Table_1_Functional Network of the Long Non-coding RNA Growth Arrest-Specific Transcript 5 and Its Interacting Proteins in Senescence.XLSX

Increasing studies show that long non-coding RNAs (lncRNAs) play essential roles in various fundamental biological processes. Long non-coding RNA growth arrest-specific transcript 5 (GAS5) showed differential expressions between young and old mouse brains in our previous RNA-Seq data, suggesting its potential role in senescence and brain aging. Examination using quantitative reverse transcription-polymerase chain reaction revealed that GAS5 had a significantly higher expression level in the old mouse brain hippocampus region than the young one. Cellular fractionation using hippocampus-derived HT22 cell line confirmed its nucleoplasm and cytoplasm subcellular localization. Overexpression or knockdown of GAS5 in HT22 cell line revealed that GAS5 inhibits cell cycle progression and promotes cell apoptosis. RNA-Seq analysis of GAS5-knockdown HT22 cells identified differentially expressed genes related to cell proliferation (e.g., DNA replication and nucleosome assembly biological processes). RNA pull-down assay using mouse brain hippocampus tissues showed that potential GAS5 interacting proteins could be enriched into several Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and some of them are involved in senescence-associated diseases such as Parkinson’s and Alzheimer’s diseases. These results contribute to understand better the underlying functional network of GAS5 and its interacting proteins in senescence at brain tissue and brain-derived cell line levels. Our study may also provide a reference for developing diagnostic and clinic biomarkers of GAS5 in senescence and brain aging.

越来越多的研究表明，长链非编码RNAs（long non-coding RNAs, lncRNAs）在多种核心生物学过程中发挥着不可或缺的作用。我们前期的RNA测序（RNA-Seq）数据显示，长链非编码RNA生长停滞特异性转录本5（long non-coding RNA growth arrest-specific transcript 5, GAS5）在年轻与年老小鼠大脑中的表达存在显著差异，提示其可能参与衰老及脑衰老过程。采用定量反转录聚合酶链式反应（quantitative reverse transcription-polymerase chain reaction）进行检测发现，GAS5在老年小鼠脑海马体区域的表达水平显著高于年轻个体。采用海马体来源的HT22细胞系进行细胞组分分离实验，证实了GAS5在细胞核质与细胞质中的亚细胞定位。在HT22细胞系中过表达或敲低GAS5的实验结果显示，GAS5可抑制细胞周期进程并促进细胞凋亡。对GAS5敲低的HT22细胞进行RNA测序分析，鉴定出了一批与细胞增殖相关的差异表达基因，涵盖DNA复制、核小体组装等生物学过程。通过小鼠脑海马体组织开展的RNA下拉实验（RNA pull-down assay）显示，潜在的GAS5互作蛋白可富集至多个京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）通路，其中部分蛋白参与了衰老相关疾病，如帕金森病与阿尔茨海默病。这些结果有助于更深入地理解GAS5及其互作蛋白在脑组织与脑来源细胞系层面的衰老相关潜在功能网络。本研究还可为开发衰老与脑衰老过程中GAS5的诊断及临床生物标志物提供参考依据。