草鱼miR-1260对鱼淋巴囊肿病毒中国株复制的调控作用OA北大核心CHSSCDCSTPCD

[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.