MicroRNA‐mediated non‐cell‐autonomous regulation of cortical radial glial transformation revealed by a Dicer1 knockout mouse model

Radial glia (RG), as neurogenic progenitors and neuronal migration scaffolds, play critical roles during cortical neurogenesis. RG transformation into astrocytes, marking the transition from developmental to physiological function of these cells, is an important step during cortical development. In this study, we aim to determine the roles of microRNAs (miRNAs) during this biological process. In a conditional Dicer1‐null mouse where Dicer1 is deleted in both RG and their neuronal progeny, we observe delayed RG transformation as revealed by the persistence of their radial processes, and reduced number and complexity of translocated RG cell bodies in the postnatal cerebral cortex. Downregulation of Notch1 signaling is crucial to RG transformation, and consistently we find that Notch1 signaling is enhanced in the Dicer1‐null cerebral cortex. In addition, we show that, among the Notch1 ligands, Jagged2 (Jag2) is preferentially upregulated in the postnatal Dicer1‐null cerebral cortex as well as primary embryonic cortical cultures with instant Dicer1 deletion. Functionally, Dicer1‐deleted postnatal cerebellar cells with elevated Jag2 expression stimulate a stronger Notch1 signaling in a RG clone L2.3 when co‐cultured than control cells. Therefore, we unravel a novel non‐cell‐autonomous mechanism that regulates RG transformation by modulating Notch1 signaling via miRNA‐mediated suppression of the Nocth1 ligand Jag2. Furthermore, we validate Jag2 as a miR‐124 target gene and demonstrate in vitro that Jag2 expression is highly sensitive to Dicer1 deletion. Finally, we propose a new concept of MiRNA‐Sensitive target genes, identification of which may unravel a unique mode of miRNA‐mediated gene expression regulation. GLIA 2015;63:860–876     

Dicer1 functions as a haploinsufficient tumor suppressor

While the global down-regulation of microRNAs (miRNAs) is a common feature of human tumors, its genetic basis is largely undefined. To explore this question, we analyzed the consequences of conditional Dicer1 mutation (Dicer1 “floxed” or Dicer1^fl) on several mouse models of cancer. Here we show Dicer1 functions as a haploinsufficient tumor suppressor gene. Deletion of a single copy of Dicer1 in tumors from Dicer1^fl/+ animals led to reduced survival compared with controls. These tumors exhibited impaired miRNA processing but failed to lose the wild-type Dicer1 allele. Moreover, tumors from Dicer1^fl/fl animals always maintained one functional Dicer1 allele. Consistent with selection against full loss of Dicer1 expression, enforced Dicer1 deletion caused inhibition of tumorigenesis. Analysis of human cancer genome copy number data reveals frequent deletion of DICER1. Importantly, however, the gene has not been reported to undergo homozygous deletion, suggesting that DICER1 is haploinsufficient in human cancer. These findings suggest Dicer1 may be an important haploinsufficient tumor suppressor gene and, furthermore, that other factors controlling miRNA biogenesis may also function in this manner.     

microRNA与肿瘤的关系

miRNA是一类分布广泛的非编码小分子RNA，其长度约为22个核苷酸，它的功能是通过负调控基因的表达从而调节细胞的生理过程，但当其表达异常时可能引起疾病甚至肿瘤的形成。过去肿瘤的研究大多数都集中于对原癌基因和抑癌基因突变的研究上。目前，发现许多肿瘤的发生很可能是受miRNA的影响，miRNA也可以起到抑癌基因或原癌基因的作用。miRNA表达的异常可能与其染色体脆性位点存在几率较高有关。本文对miRNA的结构、功能及其与肿瘤发生关系等方面进行了综述。     

MicroRNAs途径在RAW264.7细胞中作用的初步研究

目的通过检测microRNAs生成的关键酶Dicer在RAW264.7细胞不同功能状态下的表达差异研究microRNAs途径在其中产生的作用。方法采用QRT-PCR、流式细胞术等对RAW264.7细胞在不同功能分化状态下的Dicer酶进行检测。结果GMCSF与MCSF分别刺激RAW264.7细胞诱导其增殖分化后,Dicer酶的mRNA水平显著增高,但随刺激时间呈现不同趋势(P<0.01);在诱导凋亡死亡状态下,RAW264.7中Dicer酶的表达下降30%(P<0.01);LPS与IL-4分别诱导RAW264.7细胞分化为M1和M2型,Dicer的表达与M1、M2型相关炎性因子的表达有相关性。结论 MicroRNAs调控途径参与并影响RAW264.7不同功能状态。     

丙型肝炎病毒核心蛋白对Dicer切割功能的影响

目的 探讨核心蛋白与Dicer的相互作用及其对Dicer功能的影响.方法 构建丙型肝炎病毒核心蛋白真核表达质粒,采用免疫荧光染色和Western blot检测核心蛋白的表达,Western blot检测核心蛋白对细胞内Dicer表达的影响,免疫共沉淀技术检测真核细胞内核心蛋白与Dicer的相互作用.体外转录合成dsRNA,检测核心蛋白对Dicer切割dsRNA作用的影响.结果 在细胞内,核心蛋白能够明显恢复被shRNA抑制的虫荧光素酶基因的表达(P＜0.05),而对siRNA引发的RNAi无拮抗作用.在真核细胞内,加入核心蛋白后,Dicer的表达量无明显改变,并能检测到两者相互结合.在体外抑制实验中,重组表达的Dicer能切割dsRNA产生siRNA;核心蛋白加入后,dsRNA的切割受到抑制.结论 核心蛋白在真核细胞内可以结合Dicer,并通过抑制Dicer对dsRNA的切割作用,抑制RNA干扰作用.