草鱼miR-1260对鱼淋巴囊肿病毒中国株复制的调控作用

Regulation on Replication of Lymphocystis Disease Virus China,Provoked by Ctenopharyngodon idella miR-1260

【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。

【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.