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Functional analysis of DROSHA and DGCR8 microprocessor mutations as a cause of Wilms tumors

DROSHA 和 DGCR8 微处理器突变作为肾母细胞瘤病因的功能分析

基本信息

批准号：
283850712
负责人：
Professor Dr. Manfred Gessler
金额：
--
依托单位：
Lehrstuhl für Entwicklungsbiochemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2015
资助国家：
德国
起止时间：
2014-12-31 至 2020-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/283850712?language=en
关键词：
Functional analysis DROSHA DGCR8 microprocessor

项目摘要

Altered miRNA patterns are well known features of cancer, but only the recent identification of recurrent mutations in miRNA processing genes in Wilms tumors has now established deregulation of miRNA biogenesis as a driver of tumor formation. Wilms tumors are embryonal neoplasms that are derived from renal precursor cells. Especially in blastemal-type tumors, embryonic precursor cells apparently fail to terminally differentiate and remain in a highly proliferative state. By exome sequencing, we have identified recurrent mutations in the microprocessor genes DROSHA and DGCR8 in the blastemal subgroup with poor outcome. Most mutations are predicted either to inactivate the catalytic center of DROSHA or to change the RNA binding domain of DGCR8, both of which should lead to altered RNA metabolism. Together with evidence for a role of DICER1, DIS3L2 and LIN28B in rare cases of (inherited) Wilms tumors, this clearly suggests that miRNAs and their processing machinery must be critical for regulating cell fate and malignant transformation in kidney precursor cells, even if the underlying mechanism is completely unknown at this point.We plan to characterize the consequences of such mutations on clinical tumor characteristics. The focus of our work will be on in vitro analyses of the effects of mutant DROSHA/DGCR8 on cellular RNA representations and functions in cultured tumor cells as well as in mouse ES cells as genetically more tractable models. These will also be used to test the functional relevance of critical target RNAs identified in this screen to evaluate if just single genes or global effects on miRNA or even on mRNA/lncRNA may be important. To generate a model of microprocessor dependent Wilms tumor formation, we will establish mouse lines with inducible mutant DROSHA and DGCR8 cDNAs that will allow us to extend our functional studies to the in vivo situation. These experiments should provide us with unique insights into this novel pathway of miRNA driven tumor formation that should be relevant in a much broader context.

MiRNA模式改变是众所周知的癌症特征，但只有最近在Wilms肿瘤中发现miRNA加工基因的反复突变，才确立了miRNA生物发生的放松调控是肿瘤形成的驱动因素。肾母细胞瘤是一种来源于肾前体细胞的胚胎肿瘤。尤其是在胚胎型肿瘤中，胚胎前体细胞显然不能终末分化并保持高度增殖状态。通过外显子组测序，我们在胚泡亚群中发现了微处理器基因DROSHA和Dgcr8的反复突变，结果很差。据预测，大多数突变要么使DROSHA的催化中心失活，要么改变Dgcr8的RNA结合域，这两者都应该导致RNA代谢的改变。再加上DICER1、Dis3l2和Lin28b在罕见的(遗传性)肾母细胞瘤中所起作用的证据，这清楚地表明miRNAs及其加工机制对于调节肾脏前体细胞的细胞命运和恶性转化肯定是关键的，即使潜在的机制目前完全未知。我们计划表征这些突变对临床肿瘤特征的影响。我们工作的重点将是在体外分析突变的DROSHA/Dgcr8对培养的肿瘤细胞以及小鼠ES细胞中细胞RNA表达和功能的影响，作为遗传上更容易处理的模型。这些也将被用来测试在这个筛选中确定的关键靶RNA的功能相关性，以评估是否仅仅是单个基因或对miRNA甚至对mRNA/lncRNA的整体影响可能是重要的。为了建立依赖微处理器的Wilms肿瘤形成模型，我们将建立具有可诱导突变的DROSHA和Dgcr8 cDNA的小鼠系，这将使我们能够将我们的功能研究扩展到体内情况。这些实验应该为我们提供对miRNA驱动的肿瘤形成的这一新途径的独特见解，这应该在更广泛的背景下相关。