Mechanisms and role of microRNA deregulation in cancer onset and progression

MicroRNAs are key regulators of various fundamental biological processes and, although representing only a small portion of the genome, they regulate a much larger population of target genes. Mature microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotide (nt) length that control gene expression in many cellular processes. These molecules typically reduce the stability of mRNAs, including those of genes that mediate processes in tumorigenesis, such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis and invasion. MicroRNA targeting is mostly achieved through specific base-pairing interactions between the 5' end ('seed' region) of the miRNA and sites within coding and untranslated regions (UTRs) of mRNAs; target sites in the 3' UTR diminish mRNA stability. Since miRNAs frequently target hundreds of mRNAs, miRNA regulatory pathways are complex. Calin and Croce were the first to demonstrate a connection between microRNAs and increased risk of developing cancer, and meanwhile the role of microRNAs in carcinogenesis has definitively been evidenced. It needs to be considered that the complex mechanism of gene regulation by microRNAs is profoundly influenced by variation in gene sequence (polymorphisms) of the target sites. Thus, individual variability could cause patients to present differential risks regarding several diseases. Aiming to provide a critical overview of miRNA dysregulation in cancer, this article reviews the growing number of studies that have shown the importance of these small molecules and how these microRNAs can affect or be affected by genetic and epigenetic mechanisms.     

Genetic polymorphisms and microRNAs: new direction in molecular epidemiology of solid cancer

MicroRNAs (miRNAs) are small non-coding RNAs, which regulate gene expression. Single nucleotide polymorphisms (SNPs) may occur in miRNA biogenesis pathway genes, primary miRNA, pre-miRNA or a mature miRNA sequence. Such polymorphisms may be functional with respect to biogenesis and actions of mature miRNA. Specific SNPs were identified in predicted miRNA target sites within 3' untranslated regions of mRNAs. These SNPs have a potential to affect the efficiency of miRNA binding to the target sites or can create or disrupt binding sites. Resulting gene dysregulation may involve changes in phenotype and may eventually prove critical for the susceptibility to cancer and its onset as well as for estimates of prognosis and therapy response. In this review, we provide a comprehensive list of potentially functional miRNA-related SNPs and summarize their importance as candidate cancer biomarkers.     

Mutation altering the miR-184 seed region causes familial keratoconus with cataract

MicroRNAs (miRNAs) bind to complementary sequences within the 3' untranslated region (UTR) of mRNAs from hundreds of target genes, leading either to mRNA degradation or suppression of translation. We found that a mutation in the seed region of miR-184 (MIR184) is responsible for familial severe keratoconus combined with early-onset anterior polar cataract by deep sequencing of a linkage region known to contain the mutation. The mutant form fails to compete with miR-205 (MIR205) for overlapping target sites on the 3' UTRs of INPPL1 and ITGB4. Although these target genes and miR-205 are expressed widely, the phenotype is restricted to the cornea and lens because of the very high expression of miR-184 in these tissues. Our finding highlights the tissue specificity of a gene network regulated by a miRNA. Awareness of the important function of miRNAs could aid identification of susceptibility genes and new therapeutic targets for treatment of both rare and common diseases.     

Discovery of microRNA-mRNA modules via population-based probabilistic learning

MOTIVATION: MicroRNAs (miRNAs) and mRNAs constitute an important part of gene regulatory networks, influencing diverse biological phenomena. Elucidating closely related miRNAs and mRNAs can be an essential first step towards the discovery of their combinatorial effects on different cellular states. Here, we propose a probabilistic learning method to identify synergistic miRNAs involving regulation of their condition-specific target genes (mRNAs) from multiple information sources, i.e. computationally predicted target genes of miRNAs and their respective expression profiles. RESULTS: We used data sets consisting of miRNA-target gene binding information and expression profiles of miRNAs and mRNAs on human cancer samples. Our method allowed us to detect functionally correlated miRNA-mRNA modules involved in specific biological processes from multiple data sources by using a balanced fitness function and efficient searching over multiple populations. The proposed algorithm found two miRNA-mRNA modules, highly correlated with respect to their expression and biological function. Moreover, the mRNAs included in the same module showed much higher correlations when the related miRNAs were highly expressed, demonstrating our method's ability for finding coherent miRNA-mRNA modules. Most members of these modules have been reported to be closely related with cancer. Consequently, our method can provide a primary source of miRNA and target sets presumed to constitute closely related parts of gene regulatory pathways.     

Mining the 30UTR of Autism-implicated Genes for SNPs Perturbing MicroRNA Regulation

Autism spectrum disorder（ASD） refers to a group of childhood neurodevelopmental disorders with polygenic etiology. The expression of many genes implicated in ASD is tightly regulated by various factors including microRNAs（miRNAs）, a class of noncoding RNAs 22 nucleotides in length that function to suppress translation by pairing with ‘miRNA recognition elements’（MREs） present in the 30untranslated region（30UTR） of target mRNAs. This emphasizes the role played by miRNAs in regulating neurogenesis, brain development and differentiation and hence any perturbations in this regulatory mechanism might affect these processes as well. Recently, single nucleotide polymorphisms（SNPs） present within 30UTRs of mRNAs have been shown to modulate existing MREs or even create new MREs. Therefore, we hypothesized that SNPs perturbing miRNA-mediated gene regulation might lead to aberrant expression of autism-implicated genes, thus resulting in disease predisposition or pathogenesis in at least a subpopulation of ASD individuals. We developed a systematic computational pipeline that integrates data from well-established databases. By following a stringent selection criterion, we identified 9 MRE-modulating SNPs and another 12 MRE-creating SNPs in the 30UTR of autism-implicated genes. These high-confidence candidate SNPs may play roles in ASD and hence would be valuable for further functional validation.     

The Impact of 3′UTR Variants on Differential Expression of Candidate Cancer Susceptibility Genes

Variants in regulatory regions are predicted to play an important role in disease susceptibility of common diseases. Polymorphisms mapping to microRNA (miRNA) binding sites have been shown to disrupt the ability of miRNAs to target genes resulting in differential mRNA and protein expression. Skin tumor susceptibility 5 (Skts5) was identified as a locus conferring susceptibility to chemically-induced skin cancer in NIH/Ola by SPRET/Outbred F1 backcrosses. To determine if polymorphisms between the strains which mapped to putative miRNA binding sites in the 3′ untranslated region (3′UTR) of genes at Skts5 influenced expression, we conducted a systematic evaluation of 3′UTRs of candidate genes across this locus. Nine genes had polymorphisms in their 3′UTRs which fit the linkage data and eight of these contained polymorphisms suspected to interfere with or introduce miRNA binding. 3′UTRs of six genes, Bcap29, Dgkb, Hbp1, Pik3cg, Twistnb, and Tspan13 differentially affected luciferase expression, but did not appear to be differentially regulated by the evaluated miRNAs predicted to bind to only one of the two isoforms. 3′UTRs from four additional genes chosen from the locus that fit less stringent criteria were evaluated. Ifrd1 and Etv1 showed differences and contained polymorphisms predicted to disrupt or create miRNA binding sites but showed no difference in regulation by the miRNAs tested. In summary, multiple 3′UTRs with putative functional variants between susceptible and resistant strains of mice influenced differential expression independent of predicted miRNA binding.     

Interplay between microRNAs and RNA-binding proteins determines developmental processes

MicroRNAs (miRNAs) are genes involved in normal development and cancer. They inhibit gene expression by associating with 3'-Untranslated regions (3' UTRs) of messenger RNAs (mRNAs), and are thought to regulate a large proportion of protein coding genes. However, it is becoming apparent that miRNA activity is not necessarily always determined by its expression in the cell. MiRNA activity can be affected by RNA-binding proteins (RBPs). For example, the RNA-binding protein HuR associates with the 3'UTR of the CAT1 mRNA after stress, counteracting the effect of miR-122. Second, we found that the expression of an RNA-binding protein called Dead end (Dnd1) prohibits the function of several miRNAs by blocking the accessibility of target mRNAs. Dnd1 function is essential for proper development of primordial germ cells (PGCs) in zebrafish and mammals, indicating a crucial role for RBP/miRNA interplay on 3'UTRs of mRNAs in developmental decisions. In this perspective we discuss the interplay between RBPs and miRNAs in the context of germ cells and review current observations implicating RBPs in miRNA function.     

Molecular architecture of a miRNA-regulated 3' UTR

Animal genomes contain hundreds of microRNAs (miRNAs), small regulatory RNAs that control gene expression by binding to complementary sites in target mRNAs. Some rules that govern miRNA/target interaction have been elucidated but their general applicability awaits further experimentation on a case-by-case basis. We use here an assay system in transgenic nematodes to analyze the interaction of the Caenorhabditis elegans lsy-6 miRNA with 3' UTR sequences. In contrast to many previously described assay systems used to analyze miRNA/target interactions, our assay system operates within the cellular context in which lsy-6 normally functions, a single neuron in the nervous system of C. elegans. Through extensive mutational analysis, we define features in the known and experimentally validated target of lsy-6, the 3' UTR of the cog-1 homeobox gene, that are required for a functional miRNA/target interaction. We describe that both in the context of the cog-1 3' UTR and in the context of heterologous 3' UTRs, one or more seed matches are not a reliable predictor for a functional miRNA/target interaction. We rather find that two nonsequence specific contextual features beyond miRNA target sites are critical determinants of miRNA-mediated 3' UTR regulation. The contextual features reside 3' of lsy-6 binding sites in the 3' UTR and act in a combinatorial manner; mutation of each results in limited defects in 3' UTR regulation, but a combinatorial deletion results in complete loss of 3' UTR regulation. Together with two lsy-6 sites, these two contextual features are capable of imparting regulation on a heterologous 3' UTR. Moreover, the contextual features need to be present in a specific configuration relative to miRNA binding sites and could either represent protein binding sites or provide an appropriate structural context. We conclude that a given target site resides in a 3' UTR context that evolved beyond target site complementarity to support regulation by a specific miRNA. The large number of 3' UTRs that we analyzed in this study will also be useful to computational biologists in designing the next generation of miRNA/target prediction algorithms.     

Large-Scale Screens of miRNA-mRNA Interactions Unveiled That the 3′UTR of a Gene Is Targeted by Multiple miRNAs

Animal microRNA (miRNA) target prediction is still a challenge, although many prediction programs have been exploited. MiRNAs exert their function through partially binding the messenger RNAs (mRNAs; likely at 3′ untranslated regions [3′UTRs]), which makes it possible to detect the miRNA-mRNA interactions in vitro by co-transfection of miRNA and a luciferase reporter gene containing the target mRNA fragment into mammalian cells under a dual-luciferase assay system. Here, we constructed a human miRNA expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between miRNAs and the 3′UTRs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. The screening results showed that the 3′UTR of one gene can be targeted by multiple miRNAs. Among the prediction algorithms, a Bayesian phylogenetic miRNA target identification algorithm and a support vector machine (SVM) presented a relatively better performance (27% for EIMMo and 24.7% for miRDB) against the average precision (17.3%) of the nine prediction programs used here. Additionally, we noticed that a relatively high conservation level was shown at the miRNA 3′ end targeted regions, as well as the 5′ end (seed region) binding sites.     

Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets

We predict regulatory targets of vertebrate microRNAs (miRNAs) by identifying mRNAs with conserved complementarity to the seed (nucleotides 2-7) of the miRNA. An overrepresentation of conserved adenosines flanking the seed complementary sites in mRNAs indicates that primary sequence determinants can supplement base pairing to specify miRNA target recognition. In a four-genome analysis of 3' UTRs, approximately 13,000 regulatory relationships were detected above the estimate of false-positive predictions, thereby implicating as miRNA targets more than 5300 human genes, which represented 30% of our gene set. Targeting was also detected in open reading frames. In sum, well over one third of human genes appear to be conserved miRNA targets.