MicroRNA在牛病毒性腹泻病毒感染牛外周血CD14+单核细胞调控抗病毒反应的机制研究

MicroRNAs play important bio-functional roles in the regulation of antiviral innate immunity. However, the regulatory function of microRNA in the infection of bovine viral diarrhea virus (BVDV) on host immune cells remains unknown. Our preliminary analysis using qRT-PCR demonstrated that some microRNAs in bovine peripheral blood CD14+ monocytes were differentially expressed after infected with cytopathic and noncytopathic bovine viral diarrhea virus (BVDV). In the current study, the differential microRNA expression profiles in bovine peripheral blood CD14+ monocytes after infected with cytopathic and noncytopathic BVDV will be comprehensively analyzed by high-throughput sequencing technologies. The activities of immune-regulatory microRNAs to their target genes will be validated by dual-luciferase reporter assay, and the roles of microRNAs in regulation of antiviral reaction in BVDV-infected bovine peripheral blood CD14+ monocytes will be observed via a series of assays including microRNA over-expression, microRNA suppression, antiviral cellular cytokine quantitation, NF-κB and protein kinase PKR activity analysis, antiviral protein Mx detection and virus titer determination. These findings will contribute to valuable targets for new antiviral agent and immunological preparations.

MicroRNA在调控抗病毒固有免疫反应方面具有重要的生物学功能，但目前并不清楚其在牛病毒性腹泻病毒（BVDV）感染宿主免疫细胞过程中的调控机制。本项目在前期利用qRT-PCR分析获得致细胞病变型(CP)和非致细胞病变型(NCP) BVDV感染导致牛外周血CD14+ 单核细胞部分microRNA差异表达信息的基础上，进一步采用高通量测序技术全面分析细胞在CP型和NCP型BVDV感染前后的microRNA差异表达谱，以双荧光素酶报告系统验证免疫调控性microRNA对相关靶基因的作用，进而采用microRNA过表达、microRNA抑制、抗病毒细胞因子定量、NF-κB和PKR活性分析、抗病毒蛋白Mx检测、病毒滴度测定等一系列鉴定技术，研究microRNA在BVDV感染牛外周血CD14+ 单核细胞调控抗病毒反应的作用机制，为新型高效抗病毒制剂及其它免疫制剂的研制提供有价值的靶标分子。

MicroRNA在调控抗病毒固有免疫反应方面具有重要的生物学功能，但目前并不清楚其在牛病毒性腹泻病毒（BVDV）感染宿主免疫细胞过程中的调控机制。本研究以非致细胞病变型（ncp）毒株BVDV-1a GS5和致细胞病变型（cp）BVDV毒株 AV69 分别感染牛外周血CD14+细胞，建立small RNA文库，利用Illumina HiSeq测序平台测序，分析获得CD14ncp和CD14cp细胞的microRNA表达谱，在病毒感染后分别有60种和72种microRNA差异表达。通过对差异microRNA的靶基因进行GO功能富集分析、TargetScan预测,获得bta-miR-452、bta-miR-1296、bta-miR-2331-5p潜在的免疫调控性靶基因。利用双荧光素酶报告基因实验验证bta-miR-1296对靶基因MAVS、STAT1、TEC、RELA、STAT3、STAT5B、STAT6的调控以及bta-miR-452对TRIM2、bta-miR-2331-5p对TRIM11的作用，证实了bta-miR-1296对STAT1基因的抑制作用。bta-miRNA-1296模拟物及抑制剂转染实验表明，在BVDV感染的MDBK细胞bta-miR-1296对STAT1在mRNA水平的抑制作用不显著，但bta-miR-1296抑制剂可显著提高STAT1 mRNA水平、IFN-β mRNA水平以及干扰素诱导基因ISG56的 mRNA水平，并对BVDV复制具有显著抑制作用。本研究从microRNA调控功能的角度，为全面了解宿主抗BVDV反应的调控机制提供了新的思路。