MicroRNA and microRNA Clusters in the Aging Heart Figure 10. The miR-21 expression correlated with pri-mir-21 levels from 1 month to 24 months of age. doi:10.1371/journal.pone.0034688.g010 age, increased after 11 months of age and reached the highest level at 18 months of age. It then was also reduced after 18 months. Both the Ago1 and Ago2 expression levels resembled a reversed ‘‘V’’ shape during adult aging from 11 months to 20 months of age. To test the effect of Ago1 and Ago2 on the expression of miR- 21 and miR-21*, the Ago1 or Ago2 expression plasmid vector was transfected into cells derived from a cardiac muscle cell line, H9C2. As shown in Figure 14 and 15, the overexpression of Ago1 or Ago2 induced the expression of miR-21 or miR-21*, respectively. In addition, both Ago1 and Ago2 synergistically induced miR-21 and miR-21* when the mir-21 plasmid vector was co-transfected with either. Figure 11. Altered expression of miR* during adult aging. Six miR*s that were differentially expressed in old (O) vs young adult (YA) hearts. doi:10.1371/journal.pone.0034688.g011 Discussion The present study has several major findings. We found that adult aging significantly impacted the expression of 65 miRNAs in the heart; among them 55% miRNAs were increased, while 45% miRNAs were decreased in the old versus young adult hearts. Interestingly, over half of these differentially expressed miRNAs belong to 11 miRNA clusters, suggesting that these clusters participate in the complex regulation of cardiac gene expression during adult life. We also observed age-related changes in the expression of pri-miRNA transcript and Ago genes, which likely contribute to age-related changes in both miR and miR* expression in the heart. These data enhance our understanding of the complex mechanism(s) underlying the altered expression of miRNA during adult aging. In the present study, half of the miRNAs that are significantly impacted during adult aging belong to 11 miRNA clusters, suggesting the importance of understanding the structure and function of these miRNA clusters. A miRNA cluster is a group of miRNA genes located within a short distance on a chromosome. Many of the miRNA clusters may have one core promoter region and transcriptional start site shared by all miRNA genes within that cluster and were ultimately expressed within a single RNA transcript [36]. The length of the transcript typically varies from a couple of kb to over 10 kb. For instance, the polycistronic transcript of mir-1-1 and mir-133a2 cluster is 10 kb; the mir-1-2 and mir-133a1 cluster is 6 kb [24]. It has been reported that each miRNA cluster usually has 2–3 miRNA genes, with the majority of clusters having less than 10 miRNA genes [37]. However, some clusters could have more than 10 miRNA genes. For example, Hertel reported that mir-134 cluster has more than 50 miRNA genes [37]. In the present study, we found that the miR- 379,329,667,410 cluster has 38 mir genes, while the miR- 466,467,669 cluster, being one of the largest miRNA clusters in mouse genome, has 71 miRNA genes. Although a number of miRNA clusters have been reported to be associated with pathological and physiological conditions, the overall expression pattern of miRNA clusters during adult aging has not been extensively studied. Since many miRNA genes within a cluster share a common promoter or utilize a common regulatory machinery, examination of their expression pattern and regulatory PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34688