MiR-222调控Ⅰ型干扰素影响山羊副流感病毒3型复制的分子机制研究

Caprine parainfluenza virus type 3 (CPIV3) is one of the most important viral respiratory pathogens, which was first isolated from goats suffered respiratory diseases and evaluated the pathogenicity and horizontal transmission ability by our team. MicroRNAs (miRNAs) are one class of non-coding RNAs of lengths ranging from 19-24 nucleotides (nt) that play critical roles in a wide variety of biological processes. We have confirmed that miR-222 was significantly down-regulated in CPIV3 infected cells, and the ectopic expression of miR-222 regulated CPIV3 replication. Functional over-expression of miR-222 promoted typeⅠinterferons expression. However, the molecular mechanism of typeⅠinterferons suppress CPIV3 replication are unknown. In this study, miRNA pull-down assay will be performed to determine the target genes related with miR-222. The 3′ UTR reporter analysis and RNAi silencing technique will be used to identify target genes related miR-222. In addition, over-expression or decreased-expression of targets gene and miR-222 in cells, inoculation of goats with miR-222 agomir or antagomir will be done to analyze the innate immunity response. In addition, quantitative RT-PCR and western blotting, etc, are used to revealed molecular mechanisms of CPIV3 replication regulated by miR-222. Moreover, these results would be to reveal the related molecular mechanisms of CPIV3 replication regulated by miR-222, and also provide crucial scientific information and new strategies for prevention and control of CPIV3.

山羊副流感病毒3型（CPIV3）为课题组首次报道和研究的重要呼吸道病原，其致病性和传播力均较强。microRNA（miRNA）是存在于细胞内的非编码小RNA，在调控细胞和病毒的基因表达中发挥重要作用。项目申请者前期研究发现，在CPIV3感染的细胞中miR-222显著下调，过表达miR-222抑制CPIV3增殖，且miR-222参与调控Ⅰ型干扰素表达，但其作用机制尚不清楚。为此，本项目拟使用miRNA pull-down筛选miR-222调控Ⅰ型干扰素通路相关的靶基因，双荧光素酶报告系统和RNAi技术进行验证后，在细胞中过表达或抑制表达靶基因及miR-222，动物体内导入miR-222激动剂或拮抗剂，采用荧光定量RT-PCR和Western blotting等技术，研究分析miR-222调控Ⅰ型干扰素影响CPIV3复制的分子机制，以期为基于miR-222研制潜在抗病毒药物提供基础资料。

山羊副流感病毒3型（CPIV3）是影响养羊业的重要呼吸道病原，在我国羊群中广泛流行，感染率为30%-50%，但目前尚无疫苗和药物防治。微小核糖核酸（miRNA）是存在于细胞内的非编码RNA，在宿主细胞和病毒基因组表达调控途径中发挥关键作用。本项目通过高通量测序发现bta-miR-222在CPIV3感染的细胞中持续下调，过表达/抑制表达bta-miR-222能调控IFN-I的表达，影响CPIV3复制。靶基因预测和验证发现，bta-miR-222能显著降低IRF2的转录和表达量，提升IFN-I的表达水平，且siRNA敲减IRF2的MDBK细胞能显著抑制CPIV3复制。用慢病毒构建bta-miR-222稳定表达的细胞株能持续有效的抑制CPIV3增殖，进一步证实了bta-miR-222体外抑制CPIV3的作用。体内试验发现，将bta-miR-222肌肉注射豚鼠体内，qRT-PCR揭示该miRNA能在肺脏中过表达，攻毒后发现，给药组豚鼠临床症状减轻，肺部病变减少，肺脏和气管的CPIV3拷贝数显著低于对照组。 本项目揭示了bta-miR-222体内外抑制CPIV3增殖的作用，阐明了其分子机制，为基于bta-miR-222研制潜在抗病毒药物提供基础资料。