miR-34a-5p在ox-LDL引起内皮细胞损伤中的作用机制

Role and mechanism of miR-34a-5p in oxLDL-induced endothelial cell injury

目的研究miR-34a-5p在氧化型低密度脂蛋白(ox-LDL)引起内皮细胞损伤中的作用及机制。方法培养人脐静脉内皮细胞(HUVEC),分为对照组、ox-LDL组、阴性对照(NC)组、NC+ox-LDL组、miR-34a-5p敲低+ox-LDL组,采用荧光定量PCR检测miR-34a-5p的表达水平,采用western blot检测沉默信息调节因子1(Sirt1)、p53、核因子κB(NF-κB)的表达水平,采用双荧光素酶报告基因实验验证miR-34a-5p靶向Sirt1,采用试剂盒检测细胞活力A_(490)水平及凋亡率,采用酶联免疫吸附试验(ELISA)法检测一氧化氮(NO)、内皮型一氧化氮合酶(eNOS)、肿瘤坏死因子(TNF-α)、白细胞介素-6(IL-6)。结果ox-LDL组中miR-34a-5p、p53、NF-κB的表达水平高于对照组,Sirt1的表达水平低于对照组(P<0.05);Sirt1基因mRNA 3′UTR中含有miR-34a-5p的互补结合位点,miR-34a-5p组Sirt1的荧光素酶活性低于NC组(P<0.05);NC+ox-LDL组的A_(490)水平、Sirt1、NO、eNOS的表达水平低于NC组,凋亡率、miR-34a-5p、p53、NF-κB、TNF-α、IL-6的表达水平高于NC组(P<0.05);miR-34a-5p敲低+ox-LDL组的A_(490)水平、Sirt1、NO、eNOS的表达水平高于NC+ox-LDL组,凋亡率、miR-34a-5p、p53、NF-κB、TNF-α、IL-6的表达水平低于NC+ox-LDL组(P<0.05)。结论ox-LDL通过上调miR-34a-5p,靶向Sirt1引起内皮细胞损伤。

Objective To study the role and mechanism of miR-34a-5p in oxidative low-density lipoprotein(oxLDL)induced endothelial cell injury.Methods Human umbilical vein endothelial cells(HUVEC)were cultured and divided into control group,oxLDL group,negative control(NC)group,NC+oxLDL group,miR-34a-5p knockdown+oxLDL group.The expression level of miR-34a-5p was detected by fluorescent quantitative PCR;western blot was used to detect the expression levels of silent information regulator sirtuin 1(Sirt1),p53 and nuclear factor kappa-B(NF-κB);dual luciferase reporter gene assay was used to validate miR-34a-5p targeting Sirt1;cell viability and apoptosis rate of A_(490) were detected by the kit;nitric oxide(NO),endothelial nitric oxide synthase(eNOS),tumor necrosis factor(TNF-α)and interleukin 6(IL-6)were detected by enzyme-linked immunosorbent assay(ELISA).Results The expression levels of miR-34a-5p,p53,NF-κB in oxLDL group were higher than those in control group,and the expression level of Sirt1 was lower than that in control group(P<0.05);The 3'UTR of Sirt1 gene mRNA contained a complementary binding site of miR-34a-5p;the luciferase activity of Sirt1 in miR-34a-5p group was lower than that in NC group(P<0.05).The level of A_(490),the expression levels of Sirt1,NO and eNOS in NC+oxLDL group were lower than those in NC group,while the apoptosis rate,the expression level of miR-34a-5p,p53,NF-κB,TNF-α,IL-6 was higher than those of NC group(P<0.05).The level of A_(490),the expression levels of Sirt1,NO and eNOS in miR-34a-5p knockdown+oxLDL group were lower than those in NC+oxLDL group,while the apoptosis rate,the expression level of miR-34a-5p,p53,NF-κB,TNF-α,IL-6 was higher than those of NC+oxLDL group(P<0.05).Conclusion oxLDL induces endothelial cell damage by up-regulating miR-34a-5p and targeting Sirt1.