Research Article: Variability in the Incidence of miRNAs and Genes in Fragile Sites and the Role of Repeats and CpG Islands in the Distribution of Genetic Material

Date Published: June 17, 2010

Publisher: Public Library of Science

Author(s): Alessandro Laganà, Francesco Russo, Catarina Sismeiro, Rosalba Giugno, Alfredo Pulvirenti, Alfredo Ferro, Amanda Ewart Toland.

Abstract: Chromosomal fragile sites are heritable specific loci especially prone to breakage. Some of them are associated with human genetic disorders and several studies have demonstrated their importance in genome instability in cancer. MicroRNAs (miRNAs) are small non-coding RNAs responsible of post-transcriptional gene regulation and their involvement in several diseases such as cancer has been widely demonstrated. The altered expression of miRNAs is sometimes due to chromosomal rearrangements and epigenetic events, thus it is essential to study miRNAs in the context of their genomic locations, in order to find significant correlations between their aberrant expression and the phenotype.

Here we use statistical models to study the incidence of human miRNA genes on fragile sites and their association with cancer-specific translocation breakpoints, repetitive elements, and CpG islands. Our results show that, on average, fragile sites are denser in miRNAs and also in protein coding genes. However, the distribution of miRNAs and protein coding genes in fragile versus non-fragile sites depends on chromosome. We find also a positive correlation between fragility and repeats, and between miRNAs and CpG islands.

Our results show that the relationship between site fragility and miRNA density is far more complex than previously thought. For example, we find that protein coding genes seem to be following similar patterns as miRNAs, if considered their overall distribution. However, once we allow for differences at the chromosome level in our statistical analysis, we find that distribution of miRNA and protein coding genes in fragile sites is very different from that of miRNA. This is a novel result that we believe may help discover new potential correlations between the localization of miRNAs and their crucial role in biological processes and in the development of diseases.

Partial Text: Chromosomal fragile sites are heritable specific loci especially prone to breakage and rearrangements when cells are exposed to specific culture conditions or certain chemical agents such as inhibitors of DNA replication or repair [1], [2], [3]. They can be classified as rare or common, according to their frequency within the population. Rare fragile sites are present in a small fraction of the population and are usually associated with human genetic disorders, while common fragile sites are present in all individuals and, thus, represent a component of normal chromosome structure. A number of fragile sites span genes encoded by very large genomic regions. The observed rearrangements, affecting the associated genes, are usually insertions, deletions or translocations. Moreover, it has been shown that many genes involved in cancer-specific recurrent translocations are located within fragile sites [4]. This often results in the expression of altered oncogenes or the loss of tumor suppressors, contributing to the initiation of cancer [5], [6]. MicroRNAs [miRNAs] are endogenous small non coding RNAs responsible of post-transcriptional gene regulation [7]. They regulate specific target genes expression through the association with a large, multi-protein complex called RNA Induced Silencing Complex [RISC]. miRNAs into RISC recognize their targets by the binding of their bases to partially complementary sites usually located in the 3′ UTR region of their targets. However, functional miRNA binding sites can also occur within the 5′ UTR [8] or coding region [9]. miRNAs have been reported to be involved in many biological processes, including developmental timing, differentiation, proliferation, cell death, and metabolism [10], [11], [12]. Their oncogenicity has been demonstrated in a variety of cancers and their aberrant expression due to chromosomal rearrangements has been reported [13], [14]. For example, miR-15 and miR-16 are located at chromosome 13q14, a region deleted in B cell chronic lymphocytic leukemia [CLL], and it has been shown that both miRNAs are deleted or down-regulated in the majority of CLL cases [14], [15]. miRNA expression can also be regulated by epigenetic mechanisms [16]. Some miRNAs are down-regulated while others are over-expressed, and they can act as tumor-suppressor genes or oncogenes, respectively. Tumor-suppressor genes can be aberrantly methylated in cancer, and consequently down-regulated. The tumor-suppressor gene WWOX, located within the fragile site FRA16D, is correlated to multiple cancers, especially breast, prostate and ovary [17], [18]. A vastly studied mechanism of reducing WWOX at the transcriptional level is the hyper-methylation of CpG islands in its promoter and coding region [18]. Fragile sites are often characterized by repetitive sequences. Folate sensitive rare fragile sites have been found to represent loci with expansive mutations of the normally occurring CCG/CGG trinucleotide repeat sequences adjacent to a CpG island [19], [20], while non-folate sensitive rare fragile sites have been found to comprise polymorphic AT-rich minisatellite repeats [21], [22]. Fragile sites may also consist of other repetitive elements. For example, the nucleotide sequence of FRA6F is rich in repetitive elements like LINE1 and LINE2, Alu, MIR, MER and endogenous retroviral sequences and shows several DNA segments with increased helix flexibility [23]. Alu elements are the most abundant class of interspersed repeat sequences [24]. Recently, it was reported that some mammalian miRNAs are derived from genomic repeats and some of them show perfect complementarity with the MIR/LINE-2 class of repeat elements, which are present within a large number of human mRNAs and EST transcripts that contain portions of MIR and other LINE-2 elements in their 3′-untranslated regions [25]. It has been hypothesized that Alu elements within 3′-UTRs are targeted specifically by certain miRNAs [26]. Previous works showed that miRNA genes are frequently located at fragile sites and cancer-related genomic regions [6], [27], [28]. Here we present the complete mapping of the human miRNA genes on fragile sites, cancer-specific translocation breakpoints, repetitive sequences and CpG islands. The aim of this work is to highlight the potential connections between the localization of miRNAs and their role in biological processes and in the development of diseases.

Previous studies [6], [27] compared the genome positions of fragile sites and cancer susceptibility loci, with those of miRNAs in human and mouse. Results suggested a statistically significant association between the chromosomal locations of miRNAs and those of fragile sites and of regions involved in cancer. In our study we extended Calin’s work [6] to all currently known human miRNAs (today there are more than 700 known miRNA, and at the time of Calin’s work only about 200 were known). In addition, we also considered the location of protein coding genes and studied whether these followed similar patterns as miRNAs, an analysis that previous work had not considered. The results of our analysis show that fragile sites are particularly dense in miRNAs (confirming Calin’s findings) and also in protein coding genes. Our overall initial results also indicated no significant difference between miRNAs and genes in terms of their distribution in fragile versus non-fragile sites.