MicroRNAs and intestinal pathophysiology

MicroRNAs (miRNAs) represent an abundant class of endogenously expressed small RNAs, which is believed to control the expression of proteins through specific interaction with their mRNAs. MiRNAs are non-coding RNAs of 18 to 24 nucleotides that negatively regulate target mRNAs by binding to their 3'-untranslated regions (UTR). Most eukaryotic cells utilize miRNA to regulate vital functions such as cell differentiation, proliferation or apopotosis. The diversity of miRNAs and of their mRNA targets strongly indicate that they play a key role in the regulation of protein expression. To date, more than 500 different miRNAs have been identified in animals and plants. There are at least 326 miRNAs in the human genome, comprising 1-4% of all expressed human genes, which makes miRNAs one of the largest classes of gene regulators. A single miRNA can bind to and regulate many different mRNA targets and, conversely, several different miRNAs can bind to and cooperatively control a single mRNA target. The correlation between the expression of miRNAs and their effects on tumorigenesis and on the proliferation of cancer cells is beginning to gain experimental evidences. Recent studies showed that abnormal expression of miRNAs represents a common feature of cancer cells and that they can function as tumor suppressor genes or as oncogenes. Therefore, this diversity of action for miRNAs on several target genes could be one of the common mechanisms involved in the deregulation of protein expression observed during intestinal disorders. In this review, the emergent functions of miRNAs in colorectal cancer and their potential role in the intestinal inflammatory process are discussed.     

miRTar Hunter: A prediction system for identifying human microRNA target sites

MicroRNAs (miRNAs) are important regulators of gene expression and play crucial roles in many biological processes including apoptosis, differentiation, development, and tumorigenesis. Recent estimates suggest that more than 50% of human protein coding genes may be regulated by miRNAs and that each miRNA may bind to 300–400 target genes. Approximately 1,000 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. However, the targets for a majority of these miRNAs have not been identified due to the lack of large-scale experimental detection techniques. Experimental detection of miRNA target sites is a costly and time-consuming process, even though identification of miRNA targets is critical to unraveling their functions in various biological processes. To identify miRNA targets, we developed miRTar Hunter, a novel computational approach for predicting target sites regardless of the presence or absence of a seed match or evolutionary sequence conservation. Our approach is based on a dynamic programming algorithm that incorporates more sequence-specific features and reflects the properties of various types of target sites that determine diverse aspects of complementarities between miRNAs and their targets. We evaluated the performance of our algorithm on 532 known human miRNA:target pairs and 59 experimentally-verified negative miRNA:target pairs, and also compared our method with three popular programs for 481 miRNA:target pairs. miRTar Hunter outperformed three popular existing algorithms in terms of recall and precision, indicating that our unique scheme to quantify the determinants of complementary sites is effective at detecting miRNA targets. miRTar Hunter is now available at http://203.230.194.162/~kbkim.     

Selection on Synonymous Sites for Increased Accessibility around miRNA Binding Sites in Plants

Synonymous codons are widely selected for various biological mechanisms in both prokaryotes and eukaryotes. Recent evidence suggests that microRNA (miRNA) function may affect synonymous codon choices near miRNA target sites. To better understand this, we perform genome-wide analysis on synonymous codon usage around miRNA target sites in four plant genomes. We observed a general trend of increased site accessibility around miRNA target sites in plants. Guanine-cytosine (GC)-poor codons are preferred in the flank region of miRNA target sites. Within-genome analyses show significant variation among miRNA targets in species. GC content of the target gene can partly explain the variation of site accessibility among miRNA targets. miRNA targets in GC-rich genes show stronger selection signals than those in GC-poor genes. Gene's codon usage bias and the conservation level of miRNA and its target also have some effects on site accessibility, but the expression level of miRNA or its target and the mechanism of miRNA activity do not contribute to site accessibility differences among miRNA targets. We suggest that synonymous codons near miRNA targets are selected for efficient miRNA binding and proper miRNA function. Our results present a new dimension of natural selection on synonymous codons near miRNA target sites in plants, which will have important implications of coding sequence evolution.     

Impact of miRNA Sequence on miRNA Expression and Correlation between miRNA Expression and Cell Cycle Regulation in Breast Cancer Cells

The miRNAs regulate cell functions by inhibiting expression of proteins. Research on miRNAs had usually focused on identifying targets by base pairing between miRNAs and their targets. Instead of identifying targets, this paper proposed an innovative approach, namely impact significance analysis, to study the correlation between mature sequence, expression across patient samples or time and global function on cell cycle signaling of miRNAs. With three distinct types of data: The Cancer Genome Atlas miRNA expression data for 354 human breast cancer specimens, microarray of 266 miRNAs in mouse Embryonic Stem cells (ESCs), and Reverse Phase Protein Array (RPPA) transfected by 776 miRNAs in MDA-MB-231 cell line, we linked the expression and function of miRNAs by their mature sequence and discovered systematically that the similarity of miRNA expression enhances the similarity of miRNA function, which indicates the miRNA expression can be used as a supplementary factor to predict miRNA function. The results also show that both seed region and 3'' portion are associated with miRNA expression levels across human breast cancer specimens and in ESCs; miRNAs with similar seed tend to have similar 3'' portion. And we discussed that the impact of 3'' portion, including nucleotides , is not significant for miRNA function. These results provide novel insights to understand the correlation between miRNA sequence, expression and function. They can be applied to improve the prediction algorithm and the impact significance analysis can also be implemented to similar analysis for other small RNAs such as siRNAs.     

AGO-RBP crosstalk on target mRNAs: Implications in miRNA-guided gene silencing and cancer

MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) are important regulators of mRNA translation and stability in eukaryotes. While miRNAs can only bind their target mRNAs in association with Argonaute proteins (AGOs), RBPs directly bind their targets either as single entities or in complex with other RBPs to control mRNA metabolism. miRNA binding in 3′ untranslated regions (3′ UTRs) of mRNAs facilitates an intricate network of interactions between miRNA-AGO and RBPs, thus determining the fate of overlapping targets. Here, we review the current knowledge on the interplay between miRNA-AGO and multiple RBPs in different cellular contexts, the rules underlying their synergism and antagonism on target mRNAs, as well as highlight the implications of these regulatory modules in cancer initiation and progression.     

A precursor microRNA in a cancer cell nucleus

In line with their broad-based effects, microRNAs (miRNAs), small non-coding RNA molecules ~22 nucleotides long that silence target mRNAs, are thought to act as oncogenes or tumor suppressor genes based on their inhibition of tumor-suppressive and oncogenic mRNAs, respectively. We and others previously showed that global downregulation of miRNAs, a common feature of human tumors, is functionally relevant to oncogenesis as impairment of miRNA biogenesis enhanced transformation in both cancer cells and a K-Ras-driven model of lung cancer. The dysregulation of miRNA biosynthesis in cancer emerges as a cancer-specific mechanism that enhances its tumorigenic capacity. These observations are further supported by the fact that frameshift mutations of TARBP2 occur in sporadic and hereditary carcinomas with microsatellite instability and that DICER1 mutations are associated with familial pleuropulmonary blastoma. Accordingly, it was reported that reduced expression of miRNA-processing factors is associated with poor prognosis in lung cancer and ovarian cancer. Recently we have also demonstrated the presence of Exportin 5 (XPO5) inactivating mutations in tumors with microsatellite instability. This observed genetic defect is responsible for nuclear retention of pre-miRNAs, thereby reducing miRNA processing. The characterized mutant form of the XPO5 protein lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and the protein itself, as well as pre-miRNAs accumulating in the nucleus of cancer cells. Most importantly, the restoration of XPO5 function reverses the impaired export of pre-miRNAs and has tumor suppressor features. Our data suggest a cancer-specific mechanism to guide the subcellular distribution of miRNA precursors and prevent them from being processed to the active mature miRNA. The control of the miRNA biosynthesis pathway is emerging as an important mechanism in defining the spatiotemporal pattern of miRNA expression in cancer cells.     

A precursor microRNA in a cancer cell nucleus: Get me out of here!

In line with their broad-based effects, microRNAs (miRNAs), small non-coding RNA molecules ∼22 nucleotides long that silence target mRNAs, are thought to act as oncogenes or tumor suppressor genes based on their inhibition of tumor-suppressive and oncogenic mRNAs, respectively. We and others previously showed that global downregulation of miRNAs, a common feature of human tumors, is functionally relevant to oncogenesis as impairment of miRNA biogenesis enhanced transformation in both cancer cells and a K-Ras-driven model of lung cancer. The dysregulation of miRNA biosynthesis in cancer emerges as a cancer-specific mechanism that enhances its tumorigenic capacity. These observations are further supported by the fact that frameshift mutations of TARBP2 occur in sporadic and hereditary carcinomas with microsatellite instability and that DICER1 mutations are associated with familial pleuropulmonary blastoma. Accordingly, it was reported that reduced expression of miRNA-processing factors is associated with poor prognosis in lung cancer and ovarian cancer. Recently we have also demonstrated the presence of Exportin 5 (XPO5) inactivating mutations in tumors with microsatellite instability. This observed genetic defect is responsible for nuclear retention of pre-miRNAs, thereby reducing miRNA processing. The characterized mutant form of the XPO5 protein lacks a C-terminal region that contributes to the formation of the pre-miRNA/XPO5/Ran-GTP ternary complex and the protein itself, as well as pre-miRNAs accumulating in the nucleus of cancer cells. Most importantly, the restoration of XPO5 function reverses the impaired export of pre-miRNAs and has tumor suppressor features. Our data suggest a cancer-specific mechanism to guide the subcellular distribution of miRNA precursors and prevent them from being processed to the active mature miRNA. The control of the miRNA biosynthesis pathway is emerging as an important mechanism in defining the spatiotemporal pattern of miRNA expression in cancer cells.     

Mathematical modeling of combinatorial regulation suggests that apparent positive regulation of targets by miRNA could be an artifact resulting from competition for mRNA

MicroRNAs bind to and regulate the abundance and activity of target messenger RNA through sequestration, enhanced degradation, and suppression of translation. Although miRNA have a predominantly negative effect on the target protein concentration, several reports have demonstrated a positive effect of miRNA, i.e., increase in target protein concentration on miRNA overexpression and decrease in target concentration on miRNA repression. miRNA–target pair-specific effects such as protection of mRNA degradation owing to miRNA binding can explain some of these effects. However, considering such pairs in isolation might be an oversimplification of the RNA biology, as it is known that one miRNA interacts with several targets, and conversely target mRNA are subject to regulation by several miRNAs. We formulate a mathematical model of this combinatorial regulation of targets by multiple miRNA. Through mathematical analysis and numerical simulations of this model, we show that miRNA that individually have a negative effect on their targets may exhibit an apparently positive net effect when the concentration of one miRNA is experimentally perturbed by repression/overexpression in such a multi-miRNA multitarget situation. We show that this apparent unexpected effect is due to competition and will not be observed when miRNA interact noncompetitively with the target mRNA. This result suggests that some of the observed unusual positive effects of miRNA may be due to the combinatorial complexity of the system rather than due to any inherently unusual positive effect of the miRNA on its target.     

MicroRNAs and breast cancer stem cells: Potential role in breast cancer therapy

MicroRNAs (miRNAs) can control cancer and cancer stem cells (CSCs), and this topic has drawn immense attention recently. Stem cells are a tiny population of a bulk of tumor cells that have enormous potential in expansion and metastasis of the tumor. miRNA have a crucial role in the management of the function of stem cells. This role is to either promote or suppress the tumor. In this review, we investigated the function and different characteristics of CSCs and function of the miRNAs that are related to them. We also demonstrated the role and efficacy of these miRNAs in breast cancer and breast cancer stem cells (BCSC). Eventually, we revealed the metastasis, tumor formation, and their role in the apoptosis process. Also, the therapeutic potential of miRNA as an effective method for the treatment of BCSC was described. Extensive research is required to investigate the employment or suppression of these miRNAs for therapeutics approached in different cancers in the future.     

miR-210: More than a silent player in hypoxia

Multiple studies have consistently established that miR (microRNA)-210 induction is a feature of the hypoxic response in both normal and transformed cells. Here, we discuss the emerging biochemical functions of this miRNA and anticipate potential clinical applications. miR-210 is a robust target of hypoxia-inducible factor, and its overexpression has been detected in a variety of cardiovascular diseases and solid tumors. High levels of miR-210 have been linked to an in vivo hypoxic signature and associated with adverse prognosis in cancer patients. A wide spectrum of miR-210 targets have been identified, with roles in mitochondrial metabolism, angiogenesis, DNA repair, and cell survival. Such targets may broadly affect the evolution of tumors and other pathological settings, such as ischemic disorders. Harnessing the knowledge of miR-210's actions may lead to novel diagnostic and therapeutic approaches.     

A quick reality check for microRNA target prediction

The regulation of protein abundance by microRNA (miRNA)-mediated repression of mRNA translation is a rapidly growing area of interest in biochemical research. In animal cells, the miRNA seed sequence does not perfectly match that of the mRNA it targets, resulting in a large number of possible miRNA targets and varied extents of repression. Several software tools are available for the prediction of miRNA targets, yet the overlap between them is limited. Jovanovic et al. have developed and applied a targeted, quantitative approach to validate predicted miRNA target proteins. Using a proteome database, they have set up and tested selected reaction monitoring assays for approximately 20% of more than 800 predicted let-7 targets, as well as control genes in Caenorhabditis elegans. Their results demonstrate that such assays can be developed quickly and with relative ease, and applied in a high-throughput setup to verify known and identify novel miRNA targets. They also show, however, that the choice of the biological system and material has a noticeable influence on the frequency, extent and direction of the observed changes. Nonetheless, selected reaction monitoring assays, such as those developed by Jovanovic et al., represent an attractive new tool in the study of miRNA function at the organism level.     

Identification of factors involved in target RNA-directed microRNA degradation

The mechanism by which micro (mi)RNAs control their target gene expression is now well understood. It is however less clear how the level of miRNAs themselves is regulated. Under specific conditions, abundant and highly complementary target RNA can trigger miRNA degradation by a mechanism involving nucleotide addition and exonucleolytic degradation. One such mechanism has been previously observed to occur naturally during viral infection. To date, the molecular details of this phenomenon are not known. We report here that both the degree of complementarity and the ratio of miRNA/target abundance are crucial for the efficient decay of the small RNA. Using a proteomic approach based on the transfection of biotinylated antimiRNA oligonucleotides, we set to identify the factors involved in target-mediated miRNA degradation. Among the retrieved proteins, we identified members of the RNA-induced silencing complex, but also RNA modifying and degradation enzymes. We further validate and characterize the importance of one of these, the Perlman Syndrome 3′-5′ exonuclease DIS3L2. We show that this protein interacts with Argonaute 2 and functionally validate its role in target-directed miRNA degradation both by artificial targets and in the context of mouse cytomegalovirus infection.