LINC00894 regulated cerebral ischemia/reperfusion injury by stabilizing EIF5 and facilitating ATF4-mediated induction of FGF21 and ACOD1

Long intergenic non-coding RNA 894 (LINC00894) modulates cancer cell proliferation and drug resistance in several tissue types. However, its role in brain is still unclear. Using RNA-pull down combined with mass spectrometry and RNA binding protein immunoprecipitation, EIF5 was identified to physically interact with LINC00894. Furthermore, LINC00894 knockdown decreased EIF5 protein expression, whereas LINC00894 overexpression increased EIF5 protein expression in cultured cells. Additionally, LINC00894 affected the ubiquitination modification of EIF5. Adeno-associated virus (AAV) mediated LINC00894 overexpression in the brains of transient middle cerebral artery occlusion reperfusion (MCAO/R) mice and rats and inhibited the expression of activated caspase-3. Meanwhile, LINC00894 knockdown increased in the oxygen–glucose deprivation and reoxygenation (OGD/R) in vitro models, whereas its overexpression decreased apoptotic cells and expression of activated caspase-3. Further, LINC00894 overexpression was revealed to increase, whereas its knockdown shortened ATF4 protein half-life. LINC00894 knockdown also decreased the expression of glutamate-cysteine ligase catalytic subunit (GCLC) and ATF4, downregulated glutathione (GSH), and the ratio of GSH to oxidized GSH (GSH: GSSG) in vitro. RNA-seq analysis combined with qRT-PCR and immunoblot showed overexpression of LINC0084 upregulated ATF4, thereby potentially targeting fibroblast growth factor 21 (FGF21) and aconitate decarboxylase 1 (ACOD1) in the MCAO/R model. Finally, we revealed that ATF4 regulated FGF21 and ACOD1 expression by directly binding to promoters; and ectopic overexpression of FGF21 or ACOD1 in LINC00894 knockdown cells decreased activated caspase-3 expression in the OGD/R model. Our results indicated that LINC00894 regulated cerebral ischemia injury by stabilizing EIF5 and facilitating EIF5-ATF4-dependent induction of FGF21 and ACOD1长链基因间非编码RNA 894（LINC00894）可调控多种组织类型中癌细胞的增殖与耐药性，但其在脑组织中的功能仍未明确。本研究通过RNA下拉（RNA-pull down）联合质谱分析（mass spectrometry）以及RNA结合蛋白免疫沉淀（RNA binding protein immunoprecipitation）实验，鉴定出真核翻译起始因子5（EIF5）可与LINC00894发生物理相互作用。进一步研究发现，在培养细胞中，敲低LINC00894会降低EIF5的蛋白表达水平，而过表达LINC00894则可上调EIF5的蛋白表达；此外，LINC00894还可影响EIF5的泛素化修饰。通过腺相关病毒（Adeno-associated virus，AAV）介导LINC00894在短暂性大脑中动脉闭塞再灌注（transient middle cerebral artery occlusion reperfusion，MCAO/R）模型小鼠和大鼠的脑组织中过表达，可抑制活化半胱天冬酶-3（activated caspase-3）的表达。与此同时，在氧糖剥夺复氧（oxygen–glucose deprivation and reoxygenation，OGD/R）体外模型中，敲低LINC00894会加重细胞损伤，而过表达LINC00894则可减少凋亡细胞数量并降低活化半胱天冬酶-3的表达。进一步研究显示，LINC00894过表达会延长激活转录因子4（ATF4）的蛋白半衰期，而敲低LINC00894则会缩短其半衰期。在体外实验中，敲低LINC00894还会降低谷氨酸-半胱氨酸连接酶催化亚基（GCLC）与ATF4的表达，下调谷胱甘肽（GSH）水平以及GSH与氧化型谷胱甘肽（GSSG）的比值。通过RNA测序（RNA-seq）联合实时荧光定量PCR（qRT-PCR）与免疫印迹实验分析显示，在MCAO/R模型中，过表达LINC00894可上调ATF4的表达，进而可能靶向调控成纤维细胞生长因子21（FGF21）与顺乌头酸脱羧酶1（ACOD1）的表达。最后，本研究证实ATF4可通过直接结合启动子区域调控FGF21与ACOD1的表达；在敲低LINC00894的细胞中异位过表达FGF21或ACOD1，可降低OGD/R模型中活化半胱天冬酶-3的表达水平。本研究结果表明，LINC00894可通过稳定EIF5并促进EIF5-ATF4依赖性的FGF21与ACOD1诱导表达，从而调控脑缺血损伤。