Council for Agricultural Research and Economics-Research Centre for Food and Nutrition, via Ardeatina 546, 00178 Rome, Italy.
Molecules. 2020 Dec 31;26(1):163. doi: 10.3390/molecules26010163.
We previously demonstrated that apoptosis induced by tocotrienols (γ and δT3) in HeLa cells is preceded by Ca release from the endoplasmic reticulum. This event is eventually followed by the induction of specific calcium-dependent signals, leading to the expression and activation of the gene encoding for the IRE1α protein and, in turn, to the alternative splicing of the pro-apoptotic protein sXbp1 and other molecules involved in the unfolded protein response, the core pathway coping with EndoR stress. Here, we showed that treatment with T3s induces the expression of a specific set of miRNAs in HeLa cells. Data interrogation based on the intersection of this set of miRNAs with a set of genes previously differentially expressed after γT3 treatment provided a few miRNA candidates to be the effectors of EndoR-stress-induced apoptosis. To identify the best candidate to act as the effector of the Xbp1-mediated apoptotic response to γT3, we performed in silico analysis based on the evaluation of the highest ∆ in Gibbs energy of different mRNA-miRNA-Argonaute (AGO) protein complexes. The involvement of the best candidate identified in silico, miR-190b, in Xbp1 splicing was confirmed in vitro using T3-treated cells pre-incubated with the specific miRNA inhibitor, providing a preliminary indication of its role as an effector of EndoR-stress-induced apoptosis.
我们之前的研究表明，生育三烯酚（γ 和 δT3）诱导 HeLa 细胞凋亡是由内质网 Ca2+释放引发的。这一事件最终会诱导特定的钙依赖性信号，导致编码 IRE1α 蛋白的基因表达和激活，进而导致促凋亡蛋白 sXbp1 和其他参与未折叠蛋白反应的分子的选择性剪接，这是应对内质网应激的核心途径。在这里，我们表明 T3 处理会诱导 HeLa 细胞中一组特定的 miRNA 表达。基于该 miRNA 集与 γT3 处理后差异表达基因集的交集进行的数据查询，提供了一些 miRNA 候选物作为内质网应激诱导凋亡的效应物。为了确定作为 Xbp1 介导的对 γT3 凋亡反应的效应物的最佳候选物，我们基于不同 mRNA-miRNA-Argonaute（AGO）蛋白复合物的 Gibbs 自由能的最高 ∆的评估进行了计算机模拟分析。在使用特定的 miRNA 抑制剂预孵育 T3 处理的细胞后，通过体外实验证实了最佳候选物 miR-190b 在 Xbp1 剪接中的作用，这初步表明其作为内质网应激诱导凋亡的效应物的作用。
