miR-124-3p通过靶向Rab11a缓解创伤性脑损伤引起的氧化应激和炎性反应

Mechanism of miR-124-3p targeting Rab11a to alleviate oxidative stress and inflammatory response induced by traumatic brain injury

目的探讨微小RNA-124-3p(miR-124-3p)/脑Ras相关蛋白11a(Rab11a)分子轴在创伤性脑损伤(TBI)引起的氧化应激和炎性反应中的作用。方法采用过氧化氢(H2O2)处理小鼠海马神经元(HT22)细胞构建TBI细胞模型,实时荧光定量PCR(RT-qPCR)检测miR-124-3p和含半胱氨酸的天冬氨酸蛋白水解酶1(caspase 1)、白细胞介素1β(IL-1β)、白细胞介素18(IL-18)及肿瘤坏死因子α(TNF-α)的表达水平;Western blot检测硒蛋白N1(SEPN1)、谷胱甘肽过氧化物酶1(GPX1)和诱导型一氧化氮合酶(iNOS)蛋白的表达水平;双荧光素酶报告基因试验验证miR-124-3p和Rab11a之间的靶向调控关系;活性氧(ROS)检测试剂盒检测ROS水平。结果miR-124-3p在H2O2处理的HT22细胞中低表达(P<0.05);过表达miR-124-3p明显抑制H2O2诱导HT22细胞SEPN1、GPX1蛋白表达水平的下调(P<0.05)和iNOS表达水平的上调(P<0.05),以及炎症因子caspase 1、IL-1β、IL-18表达水平的上调(P<0.05);双荧光素酶报告基因证实miR-124-3p靶向作用Rab11a并下调其表达(P<0.05);实验进一步证实,与敲降Rab11a比较,同时敲降miR-124-3p和Rab11a可明显下调单独敲降Rab11a对HT22细胞氧化应激和炎性反应的抑制作用(P<0.05)。结论miR-124-3p通过靶向下调Rab11a抑制H2O2诱导HT22细胞氧化应激和炎性反应。

Objective To explore the effect of microRNA-124-3p(miR-124-3p)/brain Ras-related proteins 11a(Rab11a)molecular axis on oxidative stress and inflammatory response induced by traumatic brain injury(TBI).Methods Construction of TBI cell model by treatment of mouse hippocampal neuron(HT22)cells with hydrogen peroxide(H2O2).The expression of miR-124-3p and cysteiny l aspartate specific proteinase 1(caspase 1),interleukin-1β(IL-1β),interleukin-18(IL-18)and tumour necrosis factorα(TNF-α)expression were detected by real-time fluorescence quantitative PCR(RT-qPCR).The expressions of selenoprotein N1(SEPN1),glutathione peroxidase 1(GPX1)and inducible nitric oxide synthase(iNOS)in HT22 cells were detected by Western blot.Dual luciferase reporter assay validated the targeted regulatory relationship between miR-124-3p and Rab11a.The reactive oxygen species(ROS)level was detected by ROS detection kit.Results Low expression of miR-124-3p was observed in H2O2-treated HT22 cells(P<0.05).Over expression of miR-124-3p significantly inhibited the down-regulation of SEPN1 and GPX1,and up-regulation of iNOS expression,also up-regulation of inflammatory factors caspase 1,IL-1βand IL-18 induced by H2O2 treatment of HT22 cells(P<0.05).Dual luciferase reporter gene confirmed that miR-124-3p targeted Rab11a and down-regulated its expression(P<0.05).The experiment further confirmed that down-regulation of miR-124-3p and Rab11a simultaneously significantly down-regulated the inhibitory effect of sh-Rab11a on oxidative stress and inflammatory response in HT22 cells compared with down-regulated Rab11a alone(P<0.05).Conclusion miR-124-3p inhibits H2O2-induced oxidative stress and inflammatory response in HT22 cells by down-regulating Rab11a.