miR-146a-3p靶向RELB调控动脉粥样硬化小鼠内皮细胞的细胞因子分泌

miR-146a-3p targets RELB regulates cytokine secretion in endothelial cells of atherosclerotic mice

目的:探讨miR-146a-3p如何通过核因子κB亚基(RELB)调控动脉粥样硬化小鼠内皮细胞的细胞因子分泌。方法:高脂配方饲料饲养方案建立载脂蛋白敲除(ApoE-/-)小鼠动脉粥样硬化模型并进行血管内皮细胞的原代培养;通过实时荧光定量PCR检测动脉粥样硬化小鼠内皮组织与对照小鼠内皮组织中miR-146a-3p的表达量差异;通过TargetScanHuman分析miR-146a-3p与RELB的匹配情况,然后通过荧光素酶报告系统检测miR-146a-3p是否靶向RELB;在miR-146a-3p mimics过表达或者miR-146a-3p inhibitor敲低的情况下,通过酶联免疫吸附试验检测集落刺激因子(GCSF)、粒细胞巨噬细胞集落刺激因子(GM-CSF)、白介素-2(IL-2)、白介素-3(IL-3)、白介素-4(IL-4)、白介素-5(IL-5)、白介素-6(IL-6)、白介素-9(IL-9)、白介素-10(IL-10)、白介素-12 p40(IL-12 p40)、白介素-12 p70(IL-12 p70)、白介素-13(IL-13)、白介素-17(IL-17)、干扰素-γ(IFN-γ)、单核细胞趋化蛋白-1(MCP-1)、单核细胞趋化蛋白-5(MCP-5)、小诱导性细胞因子A5(RANTES)、肿瘤坏死因子-α(TNF-α)。结果:相比于对照组,动脉粥样硬化小鼠血管内皮组织中miR-146a-3p的表达量降低(P<0.05);miR-146a-3p靶向RELB的3'UTR;过表达miR-520a-3p后,RELB的表达水平下降,动脉粥样硬化小鼠内皮细胞分泌的细胞因子GCSF、GM-CSF、IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12 p40、IL-12 p70、IL-13、IL-17、IFN-γ、MCP-1、MCP-5、RANTES、TNF-α水平下降(P<0.05);敲低miR-520a-3p后,RELB的表达水平上升,动脉粥样硬化小鼠内皮细胞分泌的细胞因子GCSF、GM-CSF、IL-2、IL-3、IL-4、IL-5、IL-6、IL-9、IL-10、IL-12 p40、IL-12 p70、IL-13、IL-17、IFN-γ、MCP-1、MCP-5、RANTES、TNF-α水平上升(P<0.05)。结论:miR-146a-3p通过靶向核因子κB(NF-κB)信号通路的关键分子RELB抑制动脉粥样硬化小鼠血管内皮细胞的细胞因子分泌。

Objective To investigate how miR-146a-3p regulates cytokine secretion in endothelial cells of atherosclerotic mice via NF-KB Subunit RELB.Methods ApoE-/-mouse atherosclerosis model was established and primary culture of vascular endothelial cells was established.Real-time fluorescent quantitative PCR was used to detect endothelial tissue of atherosclerotic mice and endothelial tissue of control mice as well as the difference in expression of miR-146a-3p;analysis of miR-146a-3p binding to subunit RELB of NF-kB complex by TargetScanHuman,followed by detection of miR-146a-3p targeting RELB by luciferase reporter system.In the case of miR-146a-3p mimics overexpression or miR-146a-3p inhibitor knockdown,colony stimulating factor(GCSF),granulocyte-macrophage colony stimulating factor(GM-CSF),interleukin 2(IL-2),interleukin 3(IL-3),interleukin 4(IL-4),interleukin 5(IL-5),interleukin 6(IL-6),interleukin 19(IL-9),interleukin 10(IL-10),interleukin 12 p40(IL-12 p40),interleukin 12 p70(IL-12 p70),interleukin 13(IL-13),interleukin 17(IL-17),interferon gamma(IFN-γ),monocyte chemoattractant protein-1(MCP-1),monocyte chemoattractant protein-5(MCP-5),small inducible cytokine A5(RANTES),and tumor necrosis factor-alpha(TNFα)were detected by enzyme-linked immunosorbent assay.Results Compared with the control group,the expression of miR-146a-3p was decreased in vascular endothelial cells of atherosclerotic mice(P<0.05);miR-146a-3p was targeted to the 3'UTR of RELB;After overexpression of miR-520a-3p,the expression level of RELB decreased,and the cytokines secreted by endothelial cells of atherosclerotic mice were GCSF,GM-CSF,IL-2,IL-3,IL-4,IL-5,IL-6,IL-9,IL-10,IL-12 p40,IL-12 p70,IL-13,IL-17,IFN-γ,MCP-1,MCP-5,RANTES,TNFαlevels decreased(P<0.05);After the knockdown of miR-520a-3p,the expression level of RELB increased,and the cytokines secreted by endothelial cells of atherosclerotic mice including GCSF,GM-CSF,IL-2,IL-3,IL-4,IL-5,IL-6,IL-9,IL-10,IL-12 p40,IL-12 p70,IL-13,IL-17,IFN-γ,MCP-1,MCP-5,RANTES,TNFαlevels increased(P<0.05).Conclusion miR-146a-3p inhibits cytokine secretion from vascular endothelial cells in atherosclerotic mice by targeting RELB,a key molecule of the NF-kB signaling pathway.