RNA interference-based functional genomics in cancer research – an introduction

Abstract

Advances in biology are strongly influenced by developments in the technology available to researchers. In the past, these technological breakthroughs often came from other scientific disciplines. For example, the development of the microscope followed on from improvements in optics during the 17th century and enabled the discovery of cells by Robert Hooke. More recently, major new methodological advances have often come from within biology itself, such as the foundation of recombinant DNA technology based on the discovery of restriction enzymes. In the past few years, another discovery in basic biology has shown the potential to provide a technological revolution in the way that biological research is conducted. This is the discovery of gene silencing by RNA interference, first elucidated by Fire and Mello in the nematode worm in 1998. RNA interference is a natural mechanism whereby metazoan cells suppress the expression of genes when they come across double-stranded RNA molecules of the same sequence. It probably evolved to combat viruses and rogue genetic elements that utilize doublestranded RNA during their lifecycle. It is an ancient mechanism, found across the animal and plant kingdoms, and in mammals has been supplemented by other, more recent, defence systems. The discovery of RNA interference has led to the important conceptual realization that RNA molecules can have a major impact on the regulation of gene expression beyond their two previously recognized roles as genetic information intermediates (messenger RNA) and structural components in the protein expression machinery (ribosomal and transfer RNAs). In addition to this very important advance in biological understanding, RNA interference has provided biologists with a very powerful tool with which to switch off gene expression. Previously, this had been a very difficult, expensive and timeconsuming thing to achieve in many organisms, especially mammals. RNA interference has made ‘reverse genetics’ (discovering the unknown function of a known gene) much more straightforward not only in mammals, but also in flies and worms. Yet more exciting, though at the present time less well validated, is the use of a combination of RNA interference and genomics to make ‘forward genetics’ (discovering the unknown genes underlying a known phenotype) enormously more tractable. It is this last area, especially as applied to the problem of cancer, which is the focus of this issue of Oncogene Reviews. Large libraries of RNA interference reagents can be used to knock down individual gene expression on a genome-wide scale. A total of 10 groups working on various aspects of this nascent technology review its promise, and its pitfalls. The first article (Downward, 2004) describes the various ways in which RNA interference libraries can be made and used in mammalian cell culture. Two main types of library have been constructed, one using synthetic short double-stranded RNA oligonucleotides, the other vectors that when expressed in cells drive the expression of short interfering double-stranded RNA molecules. The libraries can be used for high-throughput screening on a gene-bygene basis to find those that underlie the characteristics of malignant transformation. In addition, vector-based libraries can be used in a pooled format in selective screens: these allow the identification of genes that when disrupted allow cells to survive a selective pressure, which can be useful in identifying novel tumoursuppressor genes. The construction and use of the leading vector-based mammalian RNA interference library is described by Silva et al. (2004). This collection of vectors targeting both human and mouse genes has been successfully used to investigate a number of phenomena important in carcinogenesis, including proteasome function and cell cycle regulation. It is planned to expand it to encompass the entire human genome by mid 2005. The main competing technology, the use of large synthetic oligonucleotide collections targeting mammalian genes, is described by Sachse and Echeverri (2004). While highly effective, the relatively high costs associated with these reagents and their nonrenewable nature has meant that they have been more favoured by commercial rather than academic laboratories. The application of oligonucleotide RNA interference libraries to key biological issues underlying the transformed phenotype, such as resistance to programmed cell death, is addressed by Willingham et al. (2004). The use of RNA interference libraries, whether oligonucleotide or vector based, in high-throughput screening mode can be time-consuming and costly. One very promising approach to circumventing these problems is to use the recently developed technology of reverse transfection. As described by Vanhecke and Janitz (2004), RNA interference reagents can be spotted onto glass slides in a microarray format and cells then overlaid. This has been successfully used to study the Oncogene (2004) 23, 8334–8335 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00