1、广东海洋大学学报Journal of Guangdong Ocean University第 43 卷第 4 期2023 年 7 月Vol.43 No.4Jul.2023黄鉴涛，胡海浩，马嘉霖，等.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J.广东海洋大学学报,2023,43(4):52-61.收稿日期：2023-03-28基金项目：广东省科技特派员专项（GDKTP2021029800）；国家自然科学基金（31602199）第一作者：黄鉴涛（1998），男，硕士生，主要从事水生动物病害防控。E-mail:通信作者：闫秀英（1978），女，副教授，主要从事水生动物病害防控。E
2、-mail: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黄鉴涛，胡海浩，马嘉霖，战盈瑾，闫秀英，简纪常（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）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
3、。【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K
4、EGG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
5、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
6、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。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中图分类号：S943.112；Q78文献标志码：A文章编号：1673-9159（2023）04-0052-10doi：10.3969/j.issn.1673-9159.2023.04.007Identification of Ctenopharyngodon idella miR-218 Family and ItsRegulation in Infection of Lymphocystis Disease Vi
7、rus ChinaHUANG 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 ofAquatic Animal Disease Control and Healthy Culture/Key Laboratory of Control for Diseases ofAquatic Economic Animals of Guangdo
8、ng Higher Education Institutes,Zhanjiang 524088,China）第4期黄鉴涛等：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Abstract:【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-218-5pin grass carp ov
9、ary 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 wereobtained from GCO cells challenged with LCDV-cn,and were performed by sequencing andbioinformatics analysis.Real-time quantitative PCR(qRT-PCR)w
10、as used to acquire the expression ofthe cid-miR-218 family in GCO cells challenged with LCDV-cn.Bioinformatics methods were used topredict the target genes of the cid-miR-218 family,and dual luciferase reporter gene system was used toverify the target gene of cid-miR-218-5p.Cid-miR-218-5p mimic and
11、cid-miR-218-5p inhibitor weretransfected 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 mcpgene.GO and KEGG databases were used to enrich the signal pathways that the c
12、id-miR-218 familymight be involved in regulation.【Result】In GCO cells challenged with LCDV-cn,five members ofthe 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 decreasedand incre
13、ased,and the difference was significant(P 0.05).Various bioinformatics analysis predictedthat the cid-miR-218 family had many potentially common target genes,and il-10 was a common targetgene.Results of dual luciferase reporter gene experiment showed that the luciferase activity decreased(P 0.05)aft
14、er targeting of the family member representative cid-miR-218-5p with the PmirGLO-il-10recombinant 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 weredecreased significantl
15、y(P 0.05).After down-regulation of cid-miR-218-5p,the expression of itstarget gene il-10 and the LCDV-cn mcp gene was significantly increased(P 0.05).Moreover,it waspredicted that the cid-miR-218 family was involved in regulation of tumorigenesis signaling pathwaysof viral infection.【Conclusion】In G
16、CO cells challenged with LCDV-cn,cid-miR-218-5p of the cid-miR-218 family played important regulatory roles.Cid-miR-218-5p negatively regulated the expressionof its target gene il-10,and negatively regulated the expression of the LCDV-cn mcp gene,too.Thisstudy indicated that cid-miR-218-5p and IL-10 have potentially clinical application value in thediagnosis,prevention and control of lymphocystis disease.Key words:Grass carp ovary cells;cid-miR-218 family;lymphocystis disease virus China;target