Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 p53 and is involved in a genetic pathway that promotes cell-cycle progression [12]. In recent years, more than 700 miRNAs have been iden- tified in humans [13], and this number is increasing. In a recent report by Friedman et al., the expression of a large number of target genes is predicted to be regulated by miRNAs [14]; however, relatively few of these have been verified experimentally. To overcome this problem, a series of computational methods has been developed to predict a large number of miRNA targets; e.g., Tar- getScan [14], RNAhybrid [15], MicroTar [16], PITA [17], miRanda [18], and PicTar [19]. Nevertheless, these computational approaches often provide numerous tar- get candidates with a large number of false positives because of the weak complementarity between miRNAs and 3’-UTRs [20]. Recently, a phylogenetic profiling approach has been applied to overcome this limitation. For example, studies of the evolution of orthologous tar- get sites have provided insights into the prediction of efficient miRNA targets [21]. As for miRNAs, many miRNA families are found among various bilaterian ani- mals, suggesting that several miRNAs and their target genes may have co-evolved; however, these features have yet to be systematically characterized. In this study, we hypothesized that the core regulatory relationship between miRNAs and their target genes were conserved throughout the evolution of bilaterian animals. In addition, by predicting these relationships, we sought to elucidate the core function of miRNAs in the primitive gene-regulatory network of the common bila- terian ancestor. Accordingly, we focused on five miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) that are conserved among bilaterian species (Homo sapiens (H. sapiens), Mus musculus (M. musculus), Gallus gallus (G. gallus), Drosophila melanogaster (D. melanogaster), and Caenorhabditis elegans (C. elegans)) and designed a procedure to extract conserved miRNA/target-gene pairs. We extracted evolutionarily conserved miRNA/target- gene pairs based on hybridization patterns and ortholo- gous information. In addition, we experimentally verified several candidate pairs to support our methodology. Our results suggest a functional role of three major miRNAs (let-7, miR-1, and miR-124) that regulated genes related to development, muscle formation, and cell adhesion. These results suggest a new role for the core function of miRNAs in the primitive gene-regulatory network of the common bilaterian ancestor. Results and Discussion Extraction of evolutionarily conserved miRNAs among five bilaterian animals To extract conserved miRNA/target-gene pairs, we chose five model species (H. sapiens, M. musculus, G. Page 2 of 13 gallus, D. melanogaster, and C. elegans) among bilaterian animals, for which there exists a vast array of data on both miRNAs and mRNAs [13]. Previously, several important features were described to classify miRNAs into families. It is well known that the seed sequence (the 5’ side of the miRNA sequence) is important for interaction with the target mRNAs [22]. Many miRNA target prediction software programs were developed using the features of seed sequences [14-19]. Moreover, several features have been proposed to identify con- served miRNA families, such as conservation of the mature miRNA sequence (features of the earliest miRNA classification in miRBase) [23] and information on the phylogenetic relationship among miRNAs [24]. By focusing on these features, we proposed the following criteria for extracting well-conserved miRNA families among five species: (1) complete seed sequence match- ing, (2) mature miRNA sequence identity exceeding 75%, and (3) high conservation among miRNA families, considering the phylogenetic relationship among miR- NAs (category I) [24]. Consequently, from 2,404 mature miRNA sequences, we extracted five miRNA families (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) con- served evolutionarily among the five bilaterian animals (Table 1). The sequence identity among most of the conserved miRNA families was over 80%. In particular, the sequences of let-7 and miR-1 family members showed very high mature miRNA sequence identity (exceeding 90%) among all five bilaterian species, which suggests that a strong selective pressure exists for the nucleotide sequence. Huang et al. described the extrac- tion of 15 conserved miRNA families among six bilater- ian animals (H. sapiens, M. musculus, G. gallus, D. rerio, D. melanogaster, and C. elegans) based on their original classification method [24], which included our five miRNA families. In this manuscript, we devised more stringent criteria based on nucleotide conservation to extract highly conserved miRNA families. Nematostella vectensis and Amphimedon queenslandica, which diverged before the emergence of bilaterian animals, reportedly express various types of miRNAs [25]; how- ever, none of these miRNAs are sequentially similar to the five evolutionarily conserved miRNA families found in the current study (data not shown), which suggests that these evolutionarily conserved miRNAs appeared after the divergence of bilaterian animals or were lost in N. vectensis and A. queenslandica. Filtering and enrichment of the evolutionarily conserved miRNA/target-gene pairs To extract genes targeted through evolution by the five conserved miRNAs, we designed a procedure that com- prised three screening steps (Figure 1 and Table 2). In step 1, we extracted potential target genes based on