Neurology Department, The First Affiliated Hospital, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Department of Cardiology, Wannan Medical College, Wuhu, Anhui, China.
J Cell Physiol. 2019 Nov;234(11):21166-21181. doi: 10.1002/jcp.28719. Epub 2019 Apr 29.
Lipofectamine 2000 (Lipo2000) delivery system is commonly used for short interfering RNA (siRNA) transfection, whereas the cellular responses have attracted little attention. The purpose of this study is to evaluate the effect of siRNA transfection using Lipo2000 on cellular functions and the possible underlying mechanism. Primary human umbilical vein endothelial cells (HUVECs) and adult human coronary artery endothelial cell line (HCAECs) were treated with different concentrations of a Lipo2000/negative control siRNA (NC siRNA) complex or Lipo2000 for specific durations. The cell proliferation, apoptosis rate, and protein expression of claudin5 (CLDN5) and ETS-related gene (ERG) were analyzed as indicators of cellular function. The effects of the Lipo2000/NC siRNA complex on cellular autophagy and endoplasmic reticulum (ER) unfolded protein response (UPR) were investigated by western blot and real-time polymerase chain reaction analyses; autophagy was also evaluated by transmission electron microscopy. The Lipo2000/NC siRNA complex inhibited proliferation, downregulated various proteins, and increased the apoptosis in both HUVECs and HCAECs. Both autophagy and UPR were observed in HUVECs treated with the Lipo2000/NC siRNA complex, ER stress-induced autophagy acted as a cellular protective factor against apoptosis, as inhibition of autophagy by chemical inhibitors increased the cell apoptosis rate. Chemical chaperones failed to prevent the Lipo2000/siRNA complex-induced UPR. However, knockdown of protein kinase RNA-like ER kinase and inositol-requiring protein 1, instead of activating transcription factor-6, partially ameliorated the UPR and reversed the protein level of CLDN5 and ERG downregulated by Lipo2000/NC siRNA complex. This study provides the first evidence that the Lipo2000-mediated transport of siRNA leads to an increase in UPR and ER stress-related apoptosis in endothelial cells.
脂质体 2000（Lipo2000）转染系统常用于短发夹状 RNA（siRNA）转染，但其对细胞功能的影响尚未引起关注。本研究旨在评估 Lipo2000 转染 siRNA 对细胞功能的影响及其潜在机制。将不同浓度的 Lipo2000/阴性对照 siRNA（NC siRNA）复合物或 Lipo2000 作用于原代人脐静脉内皮细胞（HUVEC）和成人冠状动脉内皮细胞系（HCAEC），作用特定时间。以 claudin5（CLDN5）和 ETS 相关基因（ERG）的蛋白表达、细胞增殖率和凋亡率为指标，分析细胞功能。通过 Western blot 和实时聚合酶链反应分析研究 Lipo2000/NC siRNA 复合物对细胞自噬和内质网（ER）未折叠蛋白反应（UPR）的影响；通过透射电子显微镜评估自噬。Lipo2000/NC siRNA 复合物抑制 HUVEC 和 HCAEC 的增殖，下调多种蛋白，增加细胞凋亡。Lipo2000/NC siRNA 复合物处理的 HUVEC 中观察到自噬和 UPR，ER 应激诱导的自噬作为细胞抗凋亡的保护因子，化学抑制剂抑制自噬增加细胞凋亡率。化学伴侣不能防止 Lipo2000/siRNA 复合物诱导的 UPR。然而，敲低蛋白激酶 RNA 样内质网激酶和肌醇需求蛋白 1，而不是激活转录因子 6，部分改善 UPR，并逆转 Lipo2000/NC siRNA 复合物下调的 CLDN5 和 ERG 蛋白水平。本研究首次证明，Lipo2000 介导的 siRNA 转运导致内皮细胞 UPR 和 ER 应激相关凋亡增加。
