果蝇ATM/E2F1/p53协同调节miRNA应答DNA损伤的作用机制研究

It is known that miRNAs are involved into DNA damage response and the maintenance of genome, and their transcription, processing, transporting are regulated by ATM, E2F1 and p53, the key factors for maintenance of genome integrity. However, systematic studies in animal levels on how ATM / E2F1 / p53 regulates miRNA in response to DNA damage coordinately is still lacking. Using the Drosophila malanogaster model animal and RNA-seq, we identified differentially expressed miRNAs after irradiation or in atm/e2f1/p53 fly mutants, which included 44 miRNAs during embryonic stage and 117 miRNAs at third instar larval stage, respectively. Furthermore, some miRNAs might be regulated coordinately by ATM/E2F1/p53. Moreover, we detected irradiation sensitivity of some established miRNA mutants, and found that 11 miRNA mutated flies were sensitive to irradiation, and in which 7 mutants showed increased apoptosis induced by DNA damage; while 2 miRNA mutants were resistant to irradiation. In this study, we will investigate the underline mechanisms of how ATM / E2F1 / p53 work coordinately to regulate miRNA in response to DNA damage, and provide further knowledge to understand genome maintenance.

miRNA广泛参与DNA损伤应答及基因组稳定性的维护，其转录、加工、运输等受到维护基因组稳定性的关键因子ATM、E2F1、p53的调节。然而，ATM/E2F1/p53如何协同调节miRNA应答DNA损伤仍缺少动物体水平的系统研究。利用果蝇模式动物和RNA-seq，本研究鉴定了应答DNA损伤或受到ATM、E2F1、p53调节的miRNA，包括44个胚胎时期及117个三期幼虫时期的miRNA；分析发现，一些miRNA受到ATM/E2F1/p53的协同调节。对部分miRNA突变果蝇DNA损伤敏感性进行检测，发现11株miRNA突变果蝇对辐照敏感，其中7株出现DNA损伤导致的凋亡增多；2株miRNA突变果蝇对辐照耐受。本研究将深入研究ATM/E2F1/p53协同调节miRNA应答DNA损伤的作用机制，从生物体水平丰富人们对机体响应DNA损伤、维护基因组稳定性的作用机制和调控网络的了解。

小非编码miRNA，其中包括miRNA和piRNA，在生命过程中发挥重要的功能。对发育过程中和参与响应DNA损伤的小非编码RNA表达的深度定量分析研究对于加深了解其扮演的角色非常关键。在本项目研究中，我们系统分析了发育过程中和辐照后miRNA和piRNA在野生型果蝇、p53突变果蝇、e2f1突变果蝇和p53 e2f1双突变果蝇的表达谱，以及生理条件和辐照后miRNA在atm突变果蝇的表达谱。通过小RNA测序和生物信息学分析，我们发现miRNA和piRNA在发育过程中的表达可以归集为4个表达群落，表现出动态的表达模式。值得注意的是，miRNA和piRNA的表达在e2f1突变果蝇和p53 e2f1双突变果蝇中在发育的多个阶段出现巨大变化，而在p53突变果蝇中没有这种变化，表明E2F1在调节miRNA和piRNA的表达的重要作用。此外，我们鉴定了辐照后在p53突变果蝇和e2f1突变果蝇中差异表达的miRNA和piRNA，以及在atm突变果蝇中差异表达的miRNA，检测了miRNA突变果蝇对辐照的敏感性和凋亡，定位了p53和E2F1在小非编码RNA的结合域。我们的数据显示，p53和E2F1在调节小非编码RNA的表达时互相协同然而作用不同，E2F1在发育过程中和辐照后对小非编码RNA的表达起到更重要的调节功能。因此，我们的研究深度分析了发育过程中和辐照后小非编码RNA的表达特征，以及p53和E2F1在其中扮演的调节角色，为了解小RNA提供了新的知识。