基于RNA-seq分析顺铂下调DRG神经元轴突导向相关基因的表达

RNA-seq analysis on cisplatin down-regulating expression of DRG neurons'axon guidance related genes

目的采用转录组测序技术分析顺铂对小鼠背根神经节(DRG)神经元轴突导向相关基因表达的影响,以进一步探讨顺铂的神经毒性机制。方法培养原代昆明小鼠DRG神经元,分为对照组和损伤组;对照组神经元直接培养24 h,损伤组神经元先培养4 h,后加入20μmol/L顺铂处理20 h;NF-200免疫荧光染色观察神经突起长度及生长锥结构;提取细胞总RNA,用RNA-seq技术分析样品,通过文献及数据库查找轴突导向直接或间接相关基因(共231个),分析两组基因表达差异;qPCR验证Cxcl12、Ext1、Gdnf、Plxna4、Nrp2、Apbb2、Sema6a,Nrp1、Nrcam、Slit1、Ank3、Ncam1、Sema7a、Dag1、Klf7、Ntn1基因表达情况。结果与对照组相比,损伤组神经突起数量少、长度短,生长锥出现倒钩状回缩;损伤组基因表达下调数量众多,其中轴突导向相关基因下调49个;各蛋白质相互作用密切,主要集中在Ntn1、Nrp1、Nrp2、Sema6a、plxna2、plxna4、Slit2、Robo1等基因之间;qPCR验证Cxcl12、Ext1、Gdnf、Plxna4、Nrp2、Apbb2、Sema6a、Nrp1、Nrcam、Slit1、Ank3、Ncam1、Sema7a、Dag1、Klf7、Ntn1基因表达下调,与转录组测序结果一致。结论顺铂神经毒性可能与众多轴突导向相关基因表达下调有关。

Objective To explore the effect of cisplatin on the expression of axon guidance related gene in mouse dorsal root ganglion(DRG)neurons using transcriptome sequencing technique,and to further explore the neurotoxic of cisplatin.Methods The primary cultures of DRG neurons was isolated from Kunming mice and divided into control group and injury group.Neurons in the control group were cultured for 24 hours,and those in the injury group were pre-cultured for 4 hours and then treated with 20μmol/L cisplatin for 20 hours.To observe the neurites and the growth cones in two groups by the NF200 immunofluorescence staining.Total RNA was extracted from the cells,and transcriptome sequencing was performed using RNA-seq technology.The axon guidance related genes were searched through literature and database(total 231),and the differential expression of related genes was analyzed.Real-time fluorescence quantitative PCR(qPCR)was used to verify the expression of related genes(Cxcl12,Ext1,Gdnf,Plxna4,Nrp2,Apbb2,Sema6a,Nrp1,Nrcam,Slit1,Ank3,Ncam1,Sema7a,Dag1,Klf7,Ntn1).Results Compared with the control group,the neurites of the injury group were significantly shortened and reduced,and the growthcones showedbarb retraction.A large number of genes were down-regulated in the injury group as compared to the control group,among them,49 genes related to axon guidance pathway were down-regulated.The proteins interacted closely and mainly focus on Ntn1,Nrp1,Nrp2,Sema6a,plxna2,plxna4,Slit2,Robo1.The qPCR verification of related genes expression was consistent with the trend of RNA-seq results.Conclusions The neurotoxicity mechanism of cisplatin may be related to the down-regulation of many axon guidance related genes.