miR-1249在BVDV诱导MDBK细胞凋亡与持续性感染发生过程中的机制研究

Mechanisms of persistent infection induced by bovine viral diarrhea virus (BVDV) is essential for the prevention and treatment of Mucosal disease. The pathogenic mechanisms are concerned, but it is not yet clear. Indeed, some research have reported that host-cellular miRNAs, modulating the expression of various viral genes, play an important role in the host-pathogen interaction network. miRNA become possibly a breakthrough for elucidating mechanisms of BVDV infection. Our previous studies showed that miR-1249 was up-regulated significantly. miR-1249, as class of modulators, may play an important role in the regulation of cell apoptosis pathway which is involved in BVDV persistent infection. To investigate the regulatory role of miR-1249 in BVDV infection and the underlying mechanisms, total RNAs from cpBVDV-infected MDBK cells and ncpBVDV-infected MDBK cells at different time points were extracted and then subjected to Transcriptome Resequencing. Target genes of miR-1249 will be predicted by using gene array technique and target-predicting software such as TargetScan. Target genes of miR-1249 will be validated by using luciferase reporter system. Regulatory role of miRNAs will be investigated in MDBK apoptosis and persistent infection induced by BVDV using RT-PCR and Flow cytometry technology. The study will determined the role of miRNA in MDBK apoptosis and persistent infection by BVDV, which lay a foundation for developing novel anti-BVDV therapeutic strategies.

牛病毒性腹泻病毒（BVDV）的持续性感染对于该病的防治至关重要，但致病机制目前尚不清楚。miRNA已被证实在病毒感染过程中起到重要调控作用。BVDV致病机制是否与miRNA有关？我们前期研究表明BVDV感染细胞后miR-1249表达显著上调，并可促进细胞凋亡。提示：miR-1249可能与BVDV持续性感染有关。为进一步阐明miR-1249调控病毒感染的分子机制，明确其在病毒感染中的作用，本项目拟首先利用高通量测序技术建立BVDV感染MDBK细胞的转录本文库，利用该文库信息和生物信息学技术预测miR-1249的调亡相关靶基因，并用荧光素酶报告系统等技术进行验证。通过qPCR、流式细胞技术等方法研究miR-1249在细胞凋亡和BVDV感染中的调节作用。本研究将从miRNA的角度揭示BVDV引起持续性感染的机制，同时为寻找新的抗病毒治疗方法和新的抗病毒治疗靶点提供理论依据。

牛病毒性腹泻病毒是牛病毒性腹泻-粘膜病的主要抗原，该病在世界各国都有比较普遍的发生和存在，对我国养殖业同样构成严重危胁。目前，国内、外对牛病毒性腹泻-粘膜病的防治都非常关注。牛病毒性腹泻病毒的持续感染机制对该病的防治至关重要，但致病机制目前尚不清楚。miRNA已被证实在病毒感染过程中起到重要调控作用。miRNA可能成为揭示BVDV致病机制的突破口。我们前期研究结果显示miR-1249可能通过调控细胞调亡参与BVDV持续性感染。为进一步阐明miR-1249调控病毒感染的分子机制，明确其在病毒感染中的作用，本项目首先利用高通量测序技术建立BVDV感染MDBK细胞的转录本文库，分别获得cp型、ncp型BVDV感染MDBK细胞2h、6h和18h基因差异表达数据；利用该文库信息和生物信息学技术预测miR-1249的调亡相关靶基因，并进一步通过荧光定量PCR、WB、用荧光素酶报告系统等技术进行验证证实miR-1249的调亡相关靶基因为Faim2；通过qPCR、流式细胞技术等方法研究证实miR-1249通过靶向Faim2基因促进细胞凋亡并调节BVDV对宿主细胞的感染。非致细胞病变型BVDV感染宿主细胞不引起miR-1249的升高，Faim2蛋白活化，以此抑制细胞凋亡，有利于病毒的潜伏，实现在宿主体内的持续性感染。因此，Faim2是抗BVDV潜在的功能基因，同时为寻找新的抗病毒治疗方法和抗病毒治疗靶点提供理论依据。