长链非编码RNA Peg13在七氟烷所致的发育期原代神经元凋亡中的作用

Role of long non-coding RNA Peg13 in apoptosis of developing primary neurons following sevoflurane injury

目的研究长链非编码 RNA(long non-coding RNA,lncRNA)Peg13(paternally expressed 13)在七氟烷所致的发育期原代神经元凋亡中的作用。方法以胎龄为 14.5 d 的小鼠胎鼠的原代神经元为研究对象,实时定量 PCR 检测七氟烷处理不同时间后 lncRNAPeg13 的表达水平。荧光原位杂交实验检测 lncRNA Peg13 在原代神经元中的定位。构建 lncRNA Peg13 过表达和敲减质粒,分别转染原代神经元。4.1%的七氟烷处理 6 h 后,荧光显微镜观察原代神经元的形态改变;采用 CCK-8 分析原代神经元的活力;采用 TUNEL、Western blotting 检测原代神经元的凋亡程度。结果在七氟烷处理的原代神经元中,lncRNA Peg13 的表达水平呈时间依赖性下降。LncRNA Peg13 在原代神经元的细胞质和轴突中广泛表达。过表达 lncRNA Peg13 可明显缓解七氟烷处理所致的神经元形态改变,导致神经元活力提高,凋亡细胞比例减少,Bcl-2、Bax 蛋白表达量的比值(Bcl-2/Bax)升高,caspase-3 的表达水平降低。敲减 lncRNAPeg13 可明显加剧七氟烷处理所致的神经元形态改变,导致神经元活力降低,凋亡细胞比例增加,Bcl-2/Bax 降低,caspase-3 的表达水平升高。结论 LncRNA Peg13 能够缓解七氟烷诱导的发育期原代神经元的凋亡。

Objective To investigate the role of long non-coding RNA (lncRNA) Peg13 (paternally expressed 13) in the apoptosis of developing primary neurons following sevoflurane injury. Methods Primary neurons were prepared from fetal mice with 14.5 d of gestational age. The expression of lncRNA Peg13 after sevoflurane treatment was detected by quantitative PCR. The localization of lncRNA Peg13 in primary neurons was detected by in situ hybridization. Peg13 overexpression and knockdown plasmids were constructed and transfected into primary neurons. The morphology of primary neurons was observed by fluorescence microscope. Cell vability was assessed by CCK-8 assay. Cell apoptosis was determined by TUNEL assay and Western blotting. Results The expression of lncRNA Peg13 in primary neurons decreased in a time-dependent manner after sevoflurane treatment. LncRNA Peg13 was widely expressed in the cytoplasm and axon of primary neuron. Overexpression of lncRNA Peg13 resulted in decreased sevoflurane-induced apoptosis. The primary neurons were restored to normal morphology with increased cell vability. The percentage of TUNEL-positive cells was decreased. The expression ratio of Bcl-2/Bax was increased and the expression of casepase-3 was decreased. However, knockdown of lncRNA Peg13 aggravated the sevoflurane-induced apoptosis. The primary neurons had visible morphological deterioration and decreased cell vability, with increased percentage of TUNEL-positive cells, decreased ratio of Bcl-2/Bax, and increased expression of casepase-3. Conclusion LncRNA Peg13 may alleviate sevoflurane- induced apoptosis in developing primary neurons.