Ask the Expert: MicroRNAs – keep your research on track

The last few years have seen a rush of discoveries within a new field of post-transcriptional gene regulation. microRNAs, or miRNAs for short, are small regulating RNAs akin to small interfering RNAs (siRNA), but which are naturally expressed in vivo.

Originally discovered in C. elegans 14 years ago, these small 20-22 nucleotide non-coding RNA molecules bind specifically to target messenger RNAs (mRNA) blocking their translation into protein or causing their degradation. So far about 480 miRNAs have been identified in humans, with the total predicted to be 1000 or more. They are evolutionarily very well conserved, many sequences being identical across species, indicating that these small RNA molecules have been involved in the regulation of mRNA function for a long time.

The importance of miRNAs in biology has likely been missed so far due to their small size, the difficulties involved in purifying and detecting them and the fact that inactivating mutations had not been discovered. However, the development of new technologies, has led to an explosion in miRNA research and a link between miRNAs and disease, especially cancer, has been firmly established. One such new technology is the miRCURY LNA product line from Exiqon. So far, miRNAs have been shown to be potentially important as cancer biomarkers as well as possible targets for therapies.

miRNAs are expressed either as single copy or multi (poly)-cistronic transcripts and can be found as independent genes or within introns (or even exons) of other genes. The primary transcript (or pri-miRNA) contains a stem-loop structure which is released from the rest of the transcript by a nuclear RNaseIII complex (Drosha-DGCR8) to form the precursor or pre-miRNA. Following export in to the cytoplasm, the pre-miRNA is further processed by another RNase complex (Dicer-TRBP) into a ~22 nt miRNA duplex. Mature miRNAs are incorporated into an effector complex known as RISC (RNA induced silencing complex) and converted into single strands ready to target mRNAs.

Each miRNA potentially has up to 200 mRNA targets and it has been predicted that most human genes will probably be influenced in some way by miRNAs. Prediction and validation of miRNA targets is however one of the more difficult tasks within the field. miRNAs bind to the 3’ untranslated region (UTR) of target mRNAs, but the binding sites are frequently not perfectly complimentary to the miRNA sequence. A seed sequence of about 6-8bp towards the 5’ end of the miRNA sequence is thought to be important for target recognition. The fact that multiple miRNAs may bind the same target adds further complexity to target prediction.

The function of miRNAs is post-transcriptional regulation of target mRNAs. It is believed that most miRNAs function by blocking mRNA translation and reducing the levels of protein produced without affecting the level of mRNA significantly. However, it also seems that some miRNAs do cause degradation of mRNAs when the binding site and miRNA sequence are perfectly complimentary. In addition it has been suggested that multiple miRNAs may act synergistically to inhibit or fine-tune protein expression.

The biological function of most miRNAs is still unknown, especially in mammals. The first miRNAs to be studied in model organism such as worms (C. elegans), fruit flies (Drosophila melanogaster) and zebra fish (Danio rerio) have important functions during development in the coordination of cell proliferation, cell death and differentiation. miRNA expression profiles vary between different tissues and during development and it has been suggested that miRNAs may have important roles in differentiation. It is no surprise therefore, that miRNAs have also been implicated in cancer and other diseases.

A few miRNAs have already been shown to play a role in cancer both as tumour suppressors and as oncogenes. The loss or overexpression of such miRNAs respectively, has implications both for diagnosis and prognosis. Many of the known miRNA genes are found in cancer associated genomic regions, and the expression profiles of miRNAs in tumours can be differentiated from those of normal tissue. Many studies conducted so far on cancer cell lines or tumour samples have found that not only are the miRNA expression profiles different, but they can also be used to classify particular cancer types. Recently, miRNA expression profiles have been shown to be more informative for cancer diagnosis than mRNA expression profiles.

Read an expanded version of this article online at www.ngpharma.com

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