【摘要】 目的探讨miRNA 370在流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)感染神经元细胞过程中的调控作用及其对病毒复制的影响。方法将SH-SY5Y细胞以MOI=1感染JEV,分别在感染后12、24、48h收集细胞,通过计数空斑数量计算病毒滴度,采用2-△△Ct法测定miRNA 370相对表达水平;将SH-SY5Y细胞转染miRNA 370mimics后感染JEV,检测miRNA 370表达变化对病毒复制变化的影响;采用双荧光素酶报告系统验证miRNA 370对其预测靶基因Forkhead box protein O1(FOXO1)的直接靶向性;转染miRNA 370mimics/inhibitors后检测FOXO1表达变化。结果 SH-SY5Y细胞感染JEV后miRNA 370表达显著降低。miRNA 370mimics/inhibators转染后检测显示,miRNA 370能影响JEV的复制水平。FOXO1作为miRNA 370潜在靶基因,在SH-SY5Y细胞感染JEV后表达降低。双荧光素酶报告系统验证,miRNA 370通过直接与FOXO1 3’-UTR区结合发挥作用。miRNA 370mimics/inhibators转染后检测显示,miRNA 370能影响FOXO1的表达和JEV的复制。结论 miRNA 370作为JEV感染的关键分子,在FOXO1相关免疫调节中发挥重要作用,具有潜在的开发应用价值。更多还原

【Abstract】 Objectives To use molecular biology,bioinformatics,and biochemistry to examine the mechanism by which miRNA 370 regulates Japanese encephalitis virus(JEV)infection and replication in neurons. Methods SHSY5 Ycells were infected with JEV at a multiplicity of infection(MOI)of 1.Cells were collected for virus titration as plaque forming units(PFUs)/ml at 12,24,and 48 hafter infection.Total RNA was extracted using Trizol Reagent,and the relative levels of expression of miRNA 370 compared to U6 were determined using qPCR and the 2-△△Ct method with specific his-miRNA 370-5 p primers.miRNA 370 was overexpressed in SH-SY5 Ycells by transfecting 100 pmol of miRNA 370 mimics using Lipofectamine 2 000 according to the manufacturer’s protocol,and the cells were infected with JEV24 hafter transfection.Infected cells and supernatant were harvested at different times after transfection,and then qRTPCR and a plaque forming test were used to detect the levels of virus replication.A dual luciferase reporter assay was performed to confirm whether miR-370 directly targets the 3’UTR of Forkhead boxprotein O1(FOXO1),a key regulator in inflammation and immunity.Expression of FOXO1 mRNA was determined using qPCR after miR-370 mimics or inhibitors were transfected into SH-SY5 Ycells. Results Expression of miRNA 370 in SH-SY5 Ycells decreased during JEV infection.FOXO1,apotential target of miRNA 370,was confirmed to directly target the 3’-UTR of FOXO1 according to a dual luciferase reporter assay.In addition,miRNA 370 affected FOXO1 production in SH-SY5 Ycells during JEV infection. Conclusion When JEV infection progresses,miRNA 370 may act as a suppressive microRNA by targeting FOXO1 and it may be a potential molecular target for novel therapies.更多还原