摘要
目的探讨microRNA-34a(miR-34a)对氧化型低密度脂蛋白(ox-LDL)诱导人主动脉内皮细胞(HAEC)凋亡的影响及作用机制。方法ox-LDL处理HAEC,实时荧光定量PCR(qRT-PCR)检测miR-34a的表达水平。Annexin V-FITC/PI双染流式细胞仪(FCM)和Caspase-3活性检测试剂盒检测细胞凋亡率。使用Targetscan等生物信息学软件预测miR-34a的潜在靶基因,双荧光素酶报告基因实验验证miR-34a与Sirt1 mRNA 3′UTR直接靶向结合。结果在ox-LDL诱导HAEC凋亡过程中miR-34a表达逐渐增加。过表达miR-34a促进HAEC凋亡,而抑制miR-34a的表达水平可以部分程度缓解ox-LDL诱导的HAEC凋亡。进一步机制研究发现,Sirt1为miR-34a的下游靶基因,沉默Sirt1基因逆转了miR-34a抑制物(miR-34a inhibitor)的抗凋亡作用。miR-34a过表达可以使乙酰化FoxO1的蛋白表达明显增加,而miR-34a的表达下调可以使乙酰化FoxO1的蛋白表达明显降低。结论下调miR-34a对ox-LDL诱导的HAEC凋亡具有抑制作用,且该作用可能与活化Sirt1/FoxO1通路有关。
Aim To investigate the role of microRNA-34a(miR-34a)in human aortic endothelial cells(HAEC)apoptosis during atherosclerosis(As)and the underlying mechanism.Methods HAEC were treated with oxidized low-density lipoprotein(ox-LDL).The expression level of miR-34a was detected using qRT-PCR.Apoptosis was determined via flow cytometry(FCM)and Caspase-3 activity assay.Prediction of the binding between miR-34a and 3′UTR of Sirt1 mRNA was performed by bioinformatics analysis and confirmed by a dual luciferase reporter assay.Results miR-34a expression was substantially up-regulated during the ox-LDL-elicited apoptosis in HAEC.Forced expression of miR-34a promoted HAEC apoptosis whereas inhibition of miR-34a could partly alleviate apoptotic cell death induced by ox-LDL.Further analysis identified Sirt1 as a direct target of miR-34a,and Sirt1 knockdown abolished the anti-apoptotic effect of miR-34a inhibitor.Moreover,overexpression of miR-34a enhanced the expression of acetylated of FoxO1,downregulation of miR-34a repressed the protein expression of acetylated-FoxO1.Conclusion Down-regulation of miR-34a inhibited HAEC apoptosis by regulating the expression of Sirt1/FoxO1 signaling pathway.
作者
肖丽
刘萍
秦冰
XIAO Li;LIU Ping;QIN Bing(Department of Neurology,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou,Guangdong 510630,China)
出处
《中国动脉硬化杂志》
CAS
2021年第3期193-200,共8页
Chinese Journal of Arteriosclerosis
基金
国家自然科学基金项目(81701167)
广东省自然科学基金项目(2017A030310360)。
