Lu Ting, Lin Zongwei, Ren Jianwei, Yao Peng, Wang Xiaowei, Wang Zhe, Zhang Qunye
Division of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China.
The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health; The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, China.
PLoS One. 2016 Mar 1;11(3):e0149751. doi: 10.1371/journal.pone.0149751. eCollection 2016.
MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.
Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.
These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.
微小RNA是一类长度约为22个核苷酸的小型非编码RNA，在几乎所有生物过程和疾病中都发挥着关键作用。荧光标记和脂质体转染是改变细胞微小RNA水平及定位其位置的两种常用方法。尽管关于DNA/RNA脂质体转染机制已有许多研究，但对于荧光标记微小RNA脂质体转染的特性、机制和特异性却知之甚少。
因此，使用脂质体转染试剂将用不同荧光染料标记的微小RNA转染到贴壁细胞和悬浮细胞中。然后，通过流式细胞仪和激光共聚焦显微镜研究非特异性结合及其机制。结果表明，即使没有脂质体转染试剂，用Cy5（花菁荧光染料）标记的微小RNA也能牢固地结合到贴壁细胞（Hela）和悬浮细胞（K562）表面。用FAM（羧基荧光素）标记的微小RNA与K562细胞的结合明显，但在Hela细胞中不显著。加入脂质体转染试剂后，由于转染效率高，大多数与Hela细胞表面结合的荧光标记微小RNA被转染到细胞内，然而，它们中的大多数仍与K562细胞表面结合。此外，能够破坏静电相互作用的高盐缓冲液不影响上述非特异性结合，但能够破坏疏水相互作用的有机溶剂则消除了这种结合。
这些结果表明，荧光标记的微小RNA可通过疏水相互作用非特异性地结合到细胞表面。仅根据细胞荧光强度来估计转染效率会导致显著误差。因此，为了结果的准确性，应根据细胞类型使用其他评估转染效率的方法和更合适的荧光染料。
