MicroRNA Discovery and Profiling in Human Embryonic Stem Cells by Deep Sequencing of Small RNA Libraries
MicroRNA Discovery and Profiling in Human Embryonic Stem Cells by Deep Sequencing of Small RNA Libraries
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DOI：
10.1634/stemcells.2008-0356
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发表时间：
2008-01-01
期刊：
影响因子：
5.2
通讯作者：
Tewari, Muneesh
中科院分区：
文献类型：
--
作者：
Bar, Merav;Wyman, Stacia K.;Tewari, Muneesh
我们使用大规模平行焦磷酸测序来发现和表征人胚胎干细胞（hESC）中表达的microRNA（miRNAs）。对来自未分化hESC和来自同基因分化培养物的小RNA cDNA文库进行测序，总共产生了425，505个高质量的序列读数。自定义数据分析管道描绘了191个先前注释的miRNAs，13个新的miRNAs和56个候选miRNAs的表达谱。Dicer敲低hESC中新miRNAs亚组的进一步表征证实了Dicer依赖性表达，为我们的结果提供了额外的验证。一组14个miRNAs（9个已知的和5个新的）被注意到在未分化的hESC中表达，然后随着分化强烈下调。对这些miRNA的预测靶点进行功能注释分析，并与使用非hESC表达的miRNA的空模型进行比较，确定了统计学上丰富的功能类别，包括染色质重塑和谱系特异性分化注释。最后，我们的数据与OCT 4，SOX 2和NANOG结合位点的全基因组染色质免疫沉淀数据的整合暗示了这些转录因子在这里确定的九种新的/候选的miRNA的调节。将我们的结果与最近在小鼠和人类ESC中进行的深度测序研究的结果进行比较表明，在这里发现的大多数新的/候选的miRNAs在其他研究中没有被鉴定出来。这些数据表明，hESC表达比以前认识到的更大的互补的miRNA，并且它们为进一步研究miRNA调节hESC生理学提供了资源。干细胞2008; 26：2496-2505
We used massively parallel pyrosequencing to discover and characterize microRNAs (miRNAs) expressed in human embryonic stem cells (hESC). Sequencing of small RNA cDNA libraries derived from undifferentiated hESC and from isogenic differentiating cultures yielded a total of 425,505 high-quality sequence reads. A custom data analysis pipeline delineated expression profiles for 191 previously annotated miRNAs, 13 novel miRNAs, and 56 candidate miRNAs. Further characterization of a subset of the novel miRNAs in Dicer-knockdown hESC demonstrated Dicer-dependent expression, providing additional validation of our results. A set of 14 miRNAs (9 known and 5 novel) was noted to be expressed in undifferentiated hESC and then strongly downregulated with differentiation. Functional annotation analysis of predicted targets of these miRNAs and comparison with a null model using non-hESC-expressed miRNAs identified statistically enriched functional categories, including chromatin remodeling and lineage-specific differentiation annotations. Finally, integration of our data with genome-wide chromatin immunoprecipitation data on OCT4, SOX2, and NANOG binding sites implicates these transcription factors in the regulation of nine of the novel/candidate miRNAs identified here. Comparison of our results with those of recent deep sequencing studies in mouse and human ESC shows that most of the novel/candidate miRNAs found here were not identified in the other studies. The data indicate that hESC express a larger complement of miRNAs than previously appreciated, and they provide a resource for additional studies of miRNA regulation of hESC physiology. STEM CELLS 2008; 26: 2496-2505