miR-142-5p对癫痫发作后神经元损伤的作用机制

Mechanism of miR-142-5p on neuronal damage after epilepsy

目的探讨miR-142-5p/APLN对癫痫发作后神经元损伤的影响及其调控机制.方法构建大鼠海马神经元癫痫模型,正常神经元细胞作为正常对照组,以此神经元细胞为癫痫细胞模型组.取癫痫大鼠神经细胞分为anti-miR-142-5p组(转染anti-miR-142-5p)、anti-miR-con组(转染anti-miR-con)、miR-142-5p组(转染miR-142-5p mimics)、miR-con组(转染miR-con),NC组(未经任何处理的细胞);anti-miR-142-5p+si-con组(共转染anti-miR-142-5p与si-con)、anti-miR-142-5p+si-APLN组(anti-miR-142-5p与si-APLN).qRT-PCR与Western blot检测两组神经细胞中miR-142-5p与APLN的表达.ELISA法检测GSH与MDA含量.流式细胞仪检测细胞凋亡能力变化.双荧光素酶报告基因鉴定miR-142-5p的靶基因.Western blot检测Bax、Bcl-2蛋白表达.结果miR-142-5p在癫痫细胞模型组神经细胞中表达上调(P<0.05).干扰miR-142-5p表达后大鼠神经细胞中GSH含量明显增加,MDA含量明显减少(P<0.05),细胞凋亡率显著降低(P<0.05),Bcl-2蛋白表达水平显著升高(P<0.05),Bax蛋白表达水平显著降低(P<0.05);双荧光素酶报告基因实验证明miR-142-5p可直接靶向结合APLN;沉默APLN的表达可部分逆转干扰miR-142-5p的表达对大鼠癫痫神经细胞的保护作用.结论干扰miR-142-5p表达可能上调APLN的表达抑制神经细胞凋亡从而减轻癫痫发作后神经元损伤.

Objective To investigate the effect of microRNA-142-5p(miR-142-5p)/Apelin(APLN)on neuronal damage after seizure and the mechanism.Methods The rat hippocampal neuron epilepsy model was constructed.Normal neuron cells were used as normal control group.Rat hippocampal neuron epilepsy model neuron cells were epilepsy cell model group.Nerve cells from epileptic rats were divided into anti-miR-142-5p group(transfected with anti-miR-142-5p),anti-miR-con group(transfected with anti-miR-con),and miR-142-5p group(Transfected miR-142-5p mimics),miR-con group(transfected miRcon),NC group(untreated cells);anti-miR-142-5p+si-con group(co-transfected anti-miR-142-5p and sicon),anti-miR-142-5p+si-APLN group(anti-miR-142-5p and si-APLN).Real-time quantitative polymerase chain reaction(RT-qPCR)and Western blot were used to detect the expression of miR-142-5p and APLN in the two groups of neurons.The content of glutathione(GSH)and malondialdehyde(MDA)was determined by enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to detect the change of neuronal apoptosis ability in each group.The dual luciferase reporter gene identified the target gene of miR-142-5p.The expression of Bax,Bcl-2 and AKT signaling pathway-related proteins was detected by western blot.Results The expression of miR-142-5p was up-regulated in the neurons of the epileptic cell model group,and the difference was statistically significant compared with the normal control group(P<0.05).Significant increase in GSH content in rat neurons after interference with miR-142-5p expression,the MDA content was significantly decreased(P<0.05),and the apoptosis rate was significantly decreased(P<0.05).The expression level of Bcl-2 protein was significantly increased(P<0.05),the expression level of Bax protein was significantly decreased(P<0.05),and the expression levels of p-AKT and p-GSK3βwere significantly increased(P<0.05).The dual luciferase reporter gene assay demonstrated that miR-142-5p can directly target APLN.Silencing of APLN expression partially reversed the protective effect of miR-142-5p expression on rat epileptic neurons.Conclusion Interfering with miR-142-5p expression inhibits neuronal apoptosis and thus reduces neuronal damage after epileptic seizures by up-regulating the expression of APLN.