BEND6过表达对小鼠神经干细胞基因转录表达的影响

Effect of BEND6 over-expression on transcriptional expression of mouse neural stem cells

目的探究在小鼠神经干细胞(NSCs)中过表达BEND6对其基因转录表达的影响,分析BEND6在小鼠神经发育过程中潜在的生物学功能。方法在贴壁培养的小鼠NSCs中分别瞬时转染pCMV-mScarlet(对照组)和pCMV-mScarlet-BEND6(实验组)质粒,24 h后在显微镜下观察细胞中红色荧光的表达以确认mScarlet和mScarlet-BEND6蛋白的表达。分别提取两组细胞的总RNA,通过全转录组二代测序并对测序数据进行质控、参考基因组比对、定量分析、差异基因表达分析和富集分析,从而表征BEND6过表达对小鼠NSCs基因转录表达的影响及其在小鼠神经发育中的潜在功能。结果成功获取并贴壁培养小鼠NSCs。全转录组测序分析发现,与对照组相比,过表达BEND6的神经干细胞中有165个差异表达基因,其中有102个基因上调,63个基因下调。富集分析显示这些差异表达基因主要与膜电位改变,突触传递,刺激反应,生长发育等生物学过程的调节有关。结论BEND6过表达能够调控小鼠NSCs中的特定基因的转录水平,可能具有调控特定生物学过程及小鼠生长发育的功能。

Objective To investigate the role of BEND6 in regulating gene expression in mouse neural stem cells(NSCs)and to explore its biological function during neural development.Methods pCMV-mScarlet(control group)and pCMV-mScarlet-BEND6(experimental group)plasmids were transiently transfected into mouse NSCs,respectively.After 24 hours,the expression of mScarlet and mScarlet-BEND6 proteins was confirmed by the presence of red fluorescence.Total RNA was then extracted from the two groups of cells for RNA-seq.Through reference genome alignment,quantitative analysis,differential gene expression analysis and enrichment analysis of the sequencing data,we were able to profile the transcriptome of NSCs upon BEND6 overexpression and to suggest a function for mouse neural development.Results Mouse NSCs were successfully isolated for primary culture.Whole transcriptome sequencing analysis revealed 165 differentially expressed genes after force-expressing BEND6,among which 102 genes were up-regulated and 63 genes were down-regulated.Enrichment analysis showed that these differentially expressed genes were highly related to the regulation of membrane potential change,synaptic transmission,stimulation response,growth and development and other biological processes.Conclusions BEND6 regulates the expression of genes related to neuronal functions in mouse NSCs,which suggests a potential mechanism in regulating neuron specific biological processes during development.