首页|草鱼miR-1260对鱼淋巴囊肿病毒中国株复制的调控作用

草鱼miR-1260对鱼淋巴囊肿病毒中国株复制的调控作用

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  • 国家科技期刊平台
  • NETL
  • NSTL
  • 万方数据
[目的]鉴定草鱼(Ctenopharyngodon idella)miR-1260(cid-miR-1260)，分析cid-miR-1260在鱼淋巴囊肿病毒中国株(Lymphocystis disease virus China，LCDV-cn)感染草鱼卵巢细胞系(Grass carp ovary cells，GCO)过程中的时序表达特性，揭示cid-miR-1260对其靶基因znf574和LCDV-cn复制的调控作用。[方法]制备LCDV-cn感染的GCO细胞，通过茎环RT-PCR和测序鉴定cid-miR-1260;采用定量PCR检测cid-miR-1260在LCDV-cn感染GCO过程中的时序表达;应用生物信息学方法预测cid-miR-1260的靶基因，并用双荧光素酶报告基因系统验证靶基因;通过转染cid-miR-1260 mimic和cid-miR-1260 inhibitor使cid-miR-1260过表达和抑制表达，并用定量PCR检测cid-miR-1260、靶基因znf574、LCDV-cn mcp基因的表达;应用Reed-Muench法测定LCDV-cn在GCO细胞中的滴度。[结果]cid-miR-1260与其他已知miR-1260仅有一个碱基差异，且cid-miR-1260在LCDV-cn感染GCO过程中呈先上升后下降的表达变化，72 h时表达量最高(P<0。05);生物信息学分析表明，znf574为cid-miR-1260的靶基因;重组质粒pmirGLO-znf574-WT与cid-miR-1260 mimic共转染后，荧光素酶活性显著降低(P<0。05)，证实znf574为cid-miR-1260的靶基因;cid-miR-1260过表达后，靶基因znf574的表达显著下降(P<0。05)，LCDV-cn mcp的表达和LCDV-cn在GCO细胞中的滴度显著上升(P<0。05);抑制cid-miR-1260表达后，靶基因znf574的表达量显著上升(P<0。05)，LCDV-cn mcp的表达量和LCDV-cn在GCO细胞中的滴度显著下降(P<0。05)。[结论]LCDV-cn感染GCO细胞后，cid-miR-1260的表达显著上调;在LCDV-cn感染中，cid-miR-1260对其靶基因znf574的表达起负调控作用，且cid-miR-1260表达与LCDV-cn的复制呈正相关，起促LCDV-cn感染作用，而锌指蛋白ZNF574具有抗LCDV-cn作用。本研究为淋巴囊肿病的防治提供新的依据和思路。
Regulation on Replication of Lymphocystis Disease Virus China,Provoked by Ctenopharyngodon idella miR-1260
[Objective]To identify Ctenopharyngodon idella miR-1260(cid-miR-1260)and its temporal expression in grass carp ovary(GCO)cells challenged with lymphocystis disease virus China(LCDV-cn),and reveal the regulation on expression of the target gene znf574 and replication of LCDV-cn in GCO cells.[Method]GCO cells challenged with LCDV-cn were prepared,and stem-loop RT-PCR and sequencing were used to identify cid-miR-1260.The temporal expression of cid-miR-1260 during the infection of LCDV-cn in GCO cells was detected by quantitative real-time PCR(qRT-PCR).The target genes of cid-miR-1260 were predicted and verified with bioinformatics methods and dual luciferase reporter gene system.The expression of cid-miR-1260 was overexpressed and inhibited by transfecting cid-miR-1260 mimic and cid-miR-1260 inhibitor,and qRT-PCR was performed to detect the expression of cid-miR-1260,the target gene znf574 and the LCDV-cn mcp gene.The titer of LCDV-cn in GCO cells was determined by Reed-Muench method.[Result]The difference between cid-miR-1260 and other known miR-1260 was found in only one base.The expression of cid-miR-1260 increased firstly and then decreased in GCO cells challenged with LCDV-cn,and the expression level was highest at 72 h(P<0.05).It was predicted with bioinformatics methods that the gene znf574 was the target gene of cid-miR-1260.After co-transfection of the recombinant plasmid pmirGLO-znf574-WT with cid-miR-1260 mimic,the luciferase activity of pmirGLO-znf574-WT was significantly decreased(P<0.05),which indicated that the target gene of cid-miR-1260 was the gene znf574.After overexpression of cid-miR-1260,the expression of the target gene znf574 was significantly decreased(P<0.05),and the expression of the LCDV-cn mcp gene and the titer of LCDV-cn in GCO cells were significantly increased(P<0.05).After inhibiting the expression of cid-miR-1260,the expression of the target gene znf574 was significantly increased(P<0.05),and the expression of the LCDV-cn mcp gene and the titer of LCDV-cn in GCO cells were significantly decreased(P<0.05).[Conclusion]The expression of cid-miR-1260 was upregulated after infection of LCDV-cn in GCO cells.cid-miR-1260 played the negative regulation on the expression of its target gene znf574 during the infection of LCDV-cn in GCO cells.The expression of cid-miR-1260 was positively corelated to the replication of LCDV-cn in GCO cells,and cid-miR-1260 promoted the infection of LCDV-cn.While,zinc finger protein ZNF574 played an antiviral role.This study provided the new basis and idea for the prevention and treatment of lymphocystis disease.

lymphocystis disease virus Chinagrass carp ovary cellsCtenopharyngodon idella miR-1260zinc finger protein gene znf574viral replication

马嘉霖、战盈瑾、周杰、黄鉴涛、闫秀英、简纪常

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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

鱼淋巴囊肿病毒中国株 草鱼卵巢细胞系 cid-miR-1260 锌指蛋白基因znf574 病毒复制

广东省科技特派员专项国家自然科学基金

GDKTP202102980031602199

2024

10.3969/j.issn.1673-9159.2024.03.004

广东海洋大学学报
广东海洋大学

广东海洋大学学报

CSTPCDCHSSCD北大核心
影响因子：0.444
ISSN：1673-9159
年,卷(期)：2024.44(3)
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