Mirna And Other Small Rnas In Cancer

The miRNA is a small, up to 22-base long, single-stranded RNA whose nucleotide sequences are evolutionally conserved from Caenorhabditis elegans to Homo sapiens (Pasquinelli et al., 2000) and that play critical roles in biological functions (Bartel, 2004; Carthew, 2006). The miRNAs are expressed as hairpin-shaped double-stranded pre-miRNAs. Sequential processing by different RNase III enzymes, Drosha and Dicer, generates mature miRNA (Cullen, 2004).

The first characterized miRNA was the lin-4 gene in C. elegans (Lee et al., 1993). The lin-4 is known as a "heterochronic" gene: its mutant shows a disorder that affects the developmental timings, for example the first-stage larva could not proceed to the second stage. The lin-4 negatively regulates LIN-14 protein in the first larval stage. Lee et al. revealed that the lin-4 transcripts were small ncRNA of 22- and 61-nt lengths and that they contained sequences complementary to a repeated sequence element in the 3' YTP of lin-14 mRNA. A following study indicated that the lin-4 suppresses translation of lin-14 (Wightman et al., 1993). Similarly, another short RNA coding gene, let-7 (Reinhart et al., 2000), is implicated in the heterochrony in worm development. The discovery of RNAi (Fire et al., 1998) and the identification of argonaute protein as a major player in the RNAi machinery (Tabara et al., 1999) led the way to connect the miRNA with the RNAi pathway (Grishok et al., 2001). The reduction of the gene expression of the factors in the RNAi pathway (dcr-1, Dicer homologue; alg-1 and alg-2, Argonaute proteins) mimics the heterochronic phenotypes of the lin-4 and let-7 mutations. It is also shown that dcr-1, alg-1, and alg-2 are essential for the maturation of those miRNAs. The disruption of the RNAi pathway also caused the developmental disorders, some of which were rescued by an injection of mature miRNA, in zebrafish (Giraldez et al., 2005).

Although the genetics of C. elegans showed clearly that the miRNA can be a molecular switch through the suppression of translation, it has become evident that miRNAs can mediate cleavage of target mRNAs (Lim et al., 2005). It is shown that an efficient translation suppression by miRNA requires multiple miRNA binding sites in the 3' UTR of the target mRNA (Doench and Sharp, 2004; Petersen et al., 2006). Moreover, the length of the target sequences of miRNA is only 8 bases so that one miRNA can have multiple targets in the cell (Lim et al., 2005).

Since miRNA has a broad range of targets in a cell (i.e., is less specific) and since the extent of suppression of the target expression by miRNA is generally moderate, the function of miRNAs should be considered as the "fine-tuning" of gene expression in mammalian cells (Bartel and Chen, 2004). The fine-tuning effect, however, is strong enough to be a selection pressure during the evolution (Farh et al., 2005; Stark et al., 2005). One example of this can be seen in the ubiquitously expressed housekeeping genes. These have relatively short 3' UTRs and a relatively small number of miRNA target sites, indicating that such important genes in cellular functions lost those miRNA target sequences during the evolution.

It has become evident that RNAi pathway plays a critical role in the suppression of transposon (Tabara et al., 1999), in virus infection (Lecellier et al., 2005; Li et al., 2002), induction of differentiation (Chen et al., 2004a; Schratt et al., 2006), and in other biological regulations. Fazi et al. (2005) reported that the transcriptional regulation of miR-223 by the two transcription factors FNI-A and C/EBPa plays important roles in development. Interestingly, human miR-122 contributes to the Hepatitis C virus replication in human liver cells (Jopling et al., 2005). One study indicates that miRNAs can have critical functions in human hereditary disorders. Tourette's syndrome (TS) is a developmental neuropsychiatric disorder characterized by chronic vocal and motor tics. A genetic background has been suggested for the etiology. A frameshift mutation of the SLTRK1 gene, the candidate susceptible gene of the disease, was found in two independent cases of the disease. The disease-specific mutant transcript of SLTRK1 lost the target site of the miRNA miR-189. Both SLTRK1 and miR-189 are coexpressed in the brain region related to the Tourette's disease. Further, wild-type SLITRK1 overexpression induces dendritic growth in primary neuronal cultures whereas mutant does not (Abelson et al., 2005).

In terms of carcinogenesis, miRNA has been known as an important player (Hammond, 2006; Zhang et al., 2007). The first report suggesting the involvement of miRNA in malignant neoplasms appeared in 2002 (Calin et al., 2002). Calin et al. showed that two miRNAs, miR-15 and miR-16, are frequently deleted or downregulated in B cell chronic lymphocytic leukemia (CLL) (in 68% of the cases). Both miRNAs are located within a 30-kb region at chromosome 13q14, a region deleted in more than half of the CLL cases (Calin et al., 2002). It is also indicated that many human miRNA genes localize at fragile sites and cancer-related genomic regions, suggesting the involvement of change of expression of miRNA in carcinogenesis (Calin et al., 2004). One of the major topics of miRNA in cancer is that let-7 miRNA may suppress the protein expression of the protooncogene RAS (Hayashita et al., 2005; Johnson et al., 2005). It is shown that the target of the let-7 family, let-60, is a homologue of RAS in C. elegans and genetic evidence with C. elegans suggested that let-7 is epistatic to the let-60/RAS (Johnson et al., 2005). The same epistatic relationship is conserved in human. Moreover, let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors (Hayashita et al., 2005).

Biological significance of miRNA expression in cancer was also shown in other ways. The expression profiling analyses of miRNA in cancer tissue revealed that the miRNA profiles reflected the developmental lineage and differentiation state of the tumors. A general downregulation of miRNAs in tumors compared to miRNA in normal tissues were also observed (Lu et al., 2005). The study used multicolored fluorescent beads detection with a fluorescently activated cell sorting system and showed that the miRNA expression profiles can be used more accurately than conventional mRNA expression profiles in cancer diagnosis. Similarly, the expression profiles of miRNAs showed association with the prognostic factors in CLL (Calin et al., 2005). A study indicated that a cluster of miRNAs, the miR-17-92 polycistron, can enhance the tumorigenicity of mouse B-cell lymphomas induced by the Eu-myc transgene (He et al., 2005). Moreover, the expression levels of the primary or mature miRNAs derived from the miR-17-92 locus are often substantially increased in these cancers in human B-cell lymphoma samples and in cell lines to normal tissues. Through functional screening, two miRNAs, miR-372 and miR-373, were identified as cooperators of oncogenic RAS in tumorigenesis of human testicular germ cell tumors (Voorhoeve et al., 2006). These miRNAs are supposed to neutralize p53-mediated CDK inhibition, possibly through direct inhibition of the expression of the tumor-suppressor LATS2. There are many other examples of miRNAs related to carcinogenesis (Calin and Croce, 2006; Esquela-Kerscher and Slack, 2006). Zhang et al. (2006) revealed that the copy numbers of miRNA containing chromosomal regions were very frequently changed in several human cancer cells.

Many small RNAs are characterized in various organisms (Kim, 2006). One important example is rasiRNA in Drosophila (Aravin et al., 2001, 2003, 2004). The function of the rasiRNA is implicated in the suppression of selfish genes in germ line cells (Vagin et al., 2006). Several scientists reported that similar small RNAs, such as piRNAs, are found in mammalian germ line cells (Aravin et al., 2006; Girard et al., 2006; Grivna et al., 2006; Watanabe et al., 2006). It is an open question whether such small RNAs play any critical role in carcinogenesis.

XV. EPIGENETICS AND ncRNA IN CARCINOGENESIS

Most of the cancers showed various amount of aberrant DNA methyla-tion at the promoter regions of tumor suppressors (Ting et al., 2006). It is known that the chromatin-mediated abnormalities occur in early stage of carcinogenesis so the epigenetic changes may play an important role in the initiation of cancer (Feinberg et al., 2006).

It is widely accepted that the expression of ncRNAs is usually accompanied with the genome imprinting (O'Neill, 2005). On the other hand, no evidence is available that supports the expression of long ncRNAs can be related to the alteration of DNA methylation states in carcinogenesis or aging. LOI is the one of the earliest known epigenetic abnormalities found in cancer (Feinberg and Tycko, 2004). Imprinting is a phenomenon appearing as the monoallelic expression in some biallelic genes in the cells. Mammalian somatic cells have two copies of each autosomal gene: one from the father and one from the mother. Such biallelic genes are usually expressed from both paternal and maternal alleles. The imprinted genes have been found to be expressed from only one of the two alleles. So, LOI appears as a disorder of this allelic restriction of the imprinted genes in cancer cells. It means that the biallelic gene expression occurs from the genes that are normally imprinted in noncancerous cells. LOI is shown to be important in cancinogenesis, although its mechanism is still largely unknown. One of the most characterized imprinted genes in terms of LOI is IGF2. The LOI of IGF2 gene will cause increase of the dose of potent growth factor, IGF2. However, the RNA sequences of H19 itself is not critical for the establishment of IGF2 imprinting (O'Neill, 2005). Actually, the LOI state of H19 and IGF2 were not always correlated among different types of cancers (Kondo et al., 1995). Because a very long ncRNA, Air, plays a critical role in the establishment of IGFR2 gene imprinting in mice (Sleutels et al., 2002), it is quite possible that unknown ncRNA plays a positive role in epigenetic disorders found in cancer cells.

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