MiR-222对山羊副流感病毒3型复制的影响

The Molecular Mechanism Study of miR-222 Inhibit Caprine Parainfluenza Virus Type 3 Replication

为探讨miR-222对山羊副流感病毒3型(CPIV3)复制的影响及其机制。本研究将miR-222模拟体转染MDBK细胞,接种CPIV3病毒液,收集上清和细胞,测定病毒效价和干扰素(IFN)含量;通过Target Scan和miRanda等生物信息学软件预测miR-222调控的靶基因,用双荧光素酶报告系统验证其靶基因,RNA干扰技术研究靶基因对CPIV3复制的影响。结果显示,miR-222模拟体能抑制CPIV3复制,转染miR-222组IFN的表达水平较高,且病毒复制效价(10^(2. 5)TCID_(50)·0. 1 mL^(-1))显著低于miRNA阴性对照组(10^(3. 3)TCID_(50)·0. 1 mL^(-1));双荧光素酶报告系统验证和qPCR检测得出,miR-222可靶向结合IRF2的3'-UTR,并降解IRF2的mRNA和抑制IRF2蛋白表达;敲除IRF2的MDBK细胞感染CPIV3后,病毒复制效价(10^(2. 0)TCID_(50)·0. 1 mL^(-1))低于正常MDBK细胞组(10^(3. 2)TCID_(50)·0. 1 mL^(-1))。本研究表明,miR-222通过靶向降解IRF2 mRNA提高IFN表达水平来抑制CPIV3复制,为基于miR-222研制潜在抗病毒药物提供基础资料。

To explore the function of miR-222on effecting the replication of caprine parainfluenza virus type3(CPIV3).The miR-222mimics were synthesized and transfected into the MDBK cells,then infected with CPIV3.Twenty-four hours after infection,the cell cultures were harvested to determine the expression of CPIV3genome and IFN mRNA.The molecular mechanism of effecting CPIV3replication was explored using bioinformatics software,reporter gene system,RT-qPCR,Western blot and RNAi analysis.The results showed that the overexpression of miR-222promote interferon(IFN)production to inhibit CPIV3replication.Prediction the targets of miR-222discovered that interferon regulatory factor2(IRF2)has a putative miR-222target site and miR-222mimics markedly decreased the luciferase level.Furthermore,transfection with miR-222mimics decreased IRF2expression in MDBK cells at both the mRNA and protein levels.Knockdown of miR-222target inhibited CPIV3replication.These findings indicate that overexpression of miR-222promote the host antiviral innate immune response by targeting IRF2.