miR-124与p53效应miRNAs联合靶向CCNA2调节细胞衰老的分子机制

MicroRNAs (miRNAs) have emerged as critical regulators of gene expression in posttranscriptional level, and exerted an important role in cellular senescence. We previously performed the miRNA microarray on physiological aging mice, and uncovered that miR-124 and several p53 responsive miRNAs were up-regulated in aging mice; and ectopic expression of these miRNAs significantly triggered cellular senescence. Moreover, the results of target prediction and mRNA microarray showed that CCNA2 might be the common targets of miR-124 and p53 responsive miRNAs. Nonetheless, the underlying mechanisms of miR-124 and p53 responsive miRNAs on cellular senescence remain largely unknown. In the present study, we will fully elucidate the effects of these miRNAs in diverse aging models in vivo and in vitro, and further investigate into whether miR-124 infeluence the expression of p53 reponsive miRNAs; additionally, we will probe into whether these miRNAs modulate cellular senescence direct through cooperatively down-regulation of the common targets by silencing or enforced expression of target genes, in order to fully illuminate the underlying molecular mechanisms of the aging-induction effects of these miRNAs. In aggregate, the present project will not only deepen the understanding of the molecular mechanisms underneath the aging process, but also provide novel insights for delaying aging and preventing aging-related diseases.

MicroRNAs（miRNAs）作为重要的基因表达调控子，在衰老进程中的作用日益受到关注。我们前期基于生理性衰老小鼠模型，利用高通量芯片技术检测发现miR-124及多个p53效应miRNAs在衰老小鼠中上调，过表达上述miRNAs可显著促进细胞衰老；此外，mRNA芯片数据与靶基因预测结果表明，衰老小鼠中下调的CCNA2等基因可能是miR-124与p53效应miRNAs的共同靶标。尽管如此，miR-124与p53效应miRNAs调节细胞衰老的分子机制尚不清楚。本项目拟在已有工作基础上，利用体内、体外多种衰老模型，明确miR-124与p53效应miRNAs在衰老进程中的作用，并探讨二者之间的表达调控关系；进而通过功能获得与缺失实验，深入研究上述miRNAs是否通过联合靶向CCNA2调节细胞衰老。本研究对于阐明miRNA影响细胞衰老的作用机制，深化认识衰老和老年性疾病的分子机制具有重要的意义。

细胞衰老是多因素参与的、一系列基因协同调节的综合结果。MicroRNAs（miRNAs）作为重要的基因表达调控子，在衰老进程中的作用日益受到关注。本项目前期鉴定发现包括miR-124、miR-29a/b/c、miR-34a和miR-194等在内的多个p53效应miRNAs在生理性衰老小鼠和复制性衰老细胞中显著上调。本项目进一步揭示miR-124、miR-194等miRNA可显著加速细胞衰老进程。高通量芯片结果显示，Ccna2等多个细胞周期和细胞增殖相关的基因在衰老小鼠组织和衰老细胞中显著下调，并且Ccna2可有效延缓细胞衰老进程。生物信息学分析和双荧光素酶报告系统结果表明，Ccna2是miR-124和miR-29a/b/c协同调控的靶基因。此外，过表达Ccna2可有效回复p53效应 miRNAs对衰老的促进作用，表明p53效应miRNAs可通过协同调控靶基因Ccna2的表达参与调节细胞衰老进程。Ccna2可与p21竞争性结合CDK2，从而调节细胞周期进程中G1/S的转换。为进一步探讨p53/miRNAs/Ccna2通路与经典的p53/p21信号通路的关系，在细胞内沉默p21的表达。结果显示，虽然沉默p21可明显延缓细胞衰老，但是过表达p53效应miRNAs或沉默Ccna2的表达，可显著促进沉默p21细胞的衰老进程。可见，p53/p53效应miRNAs/Ccna2是独立于p53/p21的衰老调节信号通路。本项目的研究揭示了一条新的细胞衰老调控通路：p53/miRNAs/Ccna2通路，该通路可与p53/p21信号通路一起，组成p53蛋白衰老调节作用的“双保险”。本研究对于阐明miRNA影响细胞衰老的作用机制，深化认识衰老和老年性疾病的分子机制具有重要的意义。