The Expression of microRNA and microRNA Clusters in the Aging Heart Xiaomin Zhang, Gohar Azhar, Jeanne Y. Wei* Donald W. Reynolds Department of Geriatrics, The University of Arkansas for Medical Sciences and Geriatric Research, Education and Clinical Center, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, United States of America Abstract Background: The microRNAs have been implicated in the process of cardiac development, cardiac hypertrophy, and heart failure. However, the impact of adult aging on cardiac expression of miRNA clusters, as well as both miRNA guide (miR) and passenger (miR*) strands has not been well established. Methods/Results: We explored the expression profile of both miR and miR* in the hearts of young adult versus old mice. We found that 65 miRNAs were differentially expressed in the old versus young adult hearts; approximately half of them were clustered miRNAs that were distributed in 11 miRNA clusters. Each miRNA cluster contained from 2 to as many as 71 miRNA genes. The majority of the clusters displayed similar expression, with most cluster members within a cluster being either increased or decreased together, suggesting that most clusters are likely to be regulated by a common signaling mechanism and that the combined expression of multiple miRNA genes in a cluster could pose an impact on a broad range of targets during aging. We also found age-related changes in the expression of miR*s. The expression of both miR and miR* correlated with that of pri-miRNA transcript over the time course from development and maturation through adult aging. Age-related changes in the expression of Ago1 and Ago2 proteins in the heart were also observed. Transfection assay revealed that both Ago1 and Ago2 synergistically induced miR-21 and miR-21* when the mir-21 plasmid was co-transfected with either. Conclusion: The data revealed age-related changes in the expression of pri-miRNA transcript, Argonaut proteins and both miR and miR* strands. The major changes occurred later in life, from middle to old age. It is likely that the expression of miR and miR* is regulated by both pri-miRNA transcription as well as Ago1 and Ago2 proteins during adult aging. Citation: Zhang X, Azhar G, Wei JY (2012) The Expression of microRNA and microRNA Clusters in the Aging Heart. PLoS ONE 7(4): e34688. doi:10.1371/ journal.pone.0034688 Editor: Yidong Bai, University of Texas Health Science Center at San Antonio, United States of America Received October 23, 2011; Accepted March 8, 2012; Published April 18, 2012 Copyright: 2012 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported in part by National Institutes of Health grants AG-018388, AG-026091, and AG-028718 from the Department of Health and Human Services, The Geriatric Research, Education, and Clinical Center (GRECC), and the Central Arkansas Veterans Healthcare System. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: weijeanne@uams.edu Introduction Adult aging is a complex biological process associated with a progressive decline in the physiological and biochemical perfor- mance of individual tissues and organs, leading to increased susceptibility to age-related disease and functional senescence [1– 4]. The functional changes observed in the older heart are usually associated with morphological changes that occur during the normal aging process, which is characterized by a loss of myocytes with subsequent hypertrophy of the remaining viable myocytes, and proliferation of cardiac fibroblasts. As myocytes are lost and fibroblasts continue to proliferate and produce collagen, the physical properties of the aging heart become altered [1,2]. The functional and morphological changes in the older heart are accompanied by changes in cardiac gene expression, which include a-MHC, b-MHC, ANF, SERCA2 etc. [5–10]. Recent studies suggest that microRNAs (miRNAs) may play a role in the regulation of gene expression in the aging process [11– 13]. MiRNAs are short (20 to 23-nucleotide), endogenous, single- stranded RNA molecules that regulate gene expression by hybridization to messenger RNAs (mRNAs) with the consequence of messenger RNA degradation or translational inhibition of targeted transcripts. Genes that encode for miRNA are distributed across chromosomes either individually, or in clusters, in which two or more miRNA genes are located within a short distance on the same segment of a chromosome. The miRNA gene is usually transcribed by RNA polymerase II into a primary miRNA (pri-miRNA) transcript, which is cleaved at a hairpin-stem by the nuclear microprocessor complex containing Drosha and DGCR8 proteins, and generates hairpin-shaped pre- miRNA. The pre-miRNA is then exported from the nucleus to the cytoplasm, where it is cleaved by Dicer to form a 22 nt double- stranded miRNA duplexes [14]. Following their production, miRNA duplexes are sorted to confer association with specific members of the Argonaute (Ago) family of proteins, which function as the core of the RNA-induced silencing complex (RISC) [15–20]. Post-transcriptional regulation of gene expression is mediated by the RISC complex, which uses one strand of the miRNA molecule (miR, the guide strand) to target relevant messenger RNAs. Once the messenger RNA is targeted by PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e34688