Laboratory for Skin Cancer Research, Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Hospital Research Centre of Laval University (CHUL/CHUQ), Laval University, Quebec, Quebec, Canada.
PLoS One. 2010 Aug 18;5(8):e12263. doi: 10.1371/journal.pone.0012263.
Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2-3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events.
通过瞬时或稳定的 RNA 干扰（RNAi）进行基因沉默，用于研究细胞凋亡，假设凋亡事件不会影响 RNAi。然而，我们最近报道，稳定的 RNAi，即通过整合到基因组中的产生 shRNA 的 DNA 载体介导的永久性基因敲低，在细胞凋亡诱导后迅速失效，这是由于 caspase-3 介导的切割和内核糖核酸酶 Dicer-1 的失活，Dicer-1 是将 shRNA 转化为 siRNA 所必需的。由于凋亡研究也越来越多地采用瞬时 RNAi 模型，即在转染 RNAi 诱导剂后几天内，基因暂时敲低后立即诱导凋亡，我们研究了凋亡对各种瞬时 RNAi 模型的影响。我们在这里报告，与稳定的 RNAi 不同，所有形式的瞬时 RNAi，无论是依赖 Dicer-1 的（通过 21mer dsRNA）还是不依赖 Dicer-1 的（通过 27mer dsRNA 或产生 shRNA 的 DNA 载体），无论是针对外源性基因 GFP 还是内源性基因多聚（ADP-核糖）聚合酶-1，在细胞凋亡开始后 2-3 天内都不会失效。我们的结果反映了瞬时 RNAi 模型中转染后早期实现和维持 RNAi 的动力学与稳定 RNAi 模型中基因敲低的晚期稳定状态之间的差异。我们的结果还提醒人们注意，在涉及细胞凋亡的研究中，应经常验证 RNAi 状态，虽然稳定的 RNAi 可安全地用于研究早期凋亡事件，但瞬时 RNAi 更适合研究早期和晚期凋亡事件。
