MicroRNAs Regulating Gene Expression during Cellular Senescence and Aging

在衰老过程中，生物体表现出基因表达模式的改变，对应激和有丝分裂刺激的反应能力日益受损。由于转录后过程对表达蛋白集合的变化具有关键的调节作用，rbp（在其他项目中描述）和非编码rna（特别是microrna和lncrna）的作用正在成为控制年龄相关基因表达模式的主要因素。为了研究ncRNA在衰老过程中的功能，我们采用了ncRNA还原（通过转染反义分子）、ncRNA过表达（通过转染前体或成熟的ncRNA分子）、通过标记ncRNA（使用生物素或MS2标签）鉴定ncRNA相关mrna以及通过各种方法（如微阵列、RT-PCR）鉴定靶mrna等方法。我们通过测量相关mrna的稳态水平和半衰期作为ncRNA丰度的函数来研究ncRNA是否会影响衰老过程中靶mrna的稳定性。我们通过调节ncRNA水平来研究ncRNA是否影响目标mRNA的翻译，随后研究mRNA与翻译多体的相对关联，并通过量化编码蛋白的初始翻译率。我们还采用报告者结构来进一步了解由ncrna调节的过程，并使用不同的衰老相关标记来检查衰老表型的变化。

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, the role of RBPs (described in other projects) and noncoding RNAs (particularly microRNAs and lncRNAs) are emerging as major factors controlling age-related gene expression patterns. To investigate ncRNA function during senescence, we employ approaches such as ncRNA reduction (by transfecting an antisense molecules), ncRNA overexpression (by transfecting precursors or mature ncRNA molecules), and identification of ncRNA-associated mRNAs by tagging the ncRNAs (using biotin or MS2 tags) and identifying target mRNAs through various methods (eg, microarray, RT-PCR). We investigate whether ncRNAs 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 ncRNA abundance. We investigate whether ncRNAs affect the translation of target mRNAs by modulating ncRNA levels, and subsequently studying the relative association 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 ncRNAs and use different senescence-associated markers to examine changes in the senescence phenotype. During the past funding period, we have reported that the senescence-associated microRNA miR-519 plays a central role in autophagy (Abdelmohsen et al., Mol. Cell Biol, 2012), that miR-130 suppresses adipogenesis by lowering PPAR production (Lee et al., Mol. Cell Biol, 2011), and that miR-146 inhibits brain metastases (Hwang et al., Molecules and Cells, 2012). We have also shown that expression of a senescence-upregulated lncRNA (lincRNA-p21) is inhibited by the microRNA let-7 (Yoon et al., Mol. Cell 2012) and that a general factor in the production of microRNAs (Drosha) is inhibited by the senescence-downregulated protein AUF1 (Abdelmohsen et al., 2012). We reviewed the contributions of microRNAs in senescence and aging in several articles (Srikantan et al., Cell Cycle, 2011; Abdelmohsen and Gorospe, 2011; Srikantan et al., Cell Cycle, 2011) as well as in one book chapter (Grammatikakis and Gorospe, in MicroRNAs in Medicine 2012).