miR-361和miR-34a在口蹄疫病毒感染PK15细胞中的免疫调控作用研究

Innate recognition of viruses by the mammalian immune system is critical in providing both the immediate antiviral effects, and in inducing appropriate classes of adaptive immune responses required for clearing viral infection. Most cells use the retinoic acid inducible gene-I (RIG-I) to recognize certain ssRNA virus infections and melanoma differentiation-associated gene 5 (MDA5) to recognize picornaviruses, including foot-and-mouth disease virus (FMDV). miRNA are involved in immune pathways of antiviral response. However, it is still unclear the role of miRNA in FMDV-induced host innate immune evasion. Our previous evidence have indicated that miR-361 and miR-34a may be involved in immune response during FMDV infection. In this project, PK-15 cell line was used to investigate the molecular mechanism of miR-361 and miR-34a response to FMDV infection. First, target effects of miR-361 and miR-34a to Traf2, Myd88, CCL22/CCL19, IRF3 and Akt2 were estimated by dual luciferase reporter assay, qPCR and Western Blot detection on the basis of overexpression and knockdown technology. Then, evaluation of key proteins and downstream cytokines of RLRs pathway was performed to by qPCR, Western Blot and ELISA to understand the roles of miR-361 and miR-34a on RLRs pathway regulation. Finally, FMDV replication and proliferation would be evaluated after overexpression and/or knockdown miR-361 and miR-34a expression, to explore theirs’ role in FMDV-induced host innate immune evasion. These results will preliminary clarify the molecular pathogenesis of FMDV-mediated cellular miRNA regulating innate immune response.

miRNA在宿主抗病毒天然免疫应答中发挥着重要的调控作用，同时RLR介导的天然免疫是FMDV感染应答的主要途径，miRNA是否通过RLRs途径调控FMDV感染是本研究的科学问题。本项目基于前期差异miRNA筛选结果，探索猪miR-361和miR-34a在介导PK15细胞抗病毒中的作用。首先，利用萤光素酶报告、qPCR和免疫印迹技术，研究miR-361/34a对靶蛋白Traf2、IRF3、Akt2等的调控作用，然后，应用qPCR、免疫印迹、ELISA等方法检测miR-361/34a对RLR信号通路关键蛋白和细胞因子的表达影响，确定miR-361/34a对RLR信号通路的调控作用，最后，利用过表达和敲降技术研究miR-361/34a对FMDV增殖的影响。为了确保结果的可靠性，实验同时在猪肺泡巨噬细胞（PAM）中进行验证，以获得miR-361/34a在介导病毒感染细胞中的分子机制。

口蹄疫病毒（FMDV）引起的口蹄疫是偶蹄动物共患的一种急性、热性、高度传染性疫病，该病爆发会造成巨大的经济损失和社会影响。miRNA在宿主抗病毒天然免疫应答中发挥着重要的调控作用，同时RLR介导的天然免疫是FMDV感染应答的主要途径，本研究致力于筛选抑制FMDV增殖的miRNA，并初步探索miRNA介导的天然免疫调控机制。本研究证实FMDV感染PK-15细胞后能在PK-15细胞中能够快速复制和增殖，并引起的细胞病变。FMDV感染抑制PK-15细胞中Dgcr8的表达，Dgcr8在细胞内源性miRNA的产生过程中发挥重要作用。干扰内源性Dgcr8表达后，显著抑制细胞内源性miRNA的表达，促进FMDV的复制，表明miRNA在FMDV感染过程中发挥重要的调控作用。miRNA表达谱测序表明FMDV感染影响细胞中miRNA表达谱的表达，大部分miRNA表达下调，miRNA靶标预测和富集分析发现，下调miRNA的靶标与免疫调控密切相关。结合试验验证和文献报道，我们筛选miR-361和miR-34a进行功能验证，构建miRNA过表达载体和合成miRNA模拟物（mimics）转染细胞，发现过表达miR-361和miR-34a显著抑制FMDV的增殖，而干扰内源性miR-361和miR-34a表达则促进FMDV的复制。进一步构建干扰素β（IFN-β）启动子和干扰素刺激应答元件（ISRE）萤光素酶表达载体，共转染发现miR-361和miR-34a激活IFN-β启动子活性和ISRE应答元件活性，酶联免疫吸附试验（ELISA）检测发现，miR-361和miR-34a促进细胞分泌IFN-β和IFN-γ，表明miR-361和miR-34a能激活细胞免疫应答。本研究初步表明，miR-361和miR-34a能够通过激活细胞免疫应答，抑制FMDV在PK-15细胞中的增殖。