草鱼miR-218鉴定及其对鱼淋巴囊肿病毒中国株感染的调控作用  

Identification of Ctenopharyngodon idella miR-218 Family and Its Regulation in Infection of Lymphocystis Disease Virus China

作　　者：黄鉴涛 胡海浩 马嘉霖 战盈瑾 闫秀英[1] 简纪常[1] HUANG Jian-tao;HU Hai-hao;MA Jia-lin;ZHAN Ying-jin;YAN Xiu-ying;JIAN Ji-chang(Fisheries College of Guangdong Ocean University/Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture/Key Laboratory of Control for Diseases of Aquatic Economic Animals of Guangdong Higher Education Institutes,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学水产学院/广东省水产动物病害防控与健康养殖重点实验室/水产经济动物病害控制广东普通高校重点实验室,广东湛江524088

出　　处：《广东海洋大学学报》2023年第4期52-61,共10页Journal of Guangdong Ocean University

基　　金：广东省科技特派员专项(GDKTP2021029800);国家自然科学基金(31602199)。

摘　　要：【目的】鉴定草鱼(Ctenopharyngodon idella)miR-218(cid-miR-218)家族成员,分析cid-miR-218在鱼淋巴囊肿病毒中国株(LCDV-cn)感染草鱼卵巢细胞系(Grass carp ovary cells,GCO)过程中的表达特性,探索cid-miR-218-5p对LCDV-cn感染的调控作用。【方法】从LCDV-cn感染的GCO细胞中,通过茎环RT-PCR获取cid-miR-218家族成员,并进行测序验证和生物信息学分析;用定量PCR分析cid-miR-218家族在LCDV-cn感染GCO过程中的表达;用生物信息学方法预测cid-miR-218家族的靶基因,并利用双荧光素酶报告基因系统验证cid-miR-218-5p的靶基因;通过转染cid-miR-218-5p mimic和cid-miR-218-5p inhibitor上调和下调cid-miR-218-5p的表达,用定量PCR分析cid-miR-218-5p、靶基因il-10和LCDV-cn mcp基因的表达情况;应用GO和KEGG数据库,对cid-miR-218家族可能参与调控的信号通路进行富集分析。【结果】在LCDV-cn感染GCO过程中,鉴定到cid-miR-218-5p、cid-miR-218a、cid-miR-218b、cid-miR-218b-1和cid-miR-218b-2等5个cid-miR-218家族成员,其中cid-miR-218-5p的表达呈先升后降再上升的变化趋势,且差异表达最显著(P<0.05);经生物信息学分析预测,cid-miR-218家族成员有较多共同的潜在靶基因,il-10是共有的靶基因。经验证,pmirGLO-il-10重组质粒与家族成员代表cid-miR-218-5p结合后,其荧光素酶活性降低(P<0.05),证明il-10是cid-miR-218-5p的靶基因。cid-miR-218-5p过表达后,il-10与LCDV-cn mcp表达均显著下降(P<0.05),而抑制cid-miR-218-5p表达后,il-10与LCDV-cn mcp表达则显著上升(P<0.05)。预测表明cid-miR-218家族参与调控病毒感染致瘤相关信号通路。【结论】在LCDV-cn感染GCO过程中,cid-miR-218家族中cid-miR-218-5p起重要调控作用;cid-miR-218-5p负调控其靶基因il-10的表达,并负调控LCDV-cn mcp基因的表达;cid-miR-218-5p和IL-10对鱼淋巴囊肿病诊断和防控有潜在的临床应用价值。【Objective】To identify the members of the Ctenopharyngodon idella miR-218(cid-miR-218)family,and analyze their expression characteristics,and explore the regulation of cid-miR-218-5p in grass carp ovary cells(GCO)challenged with lymphocystis disease virus China(LCDV-cn).【Method】By the method of stem-loop RT-PCR,the members of the cid-miR-218 family were obtained from GCO cells challenged with LCDV-cn,and were performed by sequencing and bioinformatics analysis.Real-time quantitative PCR(qRT-PCR)was used to acquire the expression of the cid-miR-218 family in GCO cells challenged with LCDV-cn.Bioinformatics methods were used to predict the target genes of the cid-miR-218 family,and dual luciferase reporter gene system was used to verify the target gene of cid-miR-218-5p.Cid-miR-218-5p mimic and cid-miR-218-5p inhibitor were transfected into GCO cells to up-regulate and down-regulate the expression of cid-miR-218-5p.qRT-PCR was used to detect the expression of cid-miR-218-5p,the target gene il-10 and the LCDV-cn mcp gene.GO and KEGG databases were used to enrich the signal pathways that the cid-miR-218 family might be involved in regulation.【Result】In GCO cells challenged with LCDV-cn,five members of the cid-miR-218 family were identified,i.e.cid-miR-218-5p,cid-miR-218a,cid-miR-218b,cid-miR218b-1 and cid-miR-218b-2.The expression of cid-miR-218-5p increased firstly,then decreased and increased,and the difference was significant(P<0.05).Various bioinformatics analysis predicted that the cid-miR-218 family had many potentially common target genes,and il-10 was a common target gene.Results of dual luciferase reporter gene experiment showed that the luciferase activity decreased(P<0.05)after targeting of the family member representative cid-miR-218-5p with the PmirGLO-il-10 recombinant plasmid,which indicated that il-10 was the target gene of cid-miR-218-5p.After up-regulation of cid-miR-218-5p,the expression of its target gene il-10 and the LCDV-cn mcp gene were decreased significantly(P<0.05).After down-re

关 键 词：草鱼卵巢细胞系 cid-miR-218家族 鱼淋巴囊肿病毒中国株 靶基因 白介素10 

分 类 号：S943.112[农业科学—水产养殖] Q78[农业科学—水产科学]