MicroRNA—implications for cancer

Small ribonucleic acid (RNA) can act as a specific regulator of gene expression. This discovery has been an exciting breakthrough in Biological Sciences of the past decade, culminating in last year’s Nobel Prize in Physiology or Medicine awarded to Andrew Fire and Craig Mello. Building on previous work mainly in plants [50], Fire et al. [23] discovered that exogenous double-stranded RNA can be used to specifically interfere with gene function. This phenomenon was called RNA interference (RNAi). They also speculated that organisms might use double-stranded RNA naturally as a way of silencing genes. It was then shown that RNA interference was mediated by 22 nucleotide single-stranded RNAs termed small interfering RNAs (siRNAs) derived from the longer double-stranded RNA precursors [87]. The small interfering RNAs were found to repress genes by eliminating the corresponding messenger RNA transcripts, and thus, preventing protein synthesis.

Over the following years, many new small functional RNAs have been found. RNA is usually thought of as messenger RNA that serves as the template for translation of genes into proteins. In contrast, functional or non-coding RNA molecules are transcribed from a DNA sequence, but not translated into protein. The encoding DNA sequence is often referred to as an RNA gene. Functional RNA genes in the human genome include transfer RNA (tRNA), ribosomal RNA (rRNA), and various other small non-coding RNAs. Several hundred genes in our genome encode small functional RNA molecules collectively called microRNAs (miRNAs). Precursors of these miRNA molecules form structures of double-stranded RNA that can activate the RNA interference machinery. MicroRNAs downregulate gene expression either by degradation of messenger RNA through the RNA interference pathway or by inhibiting protein translation.

The first miRNA was discovered in 1993 by Victor Ambros and colleagues Rosalind Lee and Rhonda Feinbaum [42]. A genetic screen in the roundworm Caenorhabditis elegans, a millimeter-long animal used as a model organism in biological research, identified genes involved in developmental timing [42]. Surprisingly, one of the genes, termed lin-4, did not encode a protein but instead a novel 22-nucleotide small RNA. Seven years later, Reinhart et al. [70] discovered a second 22-nucleotide small RNA of this type, let-7, a gene also involved in C. elegans developmental timing. The lin-4 and let-7 small regulatory RNAs soon became very exciting for two reasons. Firstly, homologs of the let-7 gene were identified in other animals including humans [65]. The conservation of let-7 across species suggested an important and fundamental biological role for this small RNA. Secondly, the mechanism of RNA interference (RNAi) was discovered at that time, and it became clear that miRNA and RNAi pathways were intricately linked and shared common components. Within the following year, more than 100 additional small regulatory RNAs similar to lin-4 and let-7 were identified in worms, the fruit fly Drosophila, and in humans [38, 40, 41]. These small non-coding RNAs were named microRNAs (miRNAs) [38, 40, 41].

Subsequently, many more short regulatory RNAs were identified in almost all multicellular organisms, including flowering plants, worms, flies, fish, frogs, mammals [38, 40, 41, 48, 71], and in single cellular algae and DNA viruses [66, 75]. To date, more than 500 human miRNAs have been experimentally identified. Computational predictions of miRNA targets suggest that up to 30% of human protein coding genes may be regulated by miRNAs [46, 68]. This makes miRNAs one of the most abundant classes of regulatory genes in humans. MicroRNAs are now perceived as a key layer of post-transcriptional control within the networks of gene regulation.

MicroRNAs are sequentially processed from longer precursor molecules that are encoded by the miRNA genes [1] (Fig. 1). MiRNA genes are referred to by the same name (termed mir) written in italics to distinguish them from the corresponding mature miRNA (termed miR) followed by a number, e.g., mir-1 or miR-1. The encoding DNA sequence is much longer than the mature miRNA. Two ribonuclease enzymes, Drosha and Dicer, subsequently process the primary transcripts (or pri-miRNA) to generate mature miRNAs. The primary transcripts contain one or more stem-loop structures of about 70 bases. Stem-loops are double-stranded RNA structures consisting of a nucleotide sequence that can fold back on itself to form a double helix with a region of imperfect base pairing that forms an open loop at the end (Fig. 1a). The ribonuclease Drosha excises the stem-loop structure to form the precursor miRNA (or pre-miRNA) [43]. After export into the cytoplasm, the pre-miRNA is cleaved by the ribonuclease Dicer to generate a short RNA duplex [6, 28]. After untwisting, one RNA strand becomes the mature single-stranded miRNA, while the complementary strand, termed miRNA*, is usually rapidly degraded (Fig. 1b).

MicroRNAs recognize their targets based on sequence complementarity [10]. The mature miRNA is partially complementary to one or more messenger RNAs. In humans, the complementary sites are usually within the 3′-untranslated region of the target messenger RNA. To become effective, the mature miRNA forms a complex with proteins, termed the RNA-induced silencing complex. The miRNA incorporated into the silencing complex can bind to the target messenger RNA by base pairing. This base pairing subsequently causes inhibition of protein translation and/or degradation of the messenger RNA (Fig. 1c). The potential mechanisms underlying this process were recently reviewed [30, 67]. Protein levels of the target gene are consequently reduced, whereas messenger RNA levels may or may not be decreased. In humans, miRNAs mainly inhibit protein translation of their target genes and only infrequently cause degradation or cleavage of the messenger RNA [1].

The biological role and in vivo functions of most mammalian miRNAs are still poorly understood. In invertebrates, miRNAs regulate developmental timing (e.g., lin-4), neuronal differentiation, cell proliferation, growth control, and programmed cell death [9, 33, 42]. In mammals, miRNAs have been found to play a role in embryogenesis and stem cell maintenance [7], hematopoietic cell differentiation [17], and brain development [59, 60]. To date, knowledge of human miRNAs has been primarily descriptive. MicroRNA expression has been found to be deregulated in a wide range of human diseases including cancer. However, it remains uncertain whether altered miRNA expression is a cause or consequence of pathological processes. The underlying mechanisms of why and how miRNAs become deregulated are largely unknown. Although bioinformatics approaches can predict thousands of genes that are potentially targeted and regulated by miRNAs based on sequence complementarity, only very few miRNA target genes have been functionally validated. Our group is currently investigating the role of miRNAs in mammary gland development and breast cancer pathogenesis. A comparison of miRNA and gene expression identified miRNAs that classify molecular breast cancer subtypes [8]. As cancer is ultimately a consequence of disordered gene expression, miRNAs have been suggested to contribute to the development of cancer [11]. This review will focus on the connection between human miRNA biology and different aspects of carcinogenesis. Various techniques available to investigate miRNAs will also be discussed.

Three important observations early in the history of miRNAs suggested a potential role in human cancer. Firstly, the earliest miRNAs discovered in the roundworm C. elegans and the fruit fly Drosophila were shown to control cell proliferation and apoptosis [9, 42]. Their deregulation may therefore contribute to proliferative diseases such as cancer. Secondly, when human miRNAs were discovered, it was noticed that many miRNA genes were located at fragile sites in the genome or regions that are commonly amplified or deleted in human cancer [14]. Thirdly, malignant tumors and tumor cell lines were found to have widespread deregulated miRNA expression compared to normal tissues [12, 24, 52]. The question remained whether the altered miRNA expression observed in cancer is a cause or consequence of malignant transformation.

Regulatory RNAs may also have therapeutic applications by which disease-causing miRNAs could be antagonized or functional miRNAs restored. The most intuitive choice of molecules to correct altered miRNA–messenger RNA interactions are RNA oligonucleotides. These oligonucleotides need to be chemically modified to allow for stability in serum and cellular uptake. Modified antisense oligonucleotides are already being developed to utilize the intrinsic RNAi pathway for delivery of gene therapy. If the delivery problem can be overcome, then miRNA therapies may also be possible.

Two studies have successfully applied 2′-O-Methyl-modified antisense RNAs to inhibit miRNA function in cultured cells [29, 57]. Recent work by Krutzfeldt et al. [36] demonstrated that modified cholesterol-conjugated antisense RNAs designated “antagomirs” could effectively inhibit miRNA function in vivo in the adult mouse. The authors applied three daily intravenous injections of antagomirs and achieved effective inhibition of four miRNAs over a period of weeks in most tissues except brain [36]. A novel approach was recently reported by Ebert et al. [21]. They developed miRNA inhibitors that can be transiently expressed in cultured mammalian cells. These competitive inhibitors termed “miRNA sponges” derepressed miRNA targets at least as strongly as chemically modified antisense oligonucleotides [21]. A different approach was taken by Tsuda et al. [83]. The authors designed synthetic miRNAs to target overexpressed tumor proteins, such as HER-2 protein. A synthetic miRNA targeting HER-2 messenger RNA successfully inhibited HER-2 protein expression in ovarian cancer cells [83]. Together, these studies hold some promise of miRNAs as future therapeutic targets.

One limitation of antisense RNA therapies is the restricted number of cells that can be targeted. Any approach to knock down a particular miRNA with antisense oligonucleotides will only result in partial knockdown. This may represent a limitation for cancer therapies. It remains to be seen whether indirectly mediated bystander effects on cancer cells that have not been directly targeted may partly overcome this limitation. In contrast, a partial effect on function may be of therapeutic value in neurodegenerative diseases, such as Parkinson’s or Alzheimer’s disease. A partial restoration of dopamine production by antisense therapy might result in a significant clinical improvement in Parkinson patients. Similarly, a partial reduction of the disease-causing proteins in Alzheimer’s disease may lead to a clinical improvement and might be achievable by RNA based or miRNA gene therapy.

All known miRNAs are registered in a public web-based registry, the “miRBase” database that provides up-to-date information on all published miRNAs [25]. Novel miRNA genes can be discovered by bioinformatics approaches searching for evolutionary conserved stem-loop structures in the genome (reviewed in [3, 5]). Experimentally, miRNAs are discovered by cloning all small RNAs from a certain tissue type or developmental stage and subsequent sequencing to identify the subgroup of small RNAs that fulfill the criteria for miRNAs [38, 51]. Both computational and experimental approaches indicate that many more miRNAs are likely to be identified [4, 5], which is reflected by the rapidly increasing number of annotated miRNAs which increased from less than 300 to more than 4,000 over the past 4 years [58].