Lemcke Heiko, Peukert Janine, Voronina Natalia, Skorska Anna, Steinhoff Gustav, David Robert
Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany.
Department of Cardiac Surgery, Reference and Translation Center for Cardiac Stem Cell Therapy (RTC), University of Rostock, Schillingallee 69, 18057 Rostock, Germany.
J Mol Cell Cardiol. 2016 Sep;98:117-27. doi: 10.1016/j.yjmcc.2016.07.008. Epub 2016 Jul 29.
Small antisense RNAs like miRNA and siRNA are of crucial importance in cardiac physiology, pathology and, moreover, can be applied as therapeutic agents for the treatment of cardiovascular diseases. Identification of novel strategies for miRNA/siRNA therapy requires a comprehensive understanding of the underlying mechanisms. Emerging data suggest that small RNAs are transferred between cells via gap junctions and provoke gene regulatory effects in the recipient cell. To elucidate the role of miRNA/siRNA as signalling molecules, suitable tools are required that will allow the analysis of these small RNAs at the cellular level. In the present study, we applied 3 dimensional fluorescence recovery after photo bleaching microscopy (3D-FRAP) to visualise and quantify the gap junctional exchange of small RNAs between neonatal cardiomyocytes in real time. Cardiomyocytes were transfected with labelled miRNA and subjected to FRAP microscopy. Interestingly, we observed recovery rates of 21% already after 13min, indicating strong intercellular shuttling of miRNA, which was significantly reduced when connexin43 was knocked down. Flow cytometry analysis confirmed our FRAP results. Furthermore, using an EGFP/siRNA reporter construct we demonstrated that the intercellular transfer does not affect proper functioning of small RNAs, leading to marker gene silencing in the recipient cell. Our results show that 3D-FRAP microscopy is a straightforward, non-invasive live cell imaging technique to evaluate the GJ-dependent shuttling of small RNAs with high spatio-temporal resolution. Moreover, the data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation where small RNAs act as signalling molecules within the intercellular network.
像微小RNA（miRNA）和小干扰RNA（siRNA）这样的小反义RNA在心脏生理学和病理学中至关重要，此外，还可作为治疗心血管疾病的治疗剂。确定miRNA/siRNA治疗的新策略需要全面了解其潜在机制。新出现的数据表明，小RNA通过间隙连接在细胞间转移，并在受体细胞中引发基因调控作用。为了阐明miRNA/siRNA作为信号分子的作用，需要合适的工具来在细胞水平分析这些小RNA。在本研究中，我们应用三维光漂白后荧光恢复显微镜技术（3D-FRAP）实时可视化和定量新生心肌细胞之间小RNA的间隙连接交换。用标记的miRNA转染心肌细胞并进行FRAP显微镜观察。有趣的是，我们观察到13分钟后恢复率就已达到21%，表明miRNA有强烈的细胞间穿梭，当连接蛋白43被敲低时，这种穿梭显著减少。流式细胞术分析证实了我们的FRAP结果。此外，使用一种增强型绿色荧光蛋白/小干扰RNA报告构建体，我们证明细胞间转移不会影响小RNA的正常功能，从而导致受体细胞中的标记基因沉默。我们的结果表明，3D-FRAP显微镜是一种直接、非侵入性的活细胞成像技术，可用于以高时空分辨率评估小RNA依赖间隙连接的穿梭。此外，3D-FRAP获得的数据证实了一种新的细胞间基因调控途径，即小RNA在细胞间网络中作为信号分子发挥作用。
