描述了微RNA转录的转录因子依赖调节
Summary
Herein we propose a strategy to study the effect of a transcription factor of interest on the microRNA transcriptome using publically available data, computational resources and high throughput data from microRNA arrays after transfecting cells with small hairpin (sh)RNA targeting a transcription factor of interest.
Abstract
而蛋白质编码基因的转录调控被广泛研究，知之甚少转录因子如何参与非编码RNA的转录，特别的微RNA。在这里，我们提出了一个战略，研究使用规范公开提供的数据，计算资源和高吞吐量数据的microRNA的转录转录因子的潜在作用。我们使用的H3K4me3的后生签名从ENCODE项目识别微RNA启动子和染色质免疫沉淀（ChIP）-sequencing数据，以确定被富含转录因子结合位点的微RNA启动子。通过转染与shRNA的靶向感兴趣的转录因子和使细胞微RNA阵列感兴趣的细胞中，我们研究这转录因子对微RNA转录的影响。作为一个说明性的例子，我们使用我们的研究STAT3的慢性Ly的微小RNA转录的影响mphocytic性白血病（CLL）细胞。
Introduction
微RNA是内生的小非编码调控RNA，通常在转录后水平起作用的mRNA表达的作为负调节。大约1000非编码20至25个核苷酸长的微小RNA在人类基因组中的1,2-找到。微RNA调节通过微RNA及其互补种子匹配序列，其通常位于3'非翻译区的靶mRNA的（UTR）的种子序列之间规范碱基配对的基因表达。总的来说，微RNA调控的蛋白质编码基因3超过30％，但仅知之甚少从微RNA的DNA的转录。它已被提出，微RNA转录的调节类似于表达4,5。特别是，类似于其在促进蛋白质编码基因的转录活性，转录因子被认为激活微RNA 6的转录。转录因子-MicroRNA相互作用已被报告为基因表达7的调制因子，并且还可以形成反馈和前馈回路。例如，Yamakuchi 等报道的反馈环路，其中的p53诱导microRNA34a，表达这反过来又抑制了p53的阻遏SIRT的翻译，并由此提高p53活性8。
而微RNA的转录因子依赖性表达的具体例子，已报道，缺乏其中提供了关于感兴趣的转录因子如何调节微小RNA-转录的表达信息的被接受的方法。本文所提出的协议的目的是提供微小RNA-转录的转录依赖性因子调控的进行了深入的说明。通过结合公开可用的数据，生物信息学工具，并使用微阵列技术，谁遵循这个算法的研究人员将能够捕获基因组规模如何的任何在任何感兴趣的细胞类型的转录因子调节微小RNA-转录的表达和在调节微RNA表达探索转录因子mRNA的推定贡献。
Protocol
Representative Results
Discussion
蛋白质编码基因的RNA聚合酶II-依赖性转录背后的机制已被广泛研究。而这些元素组成只有1％ -人类基因组的2％，从ENCODE项目证据表明，人类基因组的80％以上的可经历转录17和什么调节非编码DNA元件的转录在很大程度上仍然未知6 。
几项研究，表明聚合酶II也负责的一些非编码蛋 白的基因，包括微RNA 6的转录，导致我们开发出从公共可用的资源，?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由来自全球CLL研究基金会的资助。得克萨斯大学MD安德森癌症中心的大学是由美国国立卫生研究院的部分通过一个癌症中心支援津贴（P30CA16672）的支持。
Materials
| Lipofectamin 2000 | Life Technologies | 11668027 | |
| 0.45 um syringe filter | Thermo Scientific (Nalgene) | 190-2545 | |
| Amicon ultracentrifugal filter device with threshold of 100kDa | Merck Millipore | ||
| Polybrene | Merck Millipore | TR-1003-G | |
| TRIzol reagent | Life Tachnologies (Invitrogen) | 15596-026 | |
| 293 Cell line human | Sigma-Aldrich | 85120602 |
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