Genome-wide Characterization of miR-34a Induced Changes in Protein and mRNA Expression by a Combined Pulsed SILAC and Microarray Analysis
Genome-wide Characterization of miR-34a Induced Changes in Protein and mRNA Expression by a Combined Pulsed SILAC and Microarray Analysis
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DOI：
10.1074/mcp.m111.010462
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发表时间：
2011-08-01
影响因子：
7
通讯作者：
Hermeking, Heiko
中科院分区：
文献类型：
--
作者：
Kaller, Markus;Liffers, Sven-Thorsten;Hermeking, Heiko
编码miR-34a microRNA的基因是p53肿瘤抑制蛋白的转录靶点，在结直肠癌和许多其他肿瘤类型中会发生表观遗传失活。在这里，我们结合脉冲SILAC （pSILAC）和微阵列分析来鉴定mir -34a诱导的蛋白质和mRNA表达的变化。pSILAC允许在激活结直肠癌细胞系中的条件miR-34a等位基因后量化1206个蛋白的新生蛋白合成。大约19%的检测到的蛋白质被差异调节，113个蛋白质被下调，115个蛋白质被上调。在相应mRNA的3'-非翻译区（UTR）具有miR-34a种子匹配序列的蛋白质显示出明显的翻译抑制倾向。参与DNA复制的蛋白，如MCM蛋白和细胞增殖，在缺乏miR-34a种子匹配的间接下调蛋白中被过度表达。在大多数情况下，直接miR-34a靶点新生蛋白合成的减少与相应mRNA水平的降低相关，表明两种类型的调控相互依赖。此外，43种pSILAC未检测到的编码蛋白的mrna在miR-34a表达后下调，并且含有miR-34a种子匹配。通过报告基因试验证实了所选miR-34a靶mrna的直接调控作用。通过下调这些mrna编码的蛋白，miR-34a可能抑制糖酵解（LDHA）、wnt信号（LEF1）、入侵/迁移（AXL）和脂质代谢（ACSL1、ACSL4）。此外，miR-34a可能通过抑制其乙酰化（MTA2, HDAC1）和降解（YY1）来激活p53。综上所述，miR-34a可能通过直接或间接抑制众多关键蛋白的表达参与多种肿瘤抑制途径。中国生物医学工程学报（自然科学版），2011,31(2):444 - 444。
The gene encoding the miR-34a microRNA is a transcriptional target of the p53 tumor suppressor protein and subject to epigenetic inactivation in colorectal cancer and numerous other tumor types. Here, we combined pulsed SILAC (pSILAC) and microarray analyses to identify miR-34a-induced changes in protein and mRNA expression. pSILAC allowed to quantify the de novo protein synthesis of 1206 proteins after activation of a conditional miR-34a allele in a colorectal cancer cell line. similar to 19% of the detected proteins were differentially regulated, with 113 proteins being down-and 115 up-regulated. The proteins with a miR-34a seed-matching-sequence in the 3'-untranslated region (UTR) of the corresponding mRNA showed a clear bias toward translational repression. Proteins involved in DNA replication, e. g. the MCM proteins, and cell proliferation, were over-represented among indirectly down-regulated proteins lacking a miR-34a seed-match. The decrease in de novo protein synthesis of direct miR-34a targets correlated with reduced levels of the corresponding mRNA in most cases, indicating an interdependence of both types of regulation. In addition, 43 mRNAs encoding proteins not detected by pSILAC were down-regulated after miR-34a expression and contained miR-34a seed-matches. The direct regulation of selected miR-34a target-mRNAs was confirmed using reporter assays. Via down-regulation of the proteins encoded by these mRNAs miR-34a presumably inhibits glycolysis (LDHA), WNT-signaling (LEF1), invasion/migration (AXL) and lipid metabolism (ACSL1, ACSL4). Furthermore, miR-34a may activate p53 by inhibiting its acetylation (MTA2, HDAC1) and degradation (YY1). In summary, miR-34a presumably participates in multiple tumor suppressive pathways by directly and indirectly suppressing the expression of numerous, critical proteins. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.010462, 1-16, 2011.