面神经损伤修复过程中差异表达microRNA的初步筛选分析

Preliminary screening and analysis of differentially expressed microRNAs during the repair of facial nerve injury

目的通过高通量RNA测序(RNA-seq)技术,筛选面神经挤压性损伤修复过程(3 d、14 d)中差异表达的microRNA,并对其中部分microRNA的表达进行验证。方法建立大鼠面神经挤压性损伤模型,通过面容观测、组织学观察评价神经修复过程;收集挤压后3 d、14 d的标本各3例,进行microRNA测序分析,筛选具有显著性差异的microRNA,并通过qPCR验证上、下调最为显著的4个microRNA在不同时期的神经标本中的表达情况。结果挤压大鼠面神经后成功造成面神经损伤模型,术后即出现面瘫表现,面容观测及组织学结果均提示术后14 d面神经损伤得到一定程度的修复。RNA测序结果提示共有96个microRNA的表达显著上调,115个microRNA的表达显著下调。挑选上、下调最明显的4个microRNA进行qPCR验证,结果提示其表达与芯片检测结果趋势一致。相较于术后3 d,术后14 d标本中miR-200a-3p表达显著上调(P<0.01),miR-300-5p表达显著下调(P<0.05)。结论在面神经损伤后的不同阶段,microRNA的表达呈现出不同的特征。异常表达的microRNA(如miR-200a-3p、miR-300-5p等)可能为面神经损伤的治疗提供新的靶点。

Objective To screen differentially expressed microRNAs during the repair of facial nerve crush injury(3 d,14 d)by high-throughput RNA sequencing technology,and to verify the expression of microRNAs of interest.Methods The rat model of facial nerve crush injury was established,and the process of nerve repair was evaluated by facial appearance observation and histological staining.Three samples were collected at 3 and 14 days after compression respectively,and microRNA sequencing analysis was performed.Bioinformatics method was used to screen for microRNA with significant differences,and qPCR was used to verify the expression of the 4 microRNAs that were most significantly down-regulated and up-regulated in nerve samples at different periods.Results The model of facial nerve injury was successfully created by crushing the facial nerve trunk of rats.Facial paralysis appeared immediately after the operation.The facial appearance observation and histological staining results all indicated that the facial nerve injury was repaired to a certain extent 14 days after the operation.The RNA sequencing results showed that a total of 96 microRNAs were significantly up-regulated and 115 microRNAs were significantly down-regulated.Four microRNAs with the most obvious up-regulation and down-regulation were selected for qPCR validation,and the results showed consistency between their expression and the trend of sequence detection results.Compared to 3 days after operation,the expression of miR-200a-3p was significantly up-regulated(P<0.01)and miR-300-5p was significantly down-regulated(P<0.05)in the specimens 14 days after operation.Conclusion The expression of microRNA shows different characteristics at different stages after facial nerve injury.Abnormal expression of microRNAs(such as miR-200a-3p,miR-300-5p)may provide new targets for the treatment of facial nerve injury.