MicroRNA and microRNA Clusters in the Aging Heart Figure 8. The 11 miRNA clusters that were affected during adult aging are located on 10 chromosomes. Mouse6Chromosome contains 2 clusters (cluster #10 ‘‘mir-106a-92a-2’’ with 6 miRNA genes, and #11 ‘‘mir-222,221’’ with 2 miRNA genes). doi:10.1371/journal.pone.0034688.g008 undergo significant change from 1 month to 9 months of age, but increased thereafter, and reached the highest level at age 18 months. However, its expression level reduced to its lowest level at 24 months of age. The miR-21* expression curve during adult aging resembles a reverse ‘‘V’’ shape (Figure 12), which is similar to what was observed in the miR-21 and pri-mir-21 expression curves. These data suggest that the primary transcript expression level affects both miR-21 and miR-21* levels. Bioinformatic analysis using MicroCosm Target Scan (http:// www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/#) re- vealed that mmu-miR-21* had 879 potential target hits, while Mmu-miR-21 had 894 hits, 95% of them are not overlapped. This finding suggest that the passenger strand is not always passive, but at least in some cases, has an important regulatory role in gene expression as well. 5. Age-related change in the expression of Ago1 and Ago2 Argonaute proteins have endonuclease activity directed against messenger RNA strands that are complementary to their bound miRNA fragments. Argonaut proteins are also partially responsi- ble for selection of the guide strand and destruction of the passenger strand. To test the hypothesis that Argonaute expression may change, thereby affecting both miR and miR* expression level during adult aging, we examined the expression of Ago1 and Ago2 genes during development, maturation and adult aging by real-time RT-PCR. As shown in Figure 13, Ago1 expression level was increased from 1 to 2 months of age and remained at that level from 2 months to 11 months, then reached the highest level at age of 18 months. Afterward, its level was decreased. The Ago2 expression stayed at the same level from 1 month to 11 months of Figure 9. The expression of miRNAs correlated with that of their corresponding pri-miRNAs. The mature form of miRNAs: miR-17, miR-21, miR-27 and miR-93 were up-regulated, as were their underlying pri-miRNAs in the old (O) vs. young adult (YA) hearts. The miR-466d-3p was down- regulated, as was also its pri-mir-466d. doi:10.1371/journal.pone.0034688.g009 PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34688