Lodrini Marco, Poschmann Gereon, Schmidt Victoria, Wünschel Jasmin, Dreidax Daniel, Witt Olaf, Höfer Thomas, Meyer Helmut E, Stühler Kai, Eggert Angelika, Deubzer Hedwig E
Department of Pediatric Hematology/Oncology/Stem Cell Transplantation Charité - Universitätsmedizin Berlin , Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany.
Molecular Proteomics Laboratory, Biological Medical Research Centre, Heinrich-Heine-University Düsseldorf , Universitätsstraße 1, 40225 Düsseldorf, Germany.
J Proteome Res. 2016 Jul 1;15(7):2178-86. doi: 10.1021/acs.jproteome.6b00134. Epub 2016 Jun 9.
MYCN and HDAC2 jointly repress the transcription of tumor suppressive miR-183 in neuroblastoma. Enforced miR-183 expression induces neuroblastoma cell death and inhibits xenograft growth in mice. Here we aimed to focus more closely on the miR-183 signaling network using a label-free mass spectrometric approach. Analysis of neuroblastoma cells transfected with either control or miR-183 expression vectors identified 85 differentially expressed proteins. All six members of the minichromosome maintenance (MCM) complex, which is indispensable for initiation and elongation during DNA replication and transcriptionally activated by MYCN in neuroblastoma, emerged to be down-regulated by miR-183. Subsequent annotation category enrichment analysis revealed a ∼14-fold enrichment in the "MCM" protein module category, which highlighted this complex as a critical node in the miR-183 signaling network. Down-regulation was confirmed by Western blotting. MCMs 2-5 were predicted by in silico methods as direct miR-183 targets. Dual-luciferase reporter gene assays with 3'-UTR constructs of the randomly selected MCMs 3 and 5 experimentally confirmed them as direct targets of miR-183. Our results reveal the MCM complex to be a critical and directly regulated node within the miR-183 signaling network in MYCN-amplified neuroblastoma cells.
MYCN和HDAC2共同抑制神经母细胞瘤中肿瘤抑制性miR-183的转录。增强miR-183的表达可诱导神经母细胞瘤细胞死亡并抑制小鼠体内异种移植瘤的生长。在此，我们旨在使用无标记质谱方法更密切地关注miR-183信号网络。对用对照或miR-183表达载体转染的神经母细胞瘤细胞进行分析，鉴定出85种差异表达的蛋白质。微小染色体维持（MCM）复合体的所有六个成员，其在DNA复制过程中的起始和延伸中不可或缺，并且在神经母细胞瘤中被MYCN转录激活，结果显示被miR-183下调。随后的注释类别富集分析显示，“MCM”蛋白质模块类别富集了约14倍，这突出了该复合体作为miR-183信号网络中的关键节点。通过蛋白质免疫印迹法证实了下调。通过计算机方法预测MCMs 2-5为miR-183的直接靶点。用随机选择的MCMs 3和5的3'-UTR构建体进行的双荧光素酶报告基因测定实验证实它们是miR-183的直接靶点。我们的结果表明，MCM复合体是MYCN扩增的神经母细胞瘤细胞中miR-183信号网络内的关键且直接受调控的节点。
