流感病毒通过miR-29c抑制宿主抗病毒免疫应答的研究

发布时间：2018-04-08 07:37

本文选题：流感病毒　切入点：miRNA　出处：《吉林大学》2015年硕士论文

【摘要】：A型流感病毒（influenza A virus，IAV）是单股负链RNA病毒，其首先侵染的是宿主的呼吸系统，临床表现从轻微的上呼吸道感染到严重的肺炎。MicroRNA(miRNA)属于非编码RNA家族，在转录后水平调控靶基因表达。目前在各种生物中已经确定了成千上万种miRNA，其参与了细胞分化、增殖、凋亡和病原感染等诸多生物学进程的调控。人的miR-29家族包括miR-29a、miR-29b及miR-29c。最近的研究显示，miR-29能够调节机体的天然免疫和适应性免疫，但其在IAV感染过程中对于宿主免疫反应的作用机制尚不明确。我们前期miRNA组学的结果表明，当机体感染IAV后，miR-29家族的表达显著上调，，尤其是miR-29c。围绕miR-29c我们进行了如下的研究。第一，首先分析了先前的关于IAV不同时相感染A549细胞的miRNA表达谱数据，并应用Real-time PCR对表达差异显著的miR-29c进行了验证。预测得到了miR-29c的潜在靶基因集合，并对此进行生物信息学数据挖掘，其中由于A20是一个重要的机体炎症和抗病毒信号通路的调节蛋白，对于维持机体的免疫稳态具有重要作用，据此筛选出miR-29c/A20互作对作为研究的切入点；第二，利用双荧光素酶报告基因技术、Real-time PCR以及Western Blotting实验对miR-29c与A20之间的相互作用机制进行了确证，并证明IAV能够有效增加A20mRNA的稳定性，且通过过表达和表达抑制的系列实验证明IAV通过诱导miR-29c的表达促进了细胞A20的表达；第三，在功能学实验部分，先利用荧光素酶报告基因和WesternBlotting技术证明miR-29c/A20能有效抑制NF-κ B信号通路的激活。我们通过Real-time PCR方法对miR-29c/A20互作对在IAV感染A549细胞内TNF-α、IFN-β、 IL-1β、 IL-6、以及IL-8等细胞因子的mRNA表达变化进行分析，证明miR-29c能通过上调A20的表达来抑制IAV诱导的NF-κ B下游抗病毒和促炎细胞因子的表达。综上所述，IAV的感染可以通过上调miR-29c进而增加A20的表达来抑制宿主天然抗病毒免疫。
[Abstract]:Influenza A virus type A virus is a single negative strand RNA virus, which first infects the host's respiratory system. Its clinical manifestations range from mild upper respiratory tract infection to severe pneumonia. MicroRNAs belong to a non-coding RNA family and regulate the expression of target genes at post-transcriptional level.At present, thousands of miRNAs have been identified in various organisms, which are involved in many biological processes such as cell differentiation, proliferation, apoptosis and pathogenic infection.The human miR-29 family includes miR-29aanmiR-29b and miR-29c.Recent studies have shown that miR-29 can regulate innate and adaptive immunity, but its mechanism of host immune response in the process of IAV infection is unclear.The results of our previous miRNA studies showed that the expression of miR-29 was significantly up-regulated when the body was infected with IAV, especially miR-29c.We have done the following research around miR-29c.Firstly, the previous data of miRNA expression profile of A549 cells infected with IAV at different time were analyzed, and Real-time PCR was used to verify the expression of miR-29c.The potential target gene sets of miR-29c were predicted, and the bioinformatics data were mined. Because A20 is an important regulatory protein of inflammation and antiviral signaling pathway, it plays an important role in maintaining the immune homeostasis of the body.The interaction between miR-29c and A20 was confirmed by using double luciferase reporter gene technique: Real-time PCR and Western Blotting experiments, and it was proved that IAV could effectively increase the stability of A20mRNA.It was proved by a series of experiments of overexpression and inhibition that IAV promoted the expression of A20 by inducing the expression of miR-29c.Luciferase reporter gene and WesternBlotting technique were used to prove that miR-29c/A20 could effectively inhibit the activation of NF- 魏 B signaling pathway.We analyzed the mRNA expression of TNF- 伪 -IFN- 尾, IL-1 尾, IL-6, IL-8 and other cytokines in A549 cells infected with IAV by Real-time PCR.It is suggested that miR-29c can inhibit the expression of antiviral and pro-inflammatory cytokines induced by IAV by up-regulating A20 expression.In conclusion, miR-29c infection can inhibit host natural anti-viral immunity by upregulating miR-29c and increasing A20 expression.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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