Recurrent DICER1 hotspot mutations in endometrial tumours and their impact on microRNA biogenesis
Recurrent DICER1 hotspot mutations in endometrial tumours and their impact on microRNA biogenesis
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DOI：
10.1002/path.4569
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发表时间：
2015-10-01
影响因子：
7.3
通讯作者：
Huntsman, David G.
中科院分区：
文献类型：
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作者：
Chen, Jiamin;Wang, Yemin;Huntsman, David G.
DICER 1在microRNA（miRNA）生物合成中起着关键作用。在卵巢性索间质肿瘤中发现了DICER 1的RNase IIIb结构域内四个金属结合位点的复发性体细胞“热点”突变，此后在其他儿科肿瘤中也有描述。在这项研究中，我们在290例子宫内膜肿瘤中筛选了DICER 1的RNase IIIb结构域，并确定了6例热点突变病例，其中包括2例受直接邻近金属结合位点的非典型G1809 R突变影响的病例。使用Illumina和桑格靶向重测序，我们在几个热点突变病例中观察并验证了双等位基因DICER 1突变。通过体外DICER 1切割试验、小RNA深度测序和实时PCR，我们证明了在残基1809上添加带正电荷的侧链的突变对5 p miRNA产生的不利影响与金属结合位点的突变相似。正如预期的那样，5 p miRNA在具有热点突变的肿瘤和细胞系中整体减少。基因表达谱的途径分析表明，由于5 p miRNA的丢失而去抑制的基因与调节细胞周期的途径密切相关。使用Dicer 1-null小鼠细胞系模型，我们发现DICER 1热点突变体的表达促进细胞增殖，而野生型（WT）DICER 1抑制细胞增殖。此外，let-7家族miRNAs的靶点在上调基因中富集，表明let-7的缺失可能影响下游途径。我们的研究结果表明，DICER 1热点突变与常见的恶性肿瘤有关，并可能构成一个独特的致癌途径。版权所有（c）2015大不列颠和爱尔兰病理学会。由John Wiley & Sons有限公司出版
DICER1 plays a critical role in microRNA (miRNA) biogenesis. Recurrent somatic 'hotspot' mutations at the four metal-binding sites within the RNase IIIb domain of DICER1 were identified in ovarian sex cord-stromal tumours and have since been described in other paediatric tumours. In this study, we screened the RNase IIIb domain of DICER1 in 290 endometrial tumours and identified six cases with hotspot mutations, including two cases affected by an atypical G1809R mutation directly adjacent to a metal-binding site. Using Illumina and Sanger targeted resequencing, we observed and validated biallelic DICER1 mutations in several cases with hotspot mutations. Through in vitro DICER1 cleavage assays, small RNA deep sequencing and real-time PCR, we demonstrated that mutations adding a positively charged side chain to residue 1809 have similar detrimental effects on 5p miRNA production to mutations at the metal-binding sites. As expected, 5p miRNAs were globally reduced in tumours and cell lines with hotspot mutations. Pathway analysis of gene expression profiles indicated that genes de-repressed due to loss of 5p miRNAs are strongly associated with pathways regulating the cell cycle. Using a Dicer1-null mouse cell line model, we found that expression of DICER1 hotspot mutants promoted cell proliferation, whereas wild-type (WT) DICER1 inhibited cell proliferation. Furthermore, targets of let-7 family miRNAs are enriched among the up-regulated genes, suggesting that loss of let-7 may be impacting downstream pathways. Our results reveal that DICER1 hotspot mutations are implicated in common malignancies and may constitute a unique oncogenic pathway. Copyright (c) 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.