The Expression of MicroRNA and microRNA Clusters in the Aging Heart

Adult aging is a complex biological process associated with altered gene expression and a decline in physiological performance. The microRNAs have been implicated in cardiac development, cardiac hypertrophy and heart failure. However, the impact of adult aging on cardiac expression of both miRNA guide strand (miR) and passenger strand (miR*), as well as miRNA clusters have not been well established. We explored the expression profile of both miR and miR* in the heart. We found that 65 miRNAs were differentially expressed in old versus young adult heart; approximately half of them were clustered miRNAs that were distributed in 11 miRNA clusters. Each miRNA cluster contains from 2 to as many as 71 miRNA genes. The majority of the clusters displayed unified expression, with most cluster members within a cluster being either increased or decreased together, suggesting that most clusters are 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. Bioinformatic analysis revealed that miR-21 and miR-21* had their own silencing targets. 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. Our data suggest that a concert of effects, including transcriptional regulation of pri-miRNA transcript and altered expression of Argonaut proteins, contribute to age-related changes in the expression of both miR and miR* strands during adult aging. The major changes occurred later in life, from middle to old age. It is likely that the expression of both miR and miR* is regulated by transcription and Ago1 and Ago2 proteins. Six healthy C57BL/6 mice were used in this study. Three of them were 4 months old (young adult mice: YA), and the other three were 24 months old (old mice). Total RNA samples were isolated from mouse hearts and then shipped to Exiqon Inc., where microRNA array analysis was performed. Three array slides were used for the hybridization. Each array slide hybridized with two samples labeled with either Hy3 or Hy5, with array #1 being hybridized with samples YA-1 (Hy3) and Old-1 (Hy5), array #2 with samples YA-2 (Hy3) and Old-2 (Hy5) and array #3 with samples YA-3 (Hy5) and Old-3 (Hy3). In our publication, we reveal the detail of the experiment and statistical analysis that were provided by Exiqon Inc.成年衰老是一类复杂的生物学过程，伴随基因表达改变与生理功能衰退。微小RNA（microRNAs）已被证实参与心脏发育、心肌肥厚与心力衰竭的调控。然而，成年衰老对心脏中miRNA引导链（miRNA guide strand, miR）、伴随链（passenger strand, miR*）以及miRNA簇（miRNA clusters）的表达影响，目前尚未得到充分阐明。本研究探究了心脏中miR与miR*的表达谱。结果显示，老年小鼠心脏与年轻成年小鼠心脏相比，共有65种miRNA呈现差异表达；其中约半数为成簇分布的miRNA，隶属于11个miRNA簇。单个miRNA簇所包含的miRNA基因数量从2个至71个不等。绝大多数簇呈现协同表达模式，即簇内多数成员的表达量同步上调或下调，这提示多数miRNA簇受共同的信号通路调控，且簇内多个miRNA基因的联合表达可能在衰老过程中对广泛的靶标产生影响。本研究同时发现了miR*表达的衰老相关变化。生物信息学分析显示，miR-21与miR-21*拥有各自的沉默靶标。在从发育、成熟直至成年衰老的时间进程中，miR与miR*的表达均与pri-miRNA转录本（pri-miRNA transcript）的表达呈相关性。此外，心脏中Ago1与Ago2蛋白的表达也出现了衰老相关的改变。本研究数据表明，包括pri-miRNA转录本的转录调控以及Argonaute蛋白（Argonaute proteins）表达改变在内的多种协同效应，共同介导了成年衰老过程中miR与miR*链的衰老相关表达变化。主要的表达变化发生在生命后期，即从中年至老年阶段。miR与miR*的表达大概率受转录调控以及Ago1、Ago2蛋白的共同调节。本研究共使用6只健康C57BL/6小鼠（C57BL/6 mice），其中3只为4月龄年轻成年小鼠（young adult mice, YA），另外3只为24月龄老年小鼠。研究人员从小鼠心脏中提取总RNA样本，随后将样本寄送至Exiqon公司（Exiqon Inc.）进行miRNA芯片分析（microRNA array analysis）。实验共使用3张芯片玻片进行杂交：每张玻片同时杂交2份分别标记Hy3或Hy5的样本，其中芯片1杂交YA-1（Hy3）与Old-1（Hy5）样本，芯片2杂交YA-2（Hy3）与Old-2（Hy5）样本，芯片3杂交YA-3（Hy5）与Old-3（Hy3）样本。本研究的实验细节与由Exiqon公司提供的统计学分析内容，均已在本团队的已发表论文中详细披露。