MicroRNA and microRNA Clusters in the Aging Heart Figure 14. The effect of Ago1 and Ago2 on the expression of miR-21. In the transfection assays, the cells were transfected with empty vector (Control), or Ago1, Ago2, mir-21, Ago1 and mir-21 as well as Ago2 and mir-21. The miR-21 guide strand was upregulated by Ago1, Ago2, mir-21 gene, Ago1 and mir-21 as well as Ago2 and mir-21 gene. The data indicated that Ago1 and Ago2 synergistically upregulated miR-21 guide strand when mir-21 gene was increased. # refers to p,0.05, n = 3. ## refers to p,0.01, n = 3. doi:10.1371/journal.pone.0034688.g014 contribute to the reported and potentially other differences that may result from ‘‘aging’’ in vitro or in vivo, in the experimental models being used. The present study suggests that most miRNA clusters are likely to be regulated at the cluster level by certain common signaling pathway(s). The combined effects of changes in expression of an entire miRNA cluster warrant further investigation. In addition, whether the altered miRNA expression, alone or in clusters, might be causal or contributory to myocardial aging remains to be determined. One of the 11 miRNA clusters uncovered in the present study is the cluster miR-17–92. The miR-17–92 cluster is comprised of 6 miRNA genes: miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1 and miR-92–1, all of which are located on chromosome 14 in the mouse. The human ortholog of miR-17-92 is indeed expressed within a single expressed sequence tag (EST), chr13orf25. In addition, the sequence upstream of the putative transcriptional start site is highly conserved across vertebrate genomes and contains an extensive CpG island, indicative of a core promoter region [36]. The miR-17-92 cluster has been implicated not only in cell proliferation and tumor growth, but also in cellular senescence. Increased expression of miR-17-92 cluster is usually associated with oncogenesis, cell growth and proliferation, while decreased expression of miR-19 and other members of the miR-17-92 cluster are generally associated with cellular senescence [48–50]. However, in the older heart, decreased expression of miR-17-92 cluster had been observed only in failure-prone (C57Bl66129Sv) mouse and human failing heart, but not in healthy aging heart [51]. In addition, the change in expression of miR-17-92 cluster is only observed in cardiac myocytes, but not in fibroblasts [51]. In the present study, the miR-17-92 cluster is up-regulated in the old heart of the C57BL6 mouse that is free of disease. This increased miR-17-92 cluster expression in the present study is likely correlated with the age-related hypertrophy of the cardiac myocytes in the old heart. It would be of interest to perform Figure 15. The effect of Ago1 and Ago2 on the expression of miR-21*. In the transfection assays, the cells were transfected with empty vector (Control), or Ago1, Ago2, mir-21, Ago1 and mir-21 as well as Ago2 and mir-21. The miR-21* passenger strand was upregulated by Ago1, Ago2, mir-21 gene, Ago1 and mir-21 as well as Ago2 and mir-21 gene. The data indicated that Ago1 and Ago2 synergistically upregulated miR-21* passenger strand when mir-21 gene was increased. # refers to p,0.05, n = 3. ## refers to p,0.01, n = 3. doi:10.1371/journal.pone.0034688.g015 transfection assays to determine if up-regulation of this cluster might reproduce changes that would be similar to the observed age-related differences. However, it is possible that a broad range of genes would be affected if all 6 miRNA genes were to be simultaneously overexpressed by transfection. Future studies of this cluster will be helpful to delineate the effects on their target genes. Two other clusters, miR-23a,27a,24-2 and mir-23b-27b-24- 1, each containing three miRNA genes, were observed in the present study to be up-regulated to different degrees in the old versus young adult heart. It has been reported that the expression levels of 23a-27a-24–2 cluster members are significantly up- regulated upon treatment with isoproterenol and aldosterone, but only miR-23a participates in initiating the hypertrophic response [52]. miR-23a, miR-27b were also induced during early hypertrophic growth in response to pressure-overload [53,54]. The miRNA genes in these clusters are important for cardiac development and maturation [55,56]. The present study revealed that the miRNAs in these two clusters might also play a role in the cardiac aging process. The miR-466,467,669 cluster is one of the largest miRNA clusters in the mouse genome, containing 71 miRNA genes. It is embedded in the intron of the Scm-like with four mbt domains 2 (Sfmbt2) gene. Sfmbt2 is a mouse and human homologue of Polycomb group (PcG) gene. Sfmbt2 is located on proximal chromosome 2, in a region known to be imprinted [57,58]. Sfmbt2 is expressed preferentially from the paternal allele in early embryos and in later stage extra-embryonic tissues. This is partly due to the fact that a CpG island spanning the transcriptional start site is differentially methylated on the maternal allele during embryo- genesis [57]. Although insight into the regulatory function of miRNAs toward their messenger RNA targets are continuing to emerge, less is currently known about the regulation of miRNA gene expression and miRNA biogenesis [14,24]. To date, hundreds of miRNAs have been discovered in mammalian genomes. Approx- imately 50% of mammalian miRNAs are ‘‘intragenic miRNAs’’ that are located in introns or exons of protein coding genes; the other half of the miRNAs are ‘‘intergenic’’ miRNAs that are PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34688