微小RNA-21的下调参与了在人干细胞衍生神经元中观察到的丙泊酚诱导的神经毒性。
Down-regulation of microRNA-21 is involved in the propofol-induced neurotoxicity observed in human stem cell-derived neurons.
作者信息
Twaroski Danielle M, Yan Yasheng, Olson Jessica M, Bosnjak Zeljko J, Bai Xiaowen
机构信息
From the Departments of Physiology and Anesthesiology (D.M.T., J.M.O., Z.J.B.) and the Department of Anesthesiology (Y.Y., X.B.), Medical College of Wisconsin, Milwaukee, Wisconsin.
出版信息
Anesthesiology. 2014 Oct;121(4):786-800. doi: 10.1097/ALN.0000000000000345.
BACKGROUND
Recent studies in various animal models have suggested that anesthetics such as propofol, when administered early in life, can lead to neurotoxicity. These studies have raised significant safety concerns regarding the use of anesthetics in the pediatric population and highlight the need for a better model to study anesthetic-induced neurotoxicity in humans. Human embryonic stem cells are capable of differentiating into any cell type and represent a promising model to study mechanisms governing anesthetic-induced neurotoxicity.
METHODS
Cell death in human embryonic stem cell-derived neurons was assessed using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling staining, and microRNA expression was assessed using quantitative reverse transcription polymerase chain reaction. miR-21 was overexpressed and knocked down using an miR-21 mimic and antagomir, respectively. Sprouty 2 was knocked down using a small interfering RNA, and the expression of the miR-21 targets of interest was assessed by Western blot.
RESULTS
Propofol dose and exposure time dependently induced significant cell death (n = 3) in the neurons and down-regulated several microRNAs, including miR-21. Overexpression of miR-21 and knockdown of Sprouty 2 attenuated the increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells following propofol exposure. In addition, miR-21 knockdown increased the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate in situ nick end labeling-positive cells by 30% (n = 5). Finally, activated signal transducer and activator of transcription 3 and protein kinase B (Akt) were down-regulated, and Sprouty 2 was up-regulated following propofol exposure (n = 3).
CONCLUSIONS
These data suggest that (1) human embryonic stem cell-derived neurons represent a promising in vitro human model for studying anesthetic-induced neurotoxicity, (2) propofol induces cell death in human embryonic stem cell-derived neurons, and (3) the propofol-induced cell death may occur via a signal transducer and activator of transcription 3/miR-21/Sprouty 2-dependent mechanism.
背景
最近在各种动物模型中的研究表明，丙泊酚等麻醉剂在生命早期使用时会导致神经毒性。这些研究引发了对儿科人群使用麻醉剂的重大安全担忧，并突出了需要一个更好的模型来研究人类麻醉诱导的神经毒性。人类胚胎干细胞能够分化为任何细胞类型，是研究麻醉诱导神经毒性机制的一个有前途的模型。
方法
使用末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸原位缺口末端标记染色评估人类胚胎干细胞衍生神经元中的细胞死亡，并使用定量逆转录聚合酶链反应评估微小RNA表达。分别使用miR-21模拟物和拮抗剂使miR-21过表达和敲低。使用小干扰RNA敲低Sprouty 2，并通过蛋白质印迹评估感兴趣的miR-21靶标的表达。
结果
丙泊酚剂量和暴露时间依赖性地诱导神经元中显著的细胞死亡（n = 3），并下调包括miR-21在内的几种微小RNA。miR-21过表达和Sprouty 2敲低减弱了丙泊酚暴露后末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸原位缺口末端标记阳性细胞的增加。此外，miR-21敲低使末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸原位缺口末端标记阳性细胞数量增加了30%（n = 5）。最后，丙泊酚暴露后，活化的信号转导和转录激活因子3和蛋白激酶B（Akt）被下调，Sprouty 2被上调（n = 3）。
结论
这些数据表明：（1）人类胚胎干细胞衍生神经元是研究麻醉诱导神经毒性的一个有前途的体外人类模型；（2）丙泊酚诱导人类胚胎干细胞衍生神经元中的细胞死亡；（3）丙泊酚诱导的细胞死亡可能通过信号转导和转录激活因子3/miR-21/Sprouty 2依赖性机制发生。
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