microRNA Introduction

microRNAs (miRNA) are small endogenous RNA molecules that play a key role in development and disease through post-transcriptional regulation of gene expression. Their expression is often deregulated in human cancers including leukemia, lymphoma and glioblastoma tissues such as colon, lung, breast, prostate, thyroid, liver, and ovaries. miRNAs induce either translational repression and/or mRNA degradation. Initially, miRNAs along with some of their target genes were identified via classical genetic techniques in 1993. However, since 2001 many more miRNAs have been discovered and their number changes rapidly every year. In 2003, there were less than 100 known human miRNA whereas today their number has exceeded 1200. Approximately half of the known miRNAs are located within the introns of protein coding genes and are hypothesized to be transcribed through the promoter of their host protein coding gene. The remaining miRNAs are located in intergenic regions and although most of their transcripts are transcribed by PolII, very little is known about the characteristics of their full transcripts whose lengths may vary from a few thousand to tenths of thousands of nucleotides.
miRNAs incorporate into the RNA-Induced Silencing Complex (RISC) and guide it to specific miRNA Recognition Elements (MREs) on the mRNA molecules of target genes. Most of the MREs are known to be located in the 3' Untranslated Region (3’UTR) of protein coding genes with the exception of only a limited number of MREs which have been found in the coding sequence of target genes.

TarBase5.0: The database of experimentally supported miRNA targets
Even though several computational programs exist to predict miRNA targets, all of them still lack in terms of precision. For this, TarBase has been designed as the database which would house a manually curated collection of experimentally supported miRNA targets in several species of central scientific interest, including plants and viruses. The first version of TarBase was introduced in 2006 and received more than 140 citations. The current version of TarBase includes more than 1300 experimentally supported targets and it is considered the standard resource for verified miRNA targets.

Papadopoulos GL, Reczko M, Simossis VA, Sethupathy P, Hatzigeorgiou AG. (2009) The database of experimentally supported targets: a functional update of TarBase., Nucleic Acids Res. ,(Database issue) ,D155-8. PubMed
website: www.microrna.gr/tarbase

microT: A program for the prediction of miRNA targets
microT was originally introduced in 2004 as one of the first programs to predict genes that are targeted by miRNAs. Since then it has received significant improvement leading to several updated versions. For example, microT 3.0 is a program which is based on several parameters calculated individually for each microRNA and combines conserved and non-conserved microRNA recognition elements into a final prediction score. The program’s performance has been assessed in a recent publication in Nature by Selbach et al and it has been shown that it is the most precise program currently available. Another significant update of the program has been microT-ANN which is an algorithm that uses Artificial Neural Networks to imcorporate several features that affect miRNA targeting. The program predictions are presented through the DIANA-microT web server which provides information for the predicted microRNA:target gene interactions offering extensive connectivity to online biological resources. Target gene and microRNA functions may be elucidated through automated bibliographic searches and functional information is accessible through KEGG pathways. The web server offers links to nomenclature, sequence and protein databases and users are facilitated by being able to search for targeted genes using different nomenclatures or functional features, such as the genes possible involvement in biological pathways.

Maragkakis M, Alexiou P, Papadopoulos GL, Reczko M, Dalamagas T, Giannopoulos G, Goumas G, Koukis E, Kourtis K, Simossis VA, Sethupathy P, Vergoulis T, Koziris N, Sellis T, Tsanakas P, Hatzigeorgiou AG. (2009) Accurate microRNA target prediction correlates with protein repression levels., BMC Bioinformatics ,2009 Sep 18;10:295. PubMed
website: www.microrna.gr/microT

DIANA-mirExTra: Analysis of expression data for microRNA function
High-throughput gene expression experiments are widely used to identify the role of genes involved in biological conditions of interest. Although microRNA expression levels may not be routinely measured in high-throughput experiments, the involvement of microRNAs in the deregulation of gene expression can be computationally estimated through analysis of overrepresented motifs in their 3' untranslated region (3'UTR) sequences.

DIANA-mirExTra is an algorithm that can identify microRNA effects to the Expression levels of protein-coding Transcripts, based on the frequency of six nucleotide long motifs (hexamers) in the 3'UTR sequences of genes. Additional features include the combination of multiple hexamers corresponding to the same microRNA sequence, use of evolutionary conservation between human and mouse to increase robustness and correction of microarray data for single nucleotide compositional bias. Direct links to further functional analysis of produced results based on DIANA-mirPath are provided for all results.

In case information about miRNA expression changes is provided, the results can be filtered to display the analysis for miRNAs of interest only. On several datasets of miRNA overexpression and repression experiments, our proposed approaches have successfully identified the deregulated miRNA. Additionally, the method was successfully used for the identification of multiple microRNAs involved in the progression from early to late stage Epithelial Ovarian Cancer (EOC) using microarray data from EOC patients.

Alexiou P, Maragkakis M, Papadopoulos GL, Simmosis VA, Zhang L, Hatzigeorgiou AG (2010) The DIANA-mirExTra web server: from gene expression data to microRNA function., PLoS ONE ,2010 Feb 11;5(2):e9171. PubMed
website: www.microrna.gr/mirextra

DIANA-mirPath: Incorporating microRNAs in pathways
DIANA-mirPath is a web-based computational tool developed to identify molecular pathways potentially altered by the expression of single or multiple microRNAs. The software performs an enrichment analysis of multiple microRNA target genes comparing each set of microRNA targets to all known KEGG pathways. The combinatorial effect of co-expressed microRNAs in the modulation of a given pathway is taken into account by the simultaneous analysis of multiple microRNAs. The graphical output of the program provides an overview of the parts of the pathway modulated by microRNAs, facilitating the interpretation and presentation of the analysis results.

DIANA-mirPath also takes into account the impact of co-expressed miRNAs in biological pathways. Given the lack of systematic integration of miRNAs in biological pathways we believe that DIANA-mirPath is a substantial aid in the planning and the interpretation of wet lab experiments aiming to infer systemic functions in miRNA expression signatures.

Papadopoulos GL, Alexiou P, Maragkakis M, Reczko M, Hatzigeorgiou AG. (2009) DIANA-mirPath: Integrating human and mouse microRNAs in pathways., Bioinformatics. ,2009 Aug 1;25(15):1991-3. Epub 2009 May 12. PubMed
website: www.microrna.gr/mirpath

miRGen 2.0: a database of microRNA genomic information and regulation
miRGen 2.0 is a database which contains information on microRNA genomic locations, experimentally suggested microRNA transcripts, microRNA expression levels in several tissues, SNPs and TFBSs near microRNAs. This version of miRGen is the first attempt to build a widely accessible and user-friendly database that connects TFs and miRNAs through putative and experimentally supported functional relationships. The connections identified in the database will further our understanding of the TF mediated regulation of miRNA genes, and pave the road for the mapping of the interplay between TFs and miRNAs as regulatory molecules. The identification of SNPs on miRNA locations and their corresponding TFBSs, as well as the expression profiles of miRNAs can improve our insight into the involvement of miRNAs in developmental processes and disease.

All results are available through a user-friendly interface that allows searches for miRNAs and for TFs of interest. For mature miRNAs, it is possible to view targets predicted by DIANA-microT and for miRNAs found in the same transcript, the user can see a functional annotation of their targets on molecular pathways through the application DIANA-mirPath

Deregulation of TF-mediated gene expression has been shown to extensively affect protein coding genes, and lead to disease. MiRNA expression levels have also been shown to change significantly in different disease states . The availability of both these resources in the same database allows researchers to identify regulatory elements, such as TFs that may affect the expression of miRNAs.

P. Alexiou, T. Vergoulis, M. Gleditzsch, G. Prekas, T. Dalamagas, M. Megraw, I. Grosse, T. Sellis, A.G. Hatzigeorgiou (2009) miRGen 2.0: a database of microRNA genomic information and regulation, Nucleic Acids Research ,2010 January; 38(Database issue): D137–D141. PubMed
website: www.microrna.gr/mirgen