Nuclear protein in testis (NUT)-midline carcinoma (NMC) is a rare, aggressive disease typically presenting with a single t(15;19) translocation that results in the generation of a bromodomain-containing protein 4 (BRD4)-NUT fusion. PER-624 is a cell line generated from an NMC patient with an unusually complex karyotype that gave no initial indication of the involvement of the NUT locus. Analysis of PER-624 next-generation transcriptome sequencing (RNA-Seq) using the algorithm FusionFinder identified a novel transcript in which Exon 15 of BRD4 was fused to Exon 2 of NUT, therefore differing from all published NMC fusion transcripts. The three additional exons contained in the PER-624 fusion encode a series of polyproline repeats, with one predicted to form a helix. In the NMC cell line PER-403, we identified the 'standard' NMC fusion and two novel isoforms. Knockdown by small interfering RNA in either cell line resulted in decreased proliferation, increased cell size and expression of cytokeratins consistent with epithelial differentiation. These data demonstrate that the novel BRD4-NUT fusion in PER-624 encodes a functional protein that is central to the oncogenic mechanism in these cells. Genomic PCR indicated that in both PER-624 and PER-403, the translocation fuses an intron of BRD4 to a region upstream of the NUT coding sequence. Thus, the generation of BRD4-NUT fusion transcripts through post-translocation RNA-splicing appears to be a common feature of these carcinomas that has not previously been appreciated, with the mechanism facilitating the expression of alternative isoforms of the fusion. Finally, ectopic expression of wild-type NUT, a protein normally restricted to the testis, could be demonstrated in PER-403, indicating additional pathways for aberrant cell signaling in NMC. This study contributes to our understanding of the genetic diversity of NMC, an important step towards finding therapeutic targets for a disease that is refractory to current treatments.

译文

睾丸 (NUT) 中线癌 (NMC) 中的核蛋白是一种罕见的侵袭性疾病,通常表现为单个t(15;19) 易位,导致产生含溴结构域的蛋白4 (BRD4)-NUT融合。PER-624是由NMC患者产生的细胞系,其具有异常复杂的核型,没有给出涉及NUT基因座的初始指示。使用算法FusionFinder对每624下一代转录组测序 (RNA-Seq) 的分析鉴定了一种新的转录物,其中BRD4的外显子15与NUT的外显子2融合,因此不同于所有已发表的NMC融合转录物。每个624融合中包含的三个额外外显子编码一系列多脯氨酸重复序列,其中一个预测形成螺旋。在每403 NMC细胞系中,我们鉴定了 “标准” NMC融合和两种新的同工型。在任一细胞系中通过小干扰RNA敲低导致增殖减少,细胞大小增加和细胞角蛋白的表达与上皮分化一致。这些数据表明,PER-624中的新型BRD4-NUT融合体编码对这些细胞中的致癌机制至关重要的功能性蛋白质。基因组PCR表明,在PER-624和PER-403中,易位将BRD4的内含子融合到NUT编码序列上游的区域。因此,通过易位后RNA剪接产生BRD4-NUT融合转录物似乎是这些癌的共同特征,以前没有被认识到,其机制促进了融合的替代同工型的表达。最后,野生型NUT (一种通常限于睾丸的蛋白质) 的异位表达可以在PER-403中得到证实,这表明NMC中异常细胞信号传导的其他途径。这项研究有助于我们了解NMC的遗传多样性,这是为目前治疗方法难以治疗的疾病寻找治疗靶点的重要一步。

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