葡萄糖转运促流感病毒复制的机制研究  

Study on the mechanism of glucose transport to promoting influenza virus replication

作　　者：师娟[1] 范砚茹 刘晓丽[1] SHI Juan;FAN Yan-ru;LIU Xiao-li(Department of Laboratory,Henan Provincial People’s Hospital,Zhengzhou 450003,Henan,China)

机构地区：[1]河南省人民医院检验科,河南郑州450003

出　　处：《中国病原生物学杂志》2021年第4期455-460,共6页Journal of Pathogen Biology

摘　　要：目的探讨流感病毒H9N2复制与葡萄糖转运之间的潜在联系。方法 H9N2感染A549细胞后,检测葡萄糖转运对其复制的影响。感染H9N2后高通量测序检测调控此生理过程的潜在miRNA。通过过表达miRNA筛选影响葡萄糖转运的miRNA。miRDB在线分析和荧光素酶报告试验鉴定miRNA底物。结果 H9N2感染A549后,A549细胞葡萄糖转运水平上升(31.2±7.1 vs 123.5±24.9,t=6.138,P<0.05)。葡萄糖转运抑制剂Phloretin处理A549后,H9N2复制水平显著下降(5.2±1.0 vs 1.6±0.2,t=6.035,P<0.05)。葡萄糖转运激动剂Insulin处理A549后,H9N2复制水平显著上升(5.6±0.9 vs 14.3±2.5,t=5.493,P<0.05)。高通量测序显示,H9N2感染后37种miRNA的表达发生显著变化。当敲低miR-345-3p时,A549细胞葡萄糖转运水平显著上升(25.4±5.3 vs 175.2±31.2 t=8.239,P<0.05),H9N2的复制水平显著上升(5.2±0.9 vs 10.6±1.6 t=4.780,P<0.05)。过表达miR-345-3p后,A549细胞葡萄糖转运水平显著下降(26.4±6.1 vs 4.7±0.2,t=6.159,P<0.05),H9N2的复制水平显著下降(5.2±0.9 vs 1.1±0.1,t=7.583,P<0.05)。miRDB在线分析显示,miR-345-3p潜在靶向74个基因。过表达miR-345-3p后,其中14个基因的表达发生6倍以上的变化。当敲低EH结构域蛋白2(EH Domain Containing 2,EHD2)时,H9N2的复制水平显著下降(4.6±0.9 vs 0.5±0.1,t=8.026,P<0.05)。过表达miR-345-3p时,EHD2的表达水平下降;敲低miR-345-3p时,EHD2的表达水平上升。此外,miR-345-3p靶向EHD2的3′端非编码区。过表达miR-345-3p后,H9N2的复制水平显著上升(5.6±0.6 vs 16.3±3.2,t=5.653,P<0.05),葡萄糖转运水平上升(35.1±7.0 vs 154.2±22.2,t=8.928,P<0.05);敲低miR-345-3p后,H9N2的复制水平显著下降(6.0±0.3 vs 0.9±0.0,t=25.78,P<0.05),葡萄糖转运水平下降(42.0±5.1 vs 11.1±3.1,t=9.208,P<0.05)。H9N2感染并敲低miR-345-3p后,EHD2和GLUT4的相互作用减弱。敲低GLUT4后,H9N2的复制水平显著下降(4.8±0.2 vs 0.9±0.1,t=26.90,P<0.05)。H9N2感染并过表达miR-345-3p后,EHD2和GObjective To investigate the potential relationship between the replication of influenza virus H9 N2 and glucose transport. Methods The effect of glucose transport on the replication of A549 cells infected with H9 N2 was determined. Potential miRNAs regulating this physiological process were detected using high-throughput sequencing after H9 N2 infection. Overexpression of miRNAs was used to screen for miRNAs affecting the level of glucose transport. An analysis using miRDB and a luciferase reporting assay were used to identify miRNA substrates. Results After H9 N2 infection, the level of glucose transport in A549 cells increased(31.2±7.1 vs. 123.5±24.9, t=6.138, P<0.05). The level of H9 N2 replication decreased significantly after treatment with the glucose transport inhibitor phloretin(5.2±1.0 vs. 1.6±0.2, t=6.035, P<0.05). The level of H9 N2 replication increased significantly after treatment of A549 cells with insulin(5.6±0.9 vs. 14.3±2.5, t=5.493, P<0.05). High-throughput sequencing indicated that the expression of 37 miRNAs changed significantly after H9 N2 infection. When miR-345-3 p was knocked down, the level of glucose transport in A549 cells increased significantly(25.4±5.3 vs. 175.2±31.2 t=8.239, P<0.05), and the level of H9 N2 replication increased significantly(5.2±0.9 vs. 10.6±1.6 t=4.780, P<0.05). After overexpression of miR-345-3 p, the level of glucose transport in A549 cells decreased significantly(26.4±6.1 vs. 4.7±0.2, t=6.159, P<0.05), and the level of H9 N2 replication decreased significantly(5.2±0.9 vs. 1.1±0.1, t=7.583, P<0.05). Analysis with miRDB indicated that 74 genes were potentially targeted by miR-345-3 p. After the overexpression of miR-345-3 p, the expression of 14 of these genes changed more than 6 times. When the EH Domain Containing 2(EHD2) was knocked down, the level of H9 N2 replication decreased significantly(4.6±0.9 vs. 0.5±0.1, t=8.026, P<0.05). The level of EHD2 expression decreased when miR-345-3 p was overexpressed. When miR-345-3 p was knocked down, the

关 键 词：流感病毒 H9N2 miR-345-3p EHD2 葡萄糖转运 

分 类 号：R373.13[医药卫生—病原生物学]