草鱼miR-462通过靶向cx32.2、slc9a3.1和tbk1调控嗜水气单胞菌感染诱导的免疫应答  

miR-462 modulates cellular immune response by targeting cx32.2,slc9a3.1 and tbk1 in CIK cells infected with Aeromonas hydrophila

作　　者：王安琪 陶丽竹 周丰林 徐晓雁[1] 沈玉帮[2] 李家乐[1,2,3] WANG Anqi;TAO Lizhu;ZHOU Fenglin;XU Xiaoyan;SHEN Yubang;LI Jiale(Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China;Shanghai Engineering Research Center of Aquaculture,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学,农业农村部淡水水产种质资源重点实验室,上海201306 [2]上海海洋大学,水产科学国家级实验教学示范中心,上海201306 [3]上海海洋大学,水产养殖工程技术研究中心,上海201306

出　　处：《水产学报》2020年第9期1467-1476,共10页Journal of Fisheries of China

基　　金：现代农业产业技术体系专项(CARS-45-03);上海市工程中心提升项目(19DZ2284300)。

摘　　要：为探究miR-462在嗜水气单胞菌感染草鱼肾脏细胞(Ctenopharyngodon idella kideny,CIK)后的调控机制,实验利用荧光定量技术检测了CIK细胞感染嗜水气单胞菌后miR-462表达水平的变化;运用RNAhybrid软件预测miR-462的靶基因,利用双荧光素酶报告基因系统进行确定;此外还分析了miR-462对靶基因下游基因的调控作用。结果显示,在CIK细胞感染嗜水气单胞菌的过程中,miR-462的表达发生显著变化;cx32.2、slc9a3.1和tbk1的表达先降低后升高,与miR-462的表达模式呈负相关。双荧光素酶报告系统显示,miR-462可靶向cx32.2、slc9a3.1和tbk1的3′非编码区抑制其表达,过表达miR-462可以显著抑制cx32.2、slc9a3.1和tbk1的表达。转染miR-462模拟物后,下游slc4a4a、tnfrsf5、cxcl9和cxcl11基因的表达受到抑制。研究表明,miR-462参与调控嗜水气单胞菌感染后草鱼CIK细胞中的免疫应答。cx32.2、slc9a3.1和tbk1被鉴定为miR-462的靶基因。miR-462可通过靶向slc9a3.1和tbk1影响下游基因的功能。Bacterial septicemia is a systemic inflammatory reaction mainly caused by the infection of Aeromonas hydrophila.Excessive development of inflammation may lead to septic shock or death in fish.A large number of studies have confirmed that miRNA is involved in the regulation of immune response after bacterial infection.To explore the regulatory mechanism of miR-462 in Ctenopharyngodon idella kidney(CIK)cells infected with A.hydrophila,the expression profiles of miR-462 upon A.hydrophila infection was detected by real-time quantitative PCR;the target genes of miR-462 were predicted by RNAhybrid software,and identified by dual-luciferase reporter assay system;in addition,the regulatory effect of miR-462 on downstream genes was analyzed.The results showed that the expression of miR-462 changed significantly after A.hydrophila infection,indicating that miR-462 participated in the regulation of immune response.Dual-luciferase reporter assay revealed that cx32.2,slc9a3.1 and tbk1 are the target genes of miR-462,which is further confirmed by the overexpression and inhibition experiments of miR-462.The expression of slc4a4a,tnfrs5,cxcl9 and cxcl11 were suppressed after miR-462 antagomir was transfected,which proved that miR-462 could affect the function of the downstream genes by targeting slc9a3.1 and tbk1.Our results may provide a theoretical basis for investigating the molecular mechanism of miR-462 regulating immune response in C.idella.

关 键 词：草鱼肾脏细胞 嗜水气单胞菌 miR-462 免疫调节 

分 类 号：S941.42[农业科学—水产养殖]