miR-301a调节小鼠巨噬细胞中炎症因子的表达

MiR-301a regulates the expression of inflammatory factors in macrophages

目的探讨miR-301a对巨噬细胞炎症因子表达水平的影响,从microRNA角度阐明动脉粥样硬化的发病机制,为动脉粥样硬化的防治提供新的思路。方法高脂喂养Apo E-/-小鼠建立动脉粥样硬化模型,收集小鼠主动脉血管,利用real-time PCR检测组织中miR-301a的表达水平;利用脂质体在RAW264.7细胞中转染miR-301a mimic和miR-301a inhibitor,用real-time PCR检测细胞中miR-301a水平,并检测肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)和单核细胞趋化蛋白1(MCP-1)的表达水平,用Western blot检测NF-κB抑制因子(NKRF)蛋白水平,用免疫荧光染色检测p65细胞定位。结果在高脂喂养的Apo E-/-小鼠主动脉血管壁组织中miR-301a的表达水平升高;在RAW264.7细胞中过表达miR-301a可抑制NKRF蛋白水平,促进p65细胞核定位,升高细胞中TNF-α、IL-6和MCP-1的mRNA表达;在RAW264.7细胞中低表达miR-301a可升高NKRF蛋白水平,促进p65细胞质定位,减少细胞中TNF-α、IL-6和MCP-1的mRNA表达。结论在高脂喂养的Apo E-/-小鼠主动脉血管壁组织中miR-301a表达水平升高;在RAW264.7细胞中miR-301a通过调节NKRF的表达影响p65活性进而调控TNF-α、IL-6和MCP-1的mRNA表达,提示microRNA在动脉粥样硬化发病的炎症机制中具有重要作用。

Aim To explore the effect of miR-301a on the expression of inflammatory factors in macrophages, from the perspective of microRNA to clarify the pathogenesis of atherosclerosis, then provide a new way of prevention and treatment of atherosclerosis. Methods ApoE^-/- mice was fed with high fat diet to establish atherosclerosis model, the expression of inflammatory factors in the atery were analyzed by real-time PCR. MiR-301a mimic and miR-301a inhibitor were transfected into RAW264.7 cells, the expression of inflammatory factors was analyzed by real-time PCR, the protein level of NF-κB repressing factor （NKRF） was measured by Western blot, the cellular location of p65 was determined by immunofluorescence. Results The level of miR-301a was increased in ateries of ApoE^-/- mice. Overexpression of miR-301a decreased the protein levels of NKRF and increased the mRNA levels of inflammatory factors, p65 was located in nuleus in the RAW264.7 cells transfected with miR-301a mimic. Inhibition of miR-301a increased the protein levels of NKRF and decreased the mRNA levels of inflammatory factors, p65 was located in cytoplasm in the RAW264.7 cells transfected with miR-301a inhibitor. Conclusion MiR-301a regulates the mRNA expression of TNF-α, IL-6 and MCP-1 in RAW264.7 cells via targeting NKRF to regulate the cellular location of p65.