MicroRNAs Regulating Gene Expression during Cellular Senescence and Aging

在衰老过程中，生物体的基因表达模式发生了变化，对压力和有丝分裂刺激的反应能力日益减弱。由于转录后过程对表达蛋白质集合的变化起着关键的调节作用，因此阐明控制年龄相关基因表达模式的microRNAs(以及其他项目中描述的限制性商业惯例)是极其重要的。为了研究microRNA在衰老过程中的功能，我们采用了microRNA还原(通过转染反义RNA)、microRNA过表达(通过转染microRNA的前体)以及通过导入生物素化的microRNA并通过各种方法(如微阵列、RT-PCR)识别靶mRNAs来鉴定microRNA相关的mRNAs的方法。我们通过测量目标mRNAs的稳态水平和半衰期作为microRNA丰度的函数，来研究microRNAs是否影响靶mRNAs在衰老过程中的稳定性。我们研究了microRNAs是否通过调节microRNA水平影响目标mRNAs的翻译，随后研究了mRNAs与翻译多聚体的相对结合，并通过量化编码蛋白质的新生翻译率。我们还使用报告结构来进一步了解由microRNAs调控的过程，并使用不同的衰老相关标记来检查衰老表型的变化。 在过去的资助期间，我们已经报道在人类二倍体成纤维细胞中，microRNA miR-519在复制衰老的实现中发挥核心作用(Marasa等人，Aging，2010)。我们还发现miR-519至少部分是通过降低RBP Hur的表达水平来触发衰老的(Abdelmohsen等人，Cell Cycle，2010)。我们还参与了与埃文斯实验室的合作努力，以确定microRNA表达模式的差异作为年龄的函数(Noren等人，PLoS one，2010)。正在进行的研究正在系统地分析生物素化的microRNAs的靶mRNAs，这些mRNAs的水平随着衰老而下降或上升。人们对阐明其他非编码RNA在细胞衰老和衰老中的作用也很感兴趣。

During aging, organisms show altered gene expression patterns and have an increasingly impaired ability to respond to stress-causing and mitogenic stimuli. Since post-transcriptional processes critically regulate changes in the collections of expressed proteins, it is extremely important to elucidate the microRNAs (as well as RBPs, as described in other projects) that control age-related gene expression patterns. To investigate microRNA function during senescence, we employ approaches such as microRNA reduction (by transfecting an antisense RNA), microRNA overexpression (by transfecting a precursor of the microRNA), and the identification of microRNA-associated mRNAs by transfecting biotinylated microRNAs and identifying target mRNAs through various methods (eg, microarray, RT-PCR). We investigate whether microRNAs affect the stability of target mRNAs during senescence by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of microRNA abundance. We investigate whether microRNAs affect the translation of target mRNAs by modulating microRNA levels, and subsequently studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by microRNAs and use different senescence-associated markers to examine changes in the senescence phenotype. During the past funding period, we have reported that in human diploid fibroblasts the microRNA miR-519 plays a central role in the implementation of replicative senescence (Marasa et al., Aging, 2010). We also discovered that miR-519 triggers senescence at least in part by lowering expression levels of the RBP HuR (Abdelmohsen et al., Cell Cycle, 2010). We also participated in collaborative efforts with the Evans laboratory to identify differences in microRNA expression patterns as a function of age (Noren et al., PLoS ONE, 2010). Ongoing studies are analyzing systematically the target mRNAs of biotinylated microRNAs whose levels decline or increase with senescence. There is also a great deal of interest in elucidating other noncoding RNA with roles in cellular senescence and aging.