Mawji Imtiaz A, Marsden Philip A
Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, University of Toronto, Medical Sciences Building, Room 7358, 1 King's College Circle, Toronto, ON M5S 1A8, Canada.
Exp Biol Med (Maywood). 2006 Jun;231(6):704-8.
Endothelin-1 (ET-1) is a potent endothelial-derived vasoconstrictor and cellular mitogen. Perturbations in ET-1 levels have been observed in a number of cardiovascular and renal disorders. Steady-state ET-1 mRNA expression is regulated in the vascular endothelium by an inducible promoter and a constitutively short mRNA half-life. Recent studies have identified mRNA stabilization as a pathophysiologically relevant mechanism of ET-1 induction in vascular endothelial cells. However, mechanistic studies on posttranscriptional pathways in physiologically relevant postconfluent primary endothelial cell monolayers have remained challenging because of endothelial resistance to DNA-based gene transfer and expression. To overcome these challenges, we developed an RNA transfection method to study ET-1 posttranscriptional regulation. Reporter transcripts transfected into either preconfluent or postconfluent primary endothelial cells were rapidly and robustly expressed. RNA transfection reconstituted poly(A)-tail-dependent protein expression and ET-1 3'-UTR-dependent mRNA destabilization, suggesting that the transfected RNA accessed endothelial cell posttranscriptional pathways. Because RNA transfection uncouples transcription from expression, the influence of the ET-1 3'-UTR on posttranscriptional expression kinetics could also be monitored. Taken together, our results suggest that RNA transfection is a versatile tool to investigate ET-1 posttranscriptional regulation in endothelial cell culture models.
内皮素 -1（ET-1）是一种强效的内皮源性血管收缩剂和细胞有丝分裂原。在许多心血管和肾脏疾病中都观察到了ET-1水平的紊乱。血管内皮细胞中ET-1的稳态mRNA表达受诱导型启动子和组成型短mRNA半衰期的调控。最近的研究已确定mRNA稳定化是血管内皮细胞中ET-1诱导的一种病理生理学相关机制。然而，由于内皮细胞对基于DNA的基因转移和表达具有抗性，在生理相关的汇合后原代内皮细胞单层中对转录后途径进行机制研究仍然具有挑战性。为了克服这些挑战，我们开发了一种RNA转染方法来研究ET-1的转录后调控。转染到汇合前或汇合后原代内皮细胞中的报告转录本能够快速且稳定地表达。RNA转染重建了多聚腺苷酸尾依赖性蛋白表达以及ET-1 3'-UTR依赖性mRNA去稳定化，这表明转染的RNA进入了内皮细胞的转录后途径。由于RNA转染使转录与表达解偶联，因此还可以监测ET-1 3'-UTR对转录后表达动力学的影响。综上所述，我们的结果表明RNA转染是一种在内皮细胞培养模型中研究ET-1转录后调控的通用工具。
