基于microRNA-92a-3p靶向SIRT1调控氧糖剥夺再灌注诱导脑微血管内皮细胞炎症反应的作用机制研究

MicroRNA-92a-3p regulates OGD/R-induced inflammatory response via targeting SIRT1 in cerebral microvascular endothelial cells

摘要:

目的 探讨基于microRNA-92a-3p（miR-92a-3p）靶向调控SIRT1氧糖剥夺再灌注（OGD/R）诱导小鼠脑微血管内皮细胞（bEnd.3）炎症反应的作用机制。方法 实时荧光定量聚合酶链反应（qRT-PCR）测定miR-92a-3p、SIRT1、肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）mRNA的表达；Western blotting明确SIRT1、核转录因子-κB（NF-κB）p65、磷酸化NF-κB（p-NF-κB）p65、TNF-α和IL-1β蛋白的表达；双荧光素酶报告基因验证miR-92a-3p与SIRT1靶向关系；划痕实验检测bEnd.3细胞的迁移能力。结果 OGD/R组miR-92a-3p相对表达量较对照组升高（P <0.05）。M_SIRT1 WT+mimics+TK组荧光素酶活性较M_SIRT1 WT+mimics NC+TK组低（P <0.05）。OGD/R组SIRT1蛋白和mRNA相对表达量较对照组降低（P <0.05），OGD+92a抑制剂组较OGD+92a抑制剂内参组升高（P <0.05）。miR-92a-3p模拟物组NF-κBp65/p-NF-κBp65蛋白较模拟物内参组高，SIRT1蛋白和mRNA表达较模拟物内参组低（P <0.05）。miR-92a-3p模拟物组IL-1β、TNF-α蛋白和mRNA相对表达量较模拟物内参组高（P <0.05）。miR-92a-3p模拟物组伤口愈合率较模拟物内参组低（P <0.05）。结论 miR-92a-3p通过靶向抑制SIRT1表达，促进OGD/R诱导的脑微血管内皮细胞炎症反应。

Abstract:

Objective To explore the mechanism of microRNA-92a-3p (miR-92a-3p) on regulating oxygen-glucose deprivation/reperfusion (OGD/R)-induced inflammatory response via targeting silent information regulator 1 (SIRT1) in cerebral microvascular endothelial cells.Methods Real-time fluorescent quantitative-based PCR was applied to detect the expressions of miR-92a-3p and SIRT1, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) mRNA. Western blotting was employed to determine the protein expressions of SIRT1, nuclear factor-κB (NF-κB) p65, p-NF-κB p65, TNF-α and IL-1β. The targeting relationship between miR-92a-3p and SIRT1 was confirmed via dual-luciferase reporter assays. The migratory ability of bEnd.3 cells was determined via scratch assays.Results The relative expression of miR-92a-3p was increased in the OGD/R group compared with the control group (P < 0.05). The luciferase activity of the M_Sirt1 WT + mimics + TK group was lower than that of the M_Sirt1 WT + mimics NC+TK group (P < 0.05). The mRNA and protein expressions of SIRT1 in the OGD/R group were lower than those in the control group (P < 0.05), and they were higher in the OGD + 92a inhibitor group than those in the OGD + 92a control group (P < 0.05). Compared with the miR-92a-3p control group, the protein expressions of NF-κBp65/p-NF-κBp65 as well as the mRNA and protein expressions of IL-1β and TNF-α were higher, and the mRNA and protein expressions of SIRT1 were lower in the miR-92a-3p mimics group (P < 0.05). The wound healing rate in the miR-92a-3p mimics group was higher than that in the miR-92a-3p control group (P < 0.05).Conclusions miR-92a-3p promotes OGD/R-induced inflammatory response in cerebral microvascular endothelial cells via targeting and inhibiting the expression of SIRT1.