of a tumor with accuracy and reproducibility in a clinical setting would constitute a valuable medical tool. Indeed, miRNA profiles have been shown to be highly informative. Using a bead-based approach, Lu et al. attempted to classify 17 poorly differentiated tumors with non-diagnostic histological appearance based on their miRNA expression profiles. A clinical diagnosis on these tumors was established by anatomical context, whether directly (a primary tumor observed in a given organ) or indirectly (a metastasis of a previously identified primary tumor). They were able to correctly diagnose 12 of the 17 samples, against 1/17 correct diagnoses for an analysis using their mRNA profile [76]. While the number of transcripts analyzed was much smaller for miRNAs than for mRNAs (~200 against > 15,000), it is possible that the miRNA-based diagnoses are more accurate because of the regulatory role of these molecules, while most mRNAs will not be regulatory in nature. Further studies, however, are necessary to ascertain the value of miRNAs as diagnostic tools, and their potential value as prognostic markers, by correlating miRNA expression with type-specific parameters such as metastatic potential, proliferative index, and response to existing treatments.

Closing Remarks: miRNAs as Treatment Targets

While the field of miRNAs — and particularly the study of the roles of miRNAs in cancer — is at an early stage, their potential as targeted therapeutic tools has not gone unnoticed. A promising approach is to target oncogenic miRNAs with oligonucleotides complementary for either their mature or precursor sequence, deemed anti-miRNA oligonucleotides (AMOs). A number of chemical modifications for these oligonucleotides are available, with the purpose of making them more stable and less toxic. 2'-O-methylated AMOs are perhaps the most common form of modified RNA, conferring limited nuclease resistance, and enhancing the binding affinity to RNA. Inhibition of let-7 function with 31-nucleotide 2'-O-Me AMOs has been demonstrated in HeLa cells and C. elegans larvae [81], and 2'-O-Me AMOs have been reported to abrogate miRNA function in cellular assays [82,83], providing proof-of-principle on the blockage of miRNA function by extraneous oligomers. Another common modification to the structure of AMOs is that of the locked nucleic acid (LNA)-modified oligomers. In these RNAs, the 2' oxygen atom is linked to the 4' position through a methylene bridge, forming a rigid bicyclic structure locked into a C3'-endo (RNA) sugar conformation [84]. This modification results in greatly enhanced affinity for RNA and the formation of exceedingly strong duplexes with excellent mismatch discrimination properties [85-87]. This makes them prime candidates for AMO-based therapy, and results in rodents have been encouraging [88].

While the use of AMOs could be useful to inhibit oncogenic miRNAs, another therapeutic possibility concerns the cases in which certain miRNAs are under-expressed, such as let-7 in lung cancer, or miR-15a and mir-16-1 in CLL. In this case, synthetic miRNAs — likely modified as discussed above — in either their mature or precursor form could be delivered to the target cells to counteract the deficiency and halt malignant growth. While this approach has been attempted in cell culture [89], further testing in animal models is required.

A pressing challenge to the use of miRNAs as therapeutic tools concerns finding methods to deliver synthetic miRNAs or AMOs to the desired tissues in a targeted and effective manner. Given that these molecules cannot discriminate between healthy and malignant cells, side effects of treatment remain a concern. Likewise, since these studies are in their infancy, little is known about the pharmacokinetics of AMOs or synthetic miRNAs, limiting the discussion of their promise as therapeutic tools into the speculative arena. Upon reaching a deeper understanding of the mechanism of miRNA biogenesis and action, and the development of new delivery technologies, these small RNAs might well fulfill their promise as valuable therapeutics.

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