Cui Chunhui, Yu Jinlong, Huang Shuxin, Zhu Huiquan, Huang Zonghai
Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou, China.
Cell Physiol Biochem. 2014;33(6):1698-714. doi: 10.1159/000362952. Epub 2014 May 20.
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.
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.
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-κB 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-κB 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-kB and NFAT, respectively, but are not post-transcriptional targets for these two miRNAs. Finally, interference of these miRNAs with NF-κB and NFAT demonstrated clear phenotypes at the cellular level as manifested by the regulation of neuroblastoma cell death by miR-939 and miR-376a.
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.
背景/目的：已知微小RNA（miRNA）可在转录后水平抑制基因表达。鉴于诱饵寡脱氧核苷酸（ODN）能够隔离转录因子（TF），且诱饵ODN与miRNA之间具有相似的双链结构，我们推测miRNA可能作为内源性诱饵分子对基因表达产生转录调控作用。
采用定量实时逆转录聚合酶链反应（RT-PCR）分析来检测miRNA和mRNA表达的变化。利用荧光素酶报告基因活性测定法研究miRNA与TF之间的功能相互作用。采用电泳迁移率变动分析（EMSA）和改良的染色质免疫沉淀分析（ChIP）来研究miRNA与TF之间的物理相互作用。使用MTT细胞活力测定法和细胞DNA片段化ELISA来研究凋亡性细胞死亡。
我们在此表明，miRNA可通过其类似诱饵的作用在转录水平对基因表达进行正向或负向调控，且该机制在生理条件下起作用以产生细胞功能。我们分别在成熟的miR-939和miR-376a中鉴定出转录因子NF-κB和NFAT的假定顺式元件。我们使用EMSA和ChIP方法通过实验证实了这些miRNA与各自靶TF进行物理结合的能力。然后，我们利用荧光素酶报告基因测定法来表征miR-939/前体miR-939:NF-κB或miR-376a/前体miR-376a:NFAT相互作用对荧光素酶基因活性的特异性调控。此外，miR-939和miR-376a分别对内源性基因Bcl-xL和FasL/miR-26产生转录调控，这两个基因分别是NF-κB和NFAT的转录靶标，但不是这两种miRNA的转录后靶标。最后，这些miRNA对NF-κB和NFAT的干扰在细胞水平上表现出明显的表型，如miR-939和miR-376a对神经母细胞瘤细胞死亡的调控所示。
我们的研究确定了一种新的miRNA非经典机制，并表明在考虑miRNA的细胞功能时应考虑转录调控（激活或抑制）的类似诱饵机制。
