miR-579-3p/DEFA3调控脓毒血症的炎症反应及靶向关系验证

miR-579-3p/DEFA3 regulated inflammatory response of sepsis and verified targeting relationship

目的:研究miR-579-3p对脓毒血症小鼠炎症因子分泌和内皮细胞焦亡的影响机制。方法:基于过表达miR-579-3p转基因小鼠和敲减α-防御素3(DEFA3)基因的转基因小鼠构建脓毒血症小鼠模型;qRT-PCR检测小鼠血浆中miR-579-3p表达;ELISA检测小鼠血清中TNF-α、IL-8和IL-6的表达;流式细胞术检测小鼠内皮细胞焦亡;Western blot检测小鼠血浆中DEFA3蛋白表达;双荧光素酶报告基因实验检测miR-579-3p与DEFA3的结合力。结果:miR-579-3p、DEFA3在脓毒血症小鼠血浆中的表达水平均显著升高(P<0.05)。过表达miR-579-3p转基因脓毒血症小鼠和敲减DEFA3基因的转基因脓毒血症小鼠血清中炎症因子TNF-α、IL-8和IL-6的表达均明显降低,内皮细胞焦亡率明显降低(P<0.05)。miR-579-3p与DEFA3存在靶向作用。结论:miR-579-3p可抑制脓毒血症小鼠的炎症因子分泌和内皮细胞焦亡,其机制与靶向DEFA3相关,可为脓毒血症的治疗提供靶标。

Objective:To study the effect of miR-579-3p on inflammatory factor secretion and endothelial cell pyroptosis in sepsis mice.Methods:Sepsis mice model were constructed based on transgenic mice that overexpressed miR-579-3p and knocked down the DEFA3 gene. qRT-PCR was used to detect the expression of miR-579-3p in mice plasma;ELISA was used to detect the expressions of TNF-α,IL-8 and IL-6 in mice serum;Flow cytometry was used to detect the pyroptosis of mice endothelial cells;Western blot was used to detect the protein expression of DEFA3 in mice plasma;dual fluorescence enzymatic reporter assay was used to detect the binding of miR-579-3p to DEFA3.Results:The expression levels of miR-579-3p,DEFA3 in plasma of sepsis mice increased significantly(P<0.05). The expressions of inflammatory factors TNF-α,IL-8 and IL-6 in serum,endothelial cell pyroptosis rate were significantly reduced of miR-579-3p transgenic sepsis mice and DEFA3 gene transgenic sepsis mice. miR-579-3p had a targeting effect on DEFA3.Conclusion:miR-579-3p can inhibit the secretion of inflammatory factors and endothelial cell pyroptosis in sepsis mice,whose mechanism is related to targeting DEFA3 and will provide targets for the treatment of sepsis.