NEK8调控Ⅰ型干扰素信号通路的初步研究  

Preliminary studies on the regulation of typeⅠinterferon signaling pathway by NEK8

作　　者：潘利洁 孙悦 俞馨 余莉[1,2,3] 曹苑苑 PAN Lijie;SUN Yue;YU Xin;YU Li;CAO Yuanyuan(School of Basic Medical Sciences,Anhui Medical University,Hefei 230032,China;Anhui Province Key Laboratoryof Zoonoses,Anhui Medical University,Hefei 230032,China;The Provincial Key Laboratory of Zoonoses of High Institutionsin Anhui,Anhui Medical University,Hefei 230032,China;Clinical College,Anhui Medical University,Hefei 230031,China)

机构地区：[1]安徽医科大学基础医学院,安徽合肥230032 [2]安徽医科大学动物源性传染病安徽省重点实验室,安徽合肥230032 [3]安徽医科大学人畜共患病安徽高校省级重点实验室,安徽合肥230032 [4]安徽医科大学临床医学院,安徽合肥230031

出　　处：《安徽大学学报（自然科学版）》2024年第5期94-101,共8页Journal of Anhui University(Natural Science Edition)

基　　金：国家自然科学基金青年基金资助项目(81802003);安徽省高校科研基金资助项目(2022AH050751);安徽医科大学博士科研基金资助项目(xj201725);安徽省新时代育人质量工程项目(2022jyjxggyj224)。

摘　　要：为探究RLR通路中激酶NEK8对Ⅰ型干扰素的调控作用及其具体作用机制,笔者利用双萤光素酶报告系统检测SeV刺激下NEK8对IFN-β-Luc.,IRF3介导的启动子ISRE-Luc.以及NF-κB-Luc.的调控作用;转染不同浓度的NEK8,分析其对IFN-β-Luc.激活的调控是否是浓度依赖性的;检测NEK8对IL-6-Luc.和IRF-1-Luc.激活的调控作用来分析NEK8对IFN-β启动子调控的特异性;转染不同阶段的信号蛋白,分析NEK8在IFN-Ⅰ通路中发挥作用的具体位置;利用qRT-PCR技术分析NEK8对IFN-ⅠmRNA水平的调控作用及其特异性.结果显示:NEK8能够负调控RLR通路中IFN-β的启动子以及IRF3介导的启动子的激活,而对NF-κB启动子的激活无调控作用并发现其对IFN-β启动子的抑制作用是浓度依赖性的;NEK8对TNF-α诱导的IL-6启动子的激活以及IFN-γ诱导的IRF-1的激活均无调控作用表明其特异性负调控IFN-β-Luc.的激活;并发现其作用位点位于VISA和TBK1之间,NEK8特异性地负调控RLR通路中SeV或poly(I:C)刺激下IFN-β,CXCL10 mRNA的转录水平,而对IL-6 mRNA的转录水平无影响,并且其对TNF-α诱导的IL-6 mRNA以及IFN-γ诱导的IRF-1 mRNA水平无调控作用.该实验首次发现了在RNA病毒感染时,NEK8特异性地对IFN-β启动子的激活及其mRNA水平的负调控作用,并证明其发挥作用的位点在VISA和TBK1之间.To investigate the regulatory effect of kinase NEK8 on typeⅠinterferon in the RLR pathway and its specific mechanism,the authors employed a dual-luciferase reporter system to detect the regulation of NEK8 on IFN-β-Luc.,IRF3-mediated promoter ISRE-Luc.,and NF-κB-Luc.under SeV infection.They transfected different concentrations of NEK8 to analyze whether its regulation was concentration gradient-dependent.Additionally,the regulatory effect of NEK8 on IL-6-Luc.and IRF-1-Luc.activation was detected to analyze the specificity of NEK8 in regulating the IFN-βpromoter.Signaling proteins at different stages were transfected to analyze functional location of NEK8.The qRT-PCR was utilized to analyze the regulatory effect and specificity of NEK8 on the mRNA levels of IFN-Ⅰ.The results showed that NEK8 negatively regulated the activation of the IFN-βpromoter and the IRF3-mediated promoter in the RLR pathway,while it had no regulatory effect on the activation of the NF-κB promoter.It was also found that the inhibitory effect of NEK8 on the IFN-βpromoter was concentration dependence.NEK8 had no regulatory effect on the activation of the IL-6 promoter induced by TNF-αor the activation of IRF-1 induced by IFN-γ,indicating its specific negative regulation of IFN-βpromoter.Furthermore,the study found that NEK8 functions between VISA and TBK1.NEK8 specifically negatively regulated the transcriptional levels of IFN-βand CXCL10 mRNA under SeV or poly(I:C)stimulation in the RLR pathway,while it had no effect on the transcriptional level of IL-6 mRNA.It also had no regulatory effect on the levels of IL-6 mRNA induced by TNF-αor IRF-1 mRNA induced by IFN-γ.It's the first time that NEK8 has been specifically identified to negatively regulate the activation of the IFN-βpromoter and its mRNA levels during RNA virus infection,further validating that its functional site resides between VISA and TBK1.

关 键 词：Ⅰ型干扰素 NEK8 RIG-I 天然免疫 

分 类 号：R37[医药卫生—病原生物学]