lncRNA NEAT1调节miR-424-5p/ELK4轴对ox-LDL诱导的血管内皮细胞损伤、Lp-PLA2和CRP水平的影响

Effect of lncRNA NEAT1 on ox-LDL-induced Vascular Endothelial Cell Injury,Lp-PLA2 and CRP Levels by Regulating miR-424-5p/ELK4 Axis

目的:探讨长链非编码RNA核旁斑组装转录本1(lncRNA NEAT1)在氧化型低密度脂蛋白(ox-LDL)诱导的血管内皮细胞损伤中的分子机制和功能。方法:收集健康人和动脉粥样硬化(AS)病人血液标本并通过实时荧光定量逆转录聚合酶链式反应(qRT-PCR)检测血清中lncRNA NEAT1、微小RNA-424-5p(miR-424-5p)和ETS域蛋白4(ELK4)mRNA表达水平。体外培养人脐静脉内皮细胞(HUVEC),qRT-PCR和蛋白免疫印迹法(Western Blot)检测细胞中lncRNA NEAT1、miR-424-5p和ELK4表达情况;细胞计数试剂盒(CCK-8)法和膜联蛋白V-异硫氰酸荧光素/碘化丙啶(Annexin V-FITC/PI)法检测HUVEC细胞增殖活力和凋亡情况;酶联免疫吸附法(ELISA)测定HUVEC中乳酸脱氢酶(LDH)释放量、肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-1β含量、脂蛋白磷脂酶A2(Lp-PLA2)和C反应蛋白(CRP)水平;采用双荧光素酶报告基因测定lncRNA NEAT1、miR-424-5p和ELK4之间的靶向相互作用。进行动物实验以评估lncRNA NEAT1在体内AS进展中的作用。结果:lncRNA NEAT1在AS病人血清和ox-LDL诱导的HUVEC中显著上调(P<0.05)。lncRNA NEAT1的沉默可削弱ox-LDL引发的细胞毒性,降低Lp-PLA2和CRP水平,并减少ApoE^(-/-)小鼠的脂质异常分泌(P<0.05)。miR-424-5p是lncRNA NEAT1在调节ox-LDL诱导的HUVEC损伤中的功能介质,ELK4是miR-424-5p的直接靶标(P<0.05)。miR-424-5p的抑制或ELK4的过表达逆转了lncRNA NEAT1沉默对ox-LDL诱导的HUVEC细胞损伤的影响(P<0.05)。结论:沉默lncRNA NEAT1可通过调控miR-424-5p/ELK4轴保护HUVEC免受ox-LDL触发的细胞毒性,降低Lp-PLA2和CRP水平。

Objective:To investigate the molecular mechanism and function of long non-coding RNA nuclear paraspeckle assembly transcript 1(lncRNA NEAT1)in oxidative low-density lipoprotein(ox-LDL)-induced vascular endothelial cell injury.Methods:Blood samples of healthy people and atherosclerosis(AS)patients were collected and serum lncRNA NEAT1,micrornA-424-5p(miR-424-5p)and ETS domain protein 4(ELK4)mRNA expression levels were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction(qRT-PCR).Human umbilical vein endothelial cells(HUVEC)were cultured in vitro,and the expressions of lncRNA NEAT1,miR-424-5p and ELK4 were detected by qRT-PCR and Western Blot.Cell counting kit(CCK-8)and Annexin V-FITC/PI method were used to detect the proliferation and apoptosis of HUVEC cells.Lactate dehydrogenase(LDH)release,tumor necrosis factor-α(TNF-α)and interleukin-1βcontents,lipoprotein phospholipase A2(Lp-PLA2)and C-reactive protein(CRP)levels in HUVEC were determined by enzyme-linked immunosorbent assay(ELISA).Targeting interactions between lncRNA NEAT1,miR-424-5p and ELK4 were determined by dual luciferase reporter genes.Animal experiments were performed to evaluate the role of lncRNA NEAT1 in the progression of AS in vivo.Results:lncRNA NEAT1 was significantly up-regulated in AS patients'serum and ox-LDL-induced HUVECs(P<0.05).Silencing of lncRNA NEAT1 attenuated ox-LDL-induced cytotoxicity,reduced Lp-PLA2 and CRP levels,and reduced abnormal lipid secretion in ApoE^(-/-) mice(P<0.05).miR-424-5p mediated lncRNA NEAT1 in regulating ox-LDL-induced HUVEC injury,and ELK4 was the direct target of miR-424-5p(P<0.05).Inhibition of miR-424-5p or overexpression of ELK4 reversed the effect of lncRNA NEAT1 silencing on ox-LDL-induced HUVEC cell damage(P<0.05).Conclusion:Silencing lncRNA NEAT1 protects HUVEC from ox-LDL-triggered cytotoxicity and reduces Lp-PLA2 and CRP levels,in part by regulating the miR-424-5p/ELK4 axis.