Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation.
Induction of NANOG expression by targeting promoter sequence with small activating RNA antagonizes retinoic acid-induced differentiation.
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DOI：
10.1042/bj20111491
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发表时间：
2012-05-01
期刊：
影响因子：
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通讯作者：
Li LC
中科院分区：
文献类型：
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作者：
Wang X;Wang J;Huang V;Place RF;Li LC
RNAa（RNA激活）是小dsRNA（双链RNA）（称为saRNA（小激活RNA））靶向启动子序列以诱导基因表达的机制。这种技术代表了一种新的方法，基因过表达，而不使用外源DNA。在本研究中，我们研究了RNA是否可以调节发育相关基因NANOG的表达并操纵细胞命运。使用基于慢病毒的报告系统作为筛选工具，我们鉴定了刺激人NCCIT胚胎癌细胞中NANOG表达的合成saRNA。与saRNA双链体的错配突变定义了基因激活的序列要求。NANOG诱导的功能分析显示，saRNA处理可预测地调节几种已知下游靶基因的表达，包括FOXH1（叉头框H1）、REST（RE1沉默转录因子）、OCT 4（八聚体结合蛋白4）和REX1（表达减少蛋白1）。用RA（视黄酸）处理触发NCCIT细胞分化，降低NANOG和OCT 4表达并上调几种神经标志物[即ASCL1（无毛鳞片复合物同源物1）、NEUROD1（神经元分化1）和PAX6（配对框6）]。然而，与saRNA共处理拮抗NANOG下调和RA诱导的分化。通过慢病毒转导的NANOG的异位过表达进一步概括了saRNA结果，提供了RNAa可用于激活发育相关基因和操纵细胞命运的概念验证。
RNAa (RNA activation) is a mechanism by which small dsRNA (double-stranded RNA), termed saRNA (small activating RNA), target promoter sequences to induce gene expression. This technique represents a novel approach to gene overexpression without the use of exogenous DNA. In the present study, we investigated whether RNAa can modulate expression of the development-related gene NANOG and manipulate cell fate. Using a lentivirus-based reporter system as a screening tool, we identified synthetic saRNAs that stimulate NANOG expression in human NCCIT embryonic carcinoma cells. Mismatch mutations to saRNA duplexes define sequence requirement for gene activation. Functional analysis of NANOG induction reveals saRNA treatment predictably modulates the expression of several known downstream target genes, including FOXH1 (forkhead box H1), REST (RE1-silencing transcription factor), OCT4 (octamer-binding protein 4) and REX1 (reduced expression protein 1). Treatment with RA (retinoic acid) triggers NCCIT cell differentiation, reducing NANOG and OCT4 expression and up-regulating several neural markers [i.e. ASCL1 (achaete-scute complex homologue 1), NEUROD1 (neuronal differentiation 1) and PAX6 (paired box 6)]. However, co-treatment with saRNA antagonizes NANOG down-regulation and RA-induced differentiation. Ectopic overexpression of NANOG via lentiviral transduction further recapitulates saRNA results, providing proof-of-concept that RNAa may be utilized to activate development-related genes and manipulate cell fate.