Department of Molecular Genetics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.
PLoS One. 2012;7(8):e44095. doi: 10.1371/journal.pone.0044095. Epub 2012 Aug 30.
The miR-17-92 cluster encodes 7 miRNAs inside a single polycistronic transcript, and is known as a group of oncogenic miRNAs that contribute to tumorigenesis in several cancers. However, their direct targets remain unclear, and it has been suggested that a single miRNA is capable of reducing the production of hundreds of proteins. The majority of reports on the identification of miRNA targets are based on computational approaches or the detection of altered mRNA levels, despite the fact that most miRNAs are thought to regulate their targets primarily by translational inhibition in higher organisms. In this study, we examined the target profiles of miR-19a, miR-20a and miR-92-1 in MCF-7 breast cancer cells by a quantitative proteomic strategy to identify their direct targets. A total of 123 proteins were significantly increased after the endogenous miR-19a, miR-20a and miR-92-1 were knocked down, and were identified as potential targets by two-dimensional electrophoresis and a mass spectrometric analysis. Among the upregulated proteins, four (PPP2R2A, ARHGAP1, IMPDH1 and NPEPL1) were shown to have miR-19a or miR-20a binding sites on their mRNAs. The luciferase activity of the plasmids with each binding site was observed to decrease, and an increased luciferase activity was observed in the presence of the specific anti-miRNA-LNA. A Western blot analysis showed the expression levels of IMPDH1 and NPEPL1 to increase after treatment with anti-miR-19a, while the expression levels of PPP2R2A and ARHGAP1 did not change. The expression levels of IMPDH1 and NPEPL1 did not significantly change by anti-miR-19a-LNA at the mRNA level. These results suggest that the IMPDH1 and NPEPL1 genes are direct targets of miR-19a in breast cancer, while the exogenous expression of these genes is not associated with the growth suppression of MCF-7 cells. Furthermore, our proteomic approaches were shown to be valuable for identifying direct miRNA targets.
miR-17-92 簇在单个多顺反子转录本中编码 7 个 miRNA，被认为是一组致癌 miRNA，它们在几种癌症的肿瘤发生中发挥作用。然而，它们的直接靶标尚不清楚，并且有人认为单个 miRNA 能够减少数百种蛋白质的产生。尽管大多数 miRNA 被认为主要通过在高等生物中抑制翻译来调节其靶标，但大多数关于 miRNA 靶标的报道都是基于计算方法或检测改变的 mRNA 水平。在这项研究中，我们通过定量蛋白质组学策略研究了 MCF-7 乳腺癌细胞中 miR-19a、miR-20a 和 miR-92-1 的靶标谱，以鉴定其直接靶标。在敲低内源性 miR-19a、miR-20a 和 miR-92-1 后，共有 123 种蛋白质显著增加，并通过二维电泳和质谱分析鉴定为潜在靶标。在上调的蛋白质中，有 4 种（PPP2R2A、ARHGAP1、IMPDH1 和 NPEPL1）在其 mRNA 上具有 miR-19a 或 miR-20a 的结合位点。发现每个结合位点的质粒的荧光素酶活性降低，并且在存在特异性抗 miRNA-LNA 的情况下观察到荧光素酶活性增加。Western blot 分析显示，在用抗 miR-19a 处理后，IMPDH1 和 NPEPL1 的表达水平增加，而 PPP2R2A 和 ARHGAP1 的表达水平没有变化。在 mRNA 水平上，抗 miR-19a-LNA 对 IMPDH1 和 NPEPL1 的表达水平没有显著影响。这些结果表明，在乳腺癌中，IMPDH1 和 NPEPL1 基因是 miR-19a 的直接靶标，而这些基因的外源性表达与 MCF-7 细胞的生长抑制无关。此外，我们的蛋白质组学方法被证明对于鉴定直接 miRNA 靶标是有价值的。
