lncRNA BDNF-AS在氧糖剥夺/复氧复糖条件下的表达、定位及互作蛋白质分析

Expression,Localization and Protein-protein Interaction Analysis of lncRNA BDNF-AS Under Oxygen and Glucose Deprivation/Reoxygenation Condition

脑缺血缺氧会引起长链非编码RNA(long non-coding RNA,lncRNA)表达变化。为了探究lncRNA BDNF-AS在氧糖剥夺/复氧复糖条件下的表达、定位及互作蛋白质,将SH-SY5Y细胞缺氧缺糖8 h、复氧复糖24 h,构建氧糖剥夺/复氧复糖细胞模型;采用CCK-8(Cell Counting Kit-8)法检测细胞活力;采用qRT-PCR检测核质中lncRNA BDNF-AS表达水平;利用pull-down和质谱技术对lncRNA BDNF-AS互作蛋白质进行鉴定;利用基因本体论(Gene Ontology,GO)、京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析互作蛋白质的功能及参与的通路;利用STRING数据库分析蛋白质-蛋白质相互作用网络。结果显示:氧糖剥夺/复氧复糖条件下,lncRNA BDNF-AS表达量显著升高,且胞质表达量显著高于胞核;氧糖剥夺/复氧复糖组有120种蛋白质可能与lncRNA BDNF-AS存在潜在的相互作用。进一步的生物信息学分析表明,lncRNA-BDNF-AS可能与UBA52、NKAP、TBK1、RAB1A、RPL38等蛋白质存在互作关系。综上可知,lncRNA BDNF-AS可能通过绑定自噬和凋亡相关蛋白质影响神经细胞功能。

Cerebral ischemia and hypoxia can cause the change of long non-coding RNA(lncRNA)expression.To investigate the expression and localization of lncRNA BDNF-AS and its interacting proteins under the condition of oxygen and glucose deprivation/reoxygenation(OGD/R),SH-SY5Y cells were treated with hypoxia for 8 h and reoxygenation(R)for 24 h to establish a OGD/R cell model.The cell viability was measured by Cell Counting Kit-8(CCK-8)assay.The expression level of lncRNA-BDNF-AS was detected by qRT-PCR in both the nucleus and cytoplasm.The interacting proteins of lncRNA BDNF-AS were analyzed by using pull-down method and mass spectrometry.Functional analysis of interacting proteins was performed using Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.The protein-protein interaction network was analyzed using STRING database.Under OGD/R condition,the expression of lncRNA BDNFAS was significantly increased,and was significantly higher in the cytoplasm than in the nucleus.In the OGD/R group,there were 120 kinds of specific proteins expressed in SH-SY5Y cells.Bioinformatic analysis showed that lncRNABDNF-AS might interact with proteins including UBA52,NKAP,TBK1,RAB1A and RPL38.Therefore,lncRNABDNF-AS mayaffect neuronal functionbybindingautophagy-and apoptosis-associatedproteins.