MicroRNAs Regulating Gene Expression during Cellular Senescence and Aging

在衰老过程中，生物体显示出改变的基因表达模式，并且对引起压力和促有丝分裂刺激的反应能力日益受损。 由于转录后过程严格调节表达蛋白质集合的变化，因此阐明控制年龄相关基因表达模式的microRNA（以及其他项目中描述的RBP）非常重要。为了研究microRNA在衰老过程中的功能，我们采用了诸如microRNA减少（通过检测反义RNA）、microRNA过表达（通过检测microRNA的前体）以及通过检测生物素化的microRNA和通过各种方法（例如，微阵列、RT-PCR）鉴定靶mRNA来鉴定microRNA相关mRNA的方法。 我们通过测量目标mRNA的稳态水平和半衰期作为microRNA丰度的函数来研究microRNA是否影响衰老过程中靶mRNA的稳定性。 我们研究microRNA是否通过调节microRNA水平影响靶mRNA的翻译，随后研究mRNA与翻译多核糖体的相对关联，并通过量化编码蛋白的新生翻译速率。 我们还采用报告构建体来获得对microRNA调节的过程的进一步了解，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，我们已经报道了在人二倍体成纤维细胞中，microRNA miR-519在复制性衰老的实施中起着核心作用（Marasa et al.，Aging，2010）。 我们还发现miR-519至少部分通过降低RBP HuR的表达水平来触发衰老（Abdelmohsen et al.，Cell Cycle，2010）。 我们还参与了与Evans实验室的合作努力，以确定作为年龄函数的microRNA表达模式的差异（Noren et al.，PLoS ONE，2010）。 正在进行的研究正在系统地分析生物素化microRNA的靶mRNA，其水平随着衰老而下降或增加。 人们对阐明其他非编码RNA在细胞衰老和老化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.