Transcriptional Regulation of Gene Expression by microRNAs as Endogenous Decoys of Transcription Factors
Transcriptional Regulation of Gene Expression by microRNAs as Endogenous Decoys of Transcription Factors
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DOI：
10.1159/000362952
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发表时间：
2014-01-01
影响因子：
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通讯作者：
Huang, Zonghai
中科院分区：
文献类型：
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作者：
Cui, Chunhui;Yu, Jinlong;Huang, Zonghai
背景/目的：microRNAs(MiRNAs)是已知的产生转录后抑制基因表达的分子。鉴于诱骗寡核苷酸具有隔离转录因子的能力，以及诱骗寡核苷酸与miRNAs之间具有相似的双链结构，我们提出miRNAs可能作为内源性诱骗分子参与基因表达的转录调控。方法：采用实时荧光定量RT-PCR方法检测细胞中miRNA和mRNA表达的变化。利用荧光素酶报告基因活性实验研究了miRNAs与转录因子之间的功能相互作用。用凝胶迁移率改变分析(EMSA)和改进的染色质免疫沉淀分析(CHIP)研究了miRNAs与转录因子之间的物理相互作用。采用四甲基偶氮唑盐比色法、细胞DNA片段化酶联免疫吸附试验检测细胞凋亡率。结果：我们发现miRNAs可以通过其诱饵样作用在转录水平上负或正地调节基因的表达，这一机制在生理条件下发挥作用，以产生细胞功能。我们分别在成熟的miR-939和miR-376a中鉴定了转录因子NF-kappa B和NFAT的顺式元件。我们使用EMSA和芯片方法，通过实验建立了这些miRNAs与它们各自的靶TF物理结合的能力。然后，我们利用荧光素酶报告基因实验来表征miR-939/Pre-miR-939：NF-kappa B或miR-376a/Pre-miR-376a：NFAT相互作用对荧光素酶基因活性的特异性调节。此外，miR-939和miR-376a对内源基因Bcl-XL和FasL/miR-26产生转录调控作用，这两个基因分别是NF-kappa B和NFAT的转录靶标，但不是这两个miRNAs的转录后靶标。这些miRNAs对NF-kappa B和NFAT的干扰在细胞水平上表现出明显的表型，表现为miR-939和miR-376a对神经母细胞瘤细胞死亡的调节。结论：我们的研究发现了miRNAs的一种新的非典范机制，并建议在考虑miRNAs的细胞功能时，应考虑转录调节(激活或抑制)的诱饵样机制。版权所有(C)2014年S.Karger AG，巴塞尔
Background/Aims: MicroRNAs (miRNAs) are known to produce post transcriptional repression of gene expression. In light of the ability of decoy oligodeocynucleotides (ODNs) to sequestrate transcription factors (TFs) and the similar double stranded structure between decoy ODNs and miRNAs, we proposed that miRNAs might act as endogenous decoy molecules to produce transcriptional regulation of gene expression. Methods: Quantitative real-time RT-PCR analysis was used to measure the changes of miRNA and mRNA expression. Luciferase reporter gene activity assay was used to investigate the functional interaction between miRNAs and TFs. Electrophoresis mobility shift assay (EMSA) and modified chromatin immunoprecipitation assay (ChIP) were utilized to investigate the physical interactions between miRNAs and TFs. MTT cell viability assay and cellular DNA fragmentation ELISA were used to study apoptotic cell death. Results: We presented here that miRNAs could regulate, either negatively or positively, gene expression at the transcriptional level through its decoy-like actions and this mechanism operates under physiological conditions to produce cellular functions. We identified the putative cis-elements for transcriptional factors NF-kappa B and NFAT in the mature miR-939 and miR-376a, respectively. We experimentally established the ability of these miRNAs to physically bind their respective target TFs, using EMSA and ChIP methods. We then utilized the luciferase reporter gene assay to characterize the specific regulation of luciferase gene activities by miR-939/pre-miR-939:NF-kappa B or miR-376a/pre-miR-376a:NFAT interactions. Moreover, miR-939 and miR-376a produced transcriptional regulation of endogenous genes Bcl-xL and FasL/miR-26 that are the transcriptional targets for NF-kappa B and NFAT, respectively, but are not post-transcriptional targets for these two miRNAs. interference of these miRNAs with NF-kappa B and NFAT demonstrated clear phenotypes at the cellular level as manifested by the regulation of neuroblastoma cell death by miR-939 and miR-376a. Conclusion: Our study identified a novel non canonical mechanism of miRNAs and suggests that when considering the cellular function of miRNAs the decoy-like mechanism for transcriptional regulation (activation or repression) should be taken into account. Copyright (C) 2014 S. Karger AG, Basel