衰老相关长链非编码RNA的鉴定及其功能研究

Long non-coding RNAs (lncRNAs) play vital roles in gene expression regulation at the level of epigenetic regulation and transcriptional and posttranscriptional modification, and are key regulators of diverse biological processes such as cell growth and differentiation. However, the biological significance of lncRNAs on cellular senescence is still unknown. Based on our previous data, the expression of lncRNAs show obvious difference between young and senescent MEFs, suggesting that these differentially expressed lncRNAs may be closely associated with cellular senescence. Therefore, the aim of this study is to validate these differentially expressed lncRNAs in young and senescent MEFs by real-time qRT-PCR, and to study the relationship of these lncRNAs with physiological aging through analyzing the expression of lncRNAs in the tissues of mice and humans with different age, and then to explore their roles on cellular senescence through inhibition of the lncRNA expression in young or senescent MEFs, and to identify specific binding proteins of lncRNAs using RNA-pull down and RNA-binding protein immunoprecipitation, and then explore the underlying mechanism of these lncRNAs on cellular senescence. This study will contribute to understand the biological significance of lncRNAs on cellular senescence, and is of great importance for understanding the mechanism of aging and aging-related diseases.

LncRNA可在多种层面上（表观遗传调控、转录调控以及转录后调控等）调控基因的表达，并参与细胞增殖和分化等生物学过程。然而lncRNA在细胞衰老中的作用尚待挖掘。本项目的前期研究结果表明，在细胞衰老过程中lncRNA表达谱具有明显的差异，提示这些差异表达lncRNA可能与细胞衰老密切相关。本项目拟在已有工作基础上全面系统鉴定衰老相关lncRNA；通过研究衰老相关lncRNA在不同年龄段的人和小鼠组织中的差异表达，探讨它们与生理性衰老的关系；通过在年轻或衰老MEF细胞中改变衰老相关lncRNA的表达，观察它们是否可促进或延缓细胞衰老；利用RNA-pull down、蛋白质谱和RNA免疫共沉淀等技术筛查与lncRNA特异性结合的蛋白质，再深入探讨这些lncRNA协同结合蛋白影响细胞衰老的分子机制。本研究对于理解lncRNA参与细胞衰老的分子机制，深化认识衰老和衰老相关疾病的机制具有重要的意义。

LncRNA可通过表观遗传、转录以及转录后调控等方式影响基因表达，并参与细胞增殖和分化等生物学过程。然而lncRNA在细胞衰老中的作用尚待挖掘。本项目的前期研究结果表明，在小鼠胚胎成纤维细胞（MEFs）衰老过程中lncRNA表达谱具有明显的差异，提示这些差异表达的lncRNA可能与细胞衰老密切相关。本项目利用荧光定量RT-PCR技术验证了lncRNA芯片结果中差异表达明显的lncRNAs；通过研究衰老相关lncRNA在年轻和衰老小鼠组织中的差异表达，分析它们与生理性衰老的关系；探讨了AK156230、AK048098和AK135413等差异表达明显的lncRNAs对MEFs细胞衰老、增殖、自噬的影响，以及自噬诱导剂Rapamycin逆转AK156230影响细胞衰老与自噬能力的分子机制；利用mRNA表达谱芯片和荧光定量RT-PCR筛选抑制AK156230后差异表达的mRNAs，并结合GO分析与KEGG通路分析探讨AK156230影响细胞衰老的潜在机制。本项目还筛选血管内皮细胞衰老相关的lncRNAs；分析了LINC00973和HIF1A-AS2等血管内皮细胞衰老相关lncRNAs的亚细胞定位及其与mRNA的表达关系；探讨了LINC00973和HIF1A-AS2等lncRNAs对血管内皮细胞衰老和小管形成的影响。本项目研究结果发现，AK156230、AK135413和AK048098等lncRNAs的表达在年轻和衰老MEFs中差异明显。沉默AK156230可显著促进MEFs细胞衰老，抑制细胞增殖和自噬能力，细胞周期阻滞在G2/M期；并且能够上调p53、p21和p-p53的表达和抑制CDK1的表达，提示沉默AK156230引起的MEFs细胞衰老与G2/M期阻滞密切相关。研究还发现Rapamycin可逆转AK156230下调引起的自噬能力下降，并且明显延缓MEFs细胞衰老。mRNA表达谱和荧光定量RT-PCR结果表明，抑制AK156230促进细胞衰老与自噬和细胞周期相关基因表达的失衡有关。本项目还发现LINC00973和HIF1A-AS2等lncRNAs在年轻和衰老血管内皮细胞衰老中差异明显，并且具有促进血管内皮细胞衰老和抑制血管生成的功能。以上研究结果有助于理解lncRNA参与细胞衰老的作用及其潜在机制，对深化认识衰老和衰老相关疾病的机制具有重要的意义。