微小RNA-145-5p靶向Toll样受体4参与动脉粥样硬化泡沫细胞炎症和氧化应激反应

MiR-145-5p targeting TLR4 participates in inflammation and oxidative stress of atherosclerosis foam cells

目的 探讨微小RNA-145-5p(miR-145-5p)对动脉粥样硬化细胞模型中炎性反应和氧化应激过程的调控作用。方法 体外构建人单核细胞白血病细胞源性泡沫细胞,分为模型组和对照组,采用定量逆转录聚合酶链反应(qRT-PCR)检测细胞内miR-145-5p相对表达。采用TargetScan数据库预测miR-145-5p与Toll样受体4(TLR4)的靶向关系。将293T细胞分为野生型+模拟物(mimic)组(转染TLR4野生型质粒+miR-145-5p模拟物)、野生型+mimic NC组(转染TLR4野生型质粒+miR-145-5p模拟物阴性对照)、突变型+mimic组(转染TLR4突变型质粒+miR-145-5p模拟物)、突变型+mimic NC组(转染TLR4突变型质粒+miR-145-5p模拟物阴性对照),采用双荧光素酶实验验证miR-145-5p与TLR4的靶向关系。体外诱导泡沫细胞,分为mimic组(转染miR-145-5p模拟物)、mimic NC组(转染模拟物阴性对照)、抑制物(inhibitor)组(转染miR-145-5p抑制物)、inhibitor NC组(转染抑制物阴性对照),采用qRT-PCR、Western blot检测TLR4 mRNA及蛋白表达,酶联免疫吸附检测肿瘤坏死因子α(TNF-α)、白细胞介素(IL)1β和IL-6表达,生化相关试剂盒测定活性氧、丙二醛和超氧化物歧化酶(SOD)活性。结果 与对照组比较,模型组miR-145-5p表达明显降低(0.29±0.01 vs 1.00±0.08,t=11.180,P<0.01)。双荧光素酶实验显示,miR-145-5p mimic组荧光素酶活性较mimic NC组显著降低,差异有统计学意义(t=8.612,P<0.01)。与mimic NC组比较,mimic组TLR4 mRNA及蛋白、TNF-α、IL-1β、IL-6、活性氧、丙二醛含量明显降低,miR-145-5p表达、SOD活性明显升高(P<0.05,P<0.01)。与inhibitor NC组比较,inhibitor组TLR4 mRNA及蛋白、TNF-α、IL-1β、IL-6、活性氧、丙二醛含量明显升高,miR-145-5p表达、SOD活性明显降低(P<0.05,P<0.01)。结论 miR-145-5p通过靶向TLR4抑制动脉粥样硬化细胞模型中的炎症和氧化应激反应。

Objective To explore the role of microRNA-145-5p(miR-145-5p)in the regulation of inflammatory response and oxidative stress process in cellular model of atherosclerosis.Methods Human monocytic leukemia THP-1 cells-derived foam cells were constructed in vitro.Then,the relative expression of miR-145-5p in the model and control groups of cells were detected by qRT-PCR.TargetScan database was used to predict the targeting relationship between miR-145-5p and Toll-like receptor 4(TLR4).The 293T cells were divided into wild-type+mimic group,wild-type+mimic negative control(NC)group,and mutant+mimic group,mutant+mimic NC group,and dual luciferase assay was employed to verify the targeting relationship of miR-145-5p and TLR4.Foam cells were cultured in vitro and divided into miR-145-5p mimic group,mimic NC group,miR-145-5p inhibitor group,and inhibitor NC group according to the corresponding treatments.The expression of TLR4 at mRNA and protein levels was detected by qRT-PCR and Wes-tern blotting.The contents of TNF-α,IL-1βand IL-6 were detected by ELISA.Biochemical reagent kits were applied for generation of reactive oxygen species(ROS),content of MDA and activity of SOD.Results The expression of miR-145-5p was significantly reduced in the model group than the control group(0.29±0.01 vs 1.00±0.08,t=11.180,P<0.01).Dual luciferase assay showed that luciferase activity was significantly lower in the miR-145-5p mimic group than the mimic NC group(t=8.612,P<0.01).Compared with the mimic NC group,the mimic group had obviously lower mRNA and protein levels of TLR4 and contents of TNF-α,IL-1β,IL-6,ROS and MDA,and higher miR-145-5p expression level and SOD activity(P<0.05,P<0.01).The treatment of inhibi-tor resulted in increased TLR4 mRNA and protein levels and TNF-α,IL-1β,IL-6,ROS and MDA contents,and decreased miR-145-5p expression and SOD activity when compared with the above levels in the inhibitor NC group(P<0.05,P<0.01).Conclusion MiR-145-5p inhibits inflammation and oxidative stress in cellular model of atherosclerosis by targeting TLR4.