lncRNA XIST靶向miR-410对谷氨酸诱导视网膜神经细胞损伤的调控机制研究  

lncRNA XIST regulates glutamate-induced retinal neuronal injury via target gene miR-410 and its molecular mechanism

作　　者：王丽纯[1] 舒宝童 马宇[3] 刘意 WANG Lichun;SHU Baotong;MA Yu;LIU Yi(Department of Ophthalmology,the Second People’s Hospital of Zhengzhou,Zhengzhou 450006,HenanProvince,China;Department of Optometry,Henan Medical College,Zhengzhou 451191,Henan Province,China;Department of Ophthalmology,the Fifth Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,Henan Province,China)

机构地区：[1]郑州市第二人民医院眼科,河南省郑州市450006 [2]河南医学高等专科学校眼视光教研室,河南省郑州市451191 [3]郑州大学第五附属医院眼科,河南省郑州市450052

出　　处：《眼科新进展》2020年第10期936-941,共6页Recent Advances in Ophthalmology

摘　　要：目的探讨长链非编码RNA XIST(lncRNA XIST)对谷氨酸(Glu)诱导视网膜神经细胞损伤的分子机制。方法原代培养视网膜神经细胞,Glu处理后建立视网膜神经细胞损伤模型。流式细胞术检测Glu对细胞凋亡能力的影响;使用试剂盒检测Glu对乳酸脱氢酶(LDH)活性的影响;qRT-PCR检测Glu对lncRNA XIST、微小RNA-410(miR-410)表达水平的影响。分别将si-lncRNA XIST、si-con、miR-410模拟物(mimics)、miR-con转染至视网膜神经细胞,检测各组细胞凋亡率及LDH活性。双荧光素酶报告实验验证lncRNA XIST与miR-410的靶向关系;Western blot检测活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved Caspase-3)、活化的含半胱氨酸的天冬氨酸蛋白水解酶9(Cleaved Caspase-9)、核因子E2相关因子2(Nrf-2)、NADH脱氢酶1(NQO1)、血红素氧合酶1(HO-1)表达量。结果与正常培养的视网膜神经细胞比较,Glu诱导后视网膜神经细胞凋亡率、LDH活性及细胞凋亡蛋白Cleaved Caspase-3、Cleaved Caspase-9和lncRNA XIST的表达水平均显著升高,miR-410和Nrf-2、HO-1、NQO1蛋白的表达水平均显著降低,差异均有统计学意义(均为P<0.05)。与阴性转染相比,转染si-lncRNA XIST后细胞凋亡率、LDH活性及Cleaved Caspase-3、Cleaved Caspase-9蛋白相对表达量均显著降低,Nrf-2、HO-1、NQO1蛋白的相对表达量均显著升高,差异均有统计学意义(均为P<0.05)。lncRNA XIST可靶向结合miR-410,并可抑制miR-410的表达(P<0.05);干扰miR-410的表达可逆转沉默lncRNA XIST对Glu诱导的视网膜神经细胞损伤的影响。结论lncRNA XIST通过靶向miR-410促进Glu诱导的视网膜神经细胞损伤,且其可能是通过抑制Nrf-2信号通路活化而发挥作用。Objective To explore the molecular mechanism of long non-coding RNA XIST(lncRNA XIST)on glutamate(Glu)-induced retinal neuronal injury.Methods Primary retinal nerve cells were cultured,and a retinal nerve cell injury model was established after Glu treatment.Flow cytometry was used to detect the effect of Glu on apoptosis.The effect of Glu on lactate dehydrogenase(LDH)activity was examined according to the kit.qRT-PCR was used to detect the effect of Glu on the expression levels of lncRNA XIST and miR-410.The si-lncRNA XIST,si-con,miR-410 mimics,and miR-con were transfected into nerve cells,respectively,and the apoptosis and LDH activity were detected by the above methods.The dual luciferase reporter assay was applied to validate the targeting relationship between lncRNA XIST and miR-410.The expression levels of Cleaved Caspase-3,Cleaved Caspase-9,nuclear factor E2-related factor 2(Nrf-2),NADH dehydrogenase 1(NQO1),and heme oxygenase 1(HO-1)were detected by Western blot.Results Compared with normal cultured retinal nerve cells,after retinal nerve cells were induced by Glu,the apoptosis rate,activity of LDH,the expression levels of apoptosis proteins Cleaved Caspase-3,Cleaved Caspase-9,and lncRNA XIST were significantly increased(all P<0.05),but the expression levels of miR-410,Nrf-2,HO-1 and NQO1 were significantly reduced(all P<0.05).Compared with negative transfection,the apoptosis rate,LDH activity,and the expression levels of Cleaved Caspase-3,Cleaved Caspase-9 were significantly decreased after si-lncRNA XIST transfection(all P<0.05),but the expression levels of Nrf-2,HO-1 and NQO1 were significantly increased(all P<0.05).lncRNA XIST targeted binding to miR-410 and inhibited the expression of miR-410(P<0.05).Interference with the expression of miR-410 reversed the effect of silencing lncRNA XIST on Glu-induced retinal neuronal damage.Conclusion lncRNA XIST promotes Glu-induced retinal neuronal damage by targeting miR-410,and its mechanism may be through inhibition of Nrf-2 signaling pathway activation.

关 键 词：长链非编码RNA XIST 微小RNA-410 谷氨酸 视网膜神经细胞 细胞凋亡 小鼠 

分 类 号：R774[医药卫生—眼科]