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

李建明, 唐亮, 向勤, 杨大为, 项炬

生命科学研究 ›› 2023, Vol. 27 ›› Issue (1) : 1 -8.

非编码RNA专栏

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

李建明, 唐亮, 向勤, 杨大为, 项炬

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

LI Jianming, TANG Liang, XIANG Qin, YANG Dawei, XIANG Ju

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摘要

摘要: 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。

Abstract

Abstract: 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 con-dition 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 interac-tion network was analyzed using STRING database. Under OGD/R condition, the expression of lncRNA BDNF-AS 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 lncRNA BDNF-AS might interact with proteins including UBA52, NKAP, TBK1, RAB1A and RPL38. The-refore, lncRNA BDNF-AS may affect neuronal function by binding autophagy- and apoptosis-associated proteins.