Igwe Emeka I, Essler Silke, Al-Furoukh Natalie, Dehne Nathalie, Brüne Bernhard
Institute of Biochemistry I/ZAFES, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany.
BMC Genomics. 2009 Sep 2;10:408. doi: 10.1186/1471-2164-10-408.
Microarray analysis still is a powerful tool to identify new components of the transcriptosome. It helps to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis.
We identified 196 genes that were significantly regulated by hypoxia, 85 genes affected by nitric oxide and 292 genes induced by the cotreatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to all treatments but with different levels of expression in each group. We observed that 162 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia-regulated targets by NO.
By eliminating the interference of steady state mRNA in gene expression profiling, we obtained a smaller number of significantly regulated transcripts in our study compared to published microarray data and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signaling.
微阵列分析仍然是识别转录体新成分的有力工具。它有助于增加对诸如缺氧和一氧化氮等应激条件触发的靶点的了解。然而，由于mRNA稳定性改变对基因表达模式的影响以及mRNA半衰期的变化，转录调控事件的分析仍然难以捉摸，这些变化会影响mRNA表达水平及其周转速率。为了规避这些问题，我们专注于通过核运行分析（NRO）对新转录的（新生）mRNA进行分析，随后进行微阵列分析。
我们鉴定出196个受缺氧显著调控的基因、85个受一氧化氮影响的基因以及292个巨噬细胞同时用一氧化氮和缺氧处理诱导的基因。14个基因（Bnip3、Ddit4、Vegfa、Trib3、Atf3、Cdkn1a、Scd1、D4Ertd765e、Sesn2、Son、Nnt、Lst1、Hps6和Fxyd5）在所有处理中都有，但在每组中的表达水平不同。我们观察到，只有当细胞同时用缺氧和一氧化氮处理而非单独用任何一种处理时，162个转录本才受到调控，这表明缺氧和一氧化氮之间相互作用的重要性。此外，阵列和蛋白质组学数据均支持一氧化氮对缺氧调控靶点的一致抑制作用。
通过消除稳态mRNA在基因表达谱分析中的干扰，与已发表的微阵列数据相比，我们在研究中获得了数量更少的显著调控转录本，并鉴定出了先前未知的缺氧诱导靶点。基因分析谱证实了一氧化氮和缺氧诱导信号之间的相互作用。
