School of Computing Sciences, University of East Anglia, Norwich, NR4 7TJ, United Kingdom.
RNA. 2010 Mar;16(3):489-94. doi: 10.1261/rna.1701210. Epub 2010 Jan 13.
mRNA profiling is routinely used to identify microRNA targets, however, this high-throughput technology is not suitable for identifying targets regulated only at protein level. Here, we have developed and validated a novel methodology based on computational analysis of promoter sequences combined with mRNA microarray experiments to reveal transcription factors that are direct microRNA targets at the protein level. Using this approach we identified Smad3, a key transcription factor in the TGFbeta signaling pathway, as a direct miR-140 target. We showed that miR-140 suppressed the TGFbeta pathway through repression of Smad3 and that TGFbeta suppressed the accumulation of miR-140 forming a double negative feedback loop. Our findings establish a valid strategy for the discovery of microRNA targets regulated only at protein level, and we propose that additional targets could be identified by re-analysis of existing microarray datasets.
mRNA 谱分析通常用于鉴定 microRNA 靶标，然而，这种高通量技术并不适用于鉴定仅在蛋白质水平调节的靶标。在这里，我们开发并验证了一种基于启动子序列的计算分析与 mRNA 微阵列实验相结合的新方法，以揭示直接在蛋白质水平作为 microRNA 靶标的转录因子。使用这种方法，我们鉴定了 Smad3，即 TGFβ 信号通路中的关键转录因子，为直接的 miR-140 靶标。我们表明，miR-140 通过抑制 Smad3 抑制 TGFβ 途径，而 TGFβ 抑制 miR-140 的积累，形成双负反馈环。我们的发现为仅在蛋白质水平调节的 microRNA 靶标的发现建立了有效的策略，并且我们提出通过重新分析现有的微阵列数据集可以鉴定其他靶标。
