Takane et al. BMC Genomics 2010, 11:101 http://www.biomedcentral.com/1471-2164/11/101 the three miRNAs let-7 (eight targets), miR-1 (seven tar- gets), and miR-124 (eleven targets) compared with miR- 125/lin-4 (three targets) and miR-34 (two targets) (Addi- tional file 2). This suggests that let-7, miR-1, and miR- 124 may have played a major role in primordial miRNA gene regulation in the common bilaterian ancestor. To verify the significance of conserved miRNA-target gene pairs, we performed same sequence analysis (from step 1 to step 3) against total 25 species-specific miRNAs (5 miRNAs each from 5 species) as a control experiment supposing that these miRNAs are also conserved in other bilaterians. For example, target prediction of hsa- miR-2277, a species-specific miRNA in human was per- formed in all 5 species (step 1 and step 2) and con- served targets were extracted (step 3). As a result, 11 out of 25 non-conserved miRNAs did not show any conserved miRNA-target gene pair. Furthermore, aver- age number of the miRNA-target gene pairs of the negative control was 2.4, which is statistically lower than that of conserved miRNA-target gene pairs 6.2 based on the Welch’s t-test (P < 0.05). These results support that number of genes achieved from the prediction of con- served miRNAs target genes in this study is indeed sig- nificant. In summary, we developed a new filtering method for extracting evolutionarily conserved miRNA/ target-gene pairs, which was used to extract 31 reliable miRNA/target-gene pairs among the five families of miRNAs. We discovered that only one orthologous target gene, calponin-3 (CNN3), was conserved completely among the five bilaterian animals (Figure 3A). As for target genes conserved in four species, we found, for example, the La-related protein 4 (LARP4), ETS domain-contain- ing protein Elk-3 (ELK3), argonaute-4 (EIF2C4), transge- lin-2 (TAGLN2), and V-type proton ATPase subunit B brain isoform (ATP6V1B2) genes (Figure 3B and Addi- tional file 3). Of note, the same approximate position of the predicted target site was observed in the orthologous 3’-UTR of CNN3 (120 nucleotides (nt)), LARP4 (3,100 nt), EIF2C4 (220 nt), and TAGLN2 (50 nt) among verte- brates (Figure 3 and Additional file 3). According to Bartel et al., the distribution of miRNA target sites within the 3’-UTR is biased near the mRNA stop codon or poly-A tail compared with the middle portion of 3’- UTR [26]. Our results show that target site distribution varied according to the type of miRNA target gene. The target sites on 3’-UTR of CNN3 and TAGLN2 were biased near the stop codon, from H. sapiens to C. ele- gans (Figure 3A and Additional file 3). Regarding the other candidates, we observed all types of target site dis- tribution on 3’-UTR. A future statistical analysis of miRNA target-site distribution among conserved miRNA/target-gene pairs is required to substantiate this view. With the exception of 3’-UTR of the LARP4 gene, Page 6 of 13 most of the binding patterns of evolutionarily conserved target sites were sequentially different, without taking the seed region into consideration. The target-site bind- ing patterns within 3’-UTR of the LARP4 gene were identical between H. sapiens and M. musculus (Figure 3B), although the similarity of the two 3’-UTR sequences was ~70% (data not shown). A recent study reported on cooperative regulation by multiple miRNAs [19]. Likewise, the band 4.1-like protein 4B (EPB41L4B) gene was an orthologous target of two different types of miRNAs: miR-1 and miR-124 (Table 3). Our analysis suggests that multiple miRNA regulation may have already existed in the era of ancestral bilaterian species. Experimental validation of miRNA target genes To validate the evolutionarily conserved miRNA/target- gene candidates, we performed transfection and lucifer- ase assays on 6 of the 31 identified evolutionarily con- served miRNA/target-gene candidates. Four of the six candidates (CNN3, LARP4, TAGLN2, and ATP6V1B2) were predicted to be regulated by miR-1, while one can- didate (ELK3) was predicted to be targeted by miR-124, and the final candidate (EIF2C4) was predicted to be the target of let-7. We used the well-established downregu- lation of the delta-like protein 1 precursor (DLL1) gene by miR-34 as a positive control [22], and the LARP4/let- 7 pair, which was extracted up to step 2 in our analysis, was chosen as a non-evolutionarily conserved pair. We subcloned the 3’-UTR sequence downstream from the Renilla luciferase gene (Figure 4A) and co-transfected 100 ng of the 3’-UTR reporter construct into HeLa cells using 5, 20, and 60 pmol of miRNA Mimics (hsa-let-7a, hsa-miR-1, hsa-miR-124, hsa-miR-34a, and miRIDIAN microRNA Hairpin Inhibitor Negative Control #1) (see Methods section). We observed the downregulation of six out of six candidates and of the positive control compared with the negative controls (Figure 4B, a-g). Typical results of the reporter gene assay are shown in Figure 4B for the indicated amounts of miRNAs (5, 20, and 60 pmol). The downregulation of these candidates was significant (P < 0.01), although some of these pairs represented an inhibition of only 30% under the current conditions. Among these candidates, TAGLN2 was pre- viously suggested to be downregulated by miR-1, as assessed by microarray analysis [27]. This feature was recently confirmed using the “pulsed stable isotope labelling with amino acids in cell culture” (pSILAC) method and a reporter gene assay [28]. Regarding the let-7/LARP4 combination, the expression of LARP4 was not downregulated after let-7 transfection, which was supported statistically (Figure 4B, h). These experimental results suggest that our new method has the potential for efficiently extracting reliable miRNA/target-gene pairs and may be effective in the elucidation of the