miR-181b-5p调控TIMP3对apoE-/-鼠动脉粥样硬化炎症影响及相关因子检验

Regulatory of miR-181b-5p Targeted on TIMP3 in apo E-/- Arterial Atherosclerosis Inflammation and Detection of Inflammatory Mediators

为探究mi R-181b-5p靶向调控TIMP3对apo E^-/-鼠动脉粥样硬化炎症的影响,本研究基于生物信息学预测筛选,并采用双荧光素酶报告基因系统验证mi R-181b-5p靶基因。同时在ox-LDL刺激THP-1巨噬细胞及apo E^-/-动脉粥样硬化模型小鼠中检测mi R-181b-5p靶基因m RNA及蛋白水平的表达情况,并且检测转染mi R-181b-5p后细胞水平和动物体内炎症因子TNF-α、INF-γ、IL-1β、IL-18、CCL2的表达变化。此外,通过病理切片方法检测转染mi R-181b-5p后小鼠主动脉窦处病变情况。研究通过双荧光素酶报告基因系统验证TIMP3为mi R-181b-5p靶基因,并在ox-LDL刺激THP-1巨噬细胞及apo E^-/-动脉粥样硬化模型小鼠中证明了mi R-181b-5p可以降低TIMP3转录水平从而降低其蛋白水平表达,同时研究发现mi R-181b-5p在细胞水平和动物体内均会增强炎症因子TNF-α、INF-γ、IL-1β、IL-18、CCL2的表达,并可以增加小鼠主动脉窦处病变面积。研究认为在动脉粥样硬化发生中,mi R-181b-5p可以增加病变面积并且提升相关炎症因子的表达、分泌,其作用机理很可能是通过抑制靶基因TIMP3表达,进而降低TIMP3蛋白水平实现的,为研究动脉粥样硬化机制提供了理论依据。

To explore effect of miR-181 b-5 p targeted regulating on TIMP3 in apo E^-/-arterial atherosclerosis inflammation,the target gene of mi R-181 b-5 p was validated by bioinformatics prediction screening,double luciferase reporter gene system was applied.After that,mi R-181 b-5 p target gene m RNA and protein expression levels were detected in ox-LDL-stimulated THP-1 macrophages and apo E^-/-atherosclerotic mice model.The expression variation of TNF-α,INF-γ,IL-1β,IL-18 and CCL2 in cellular level and in vivo were detected after mi R-181 b-5 p transfection.What＇s more,the lesion in aortic sinus was detected by pathological section after mi R-181 b-5 p transfection.The dual-luciferase reporter system verified mi R-181 b-5 p target gene-TIMP3.It was also demonstrated that mi R-181 b-5 p could degrade TIMP3 mRNA and then reduce protein expression level in ox-LDL-stimulated THP-1 macrophages and apo E^-/-atherosclerotic mice model.miR-181 b-5 p can enhance the expression of TNF-α,INF-γ,IL-1β,IL-18 and CCL2 in the cellular level and in vivo.In addition,mi R-181 b-5 p can enhance the lesion area in mice aortic sinus significantly.In the development of atherosclerosis,mi R-181 b-5 p can increase the lesion area and enhance the secretion of related inflammatory factors.The functional mechanism of mi R-181 b-5 p is likely to be TIMP3 m RNA degradation and TIMP3 protein reduction,which provide theoretical basis for atherosclerosis.