cid-miR-146a在LCDV-cn感染中的差异表达及其调控作用  

Differential Expression and Regulation of cid-miR-146a During Infection of LCDV-cn

作　　者：胡海浩 黄鉴涛 马嘉霖 杨硕 闫秀英[1] 简纪常[1] HU Hai-hao;HUANG Jian-tao;MA Jia-lin;YANG Shuo;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》2022年第4期31-41,共11页Journal of Guangdong Ocean University

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

摘　　要：【目的】在鱼淋巴囊肿中国病毒株(LCDV-cn)感染草鱼卵巢细胞系(GCO)过程中,分析cid-miR-146a的表达特性,探索cid-miR-146a的调控作用。【方法】采用颈环RT-PCR方法,从LCDV-cn感染的GCO细胞中获得cidmiR-146a;在LCDV-cn感染GCO过程中,用定量PCR方法获取cid-miR-146a的表达变化;用生物信息学方法预测和双荧光素酶系统验证cid-miR-146a的靶基因;转染cid-miR-146a mimic和cid-miR-146a inhibitor对cid-miR-146a进行上调和下调,用定量PCR检测cid-miR-146a、靶基因Flt1和LCDV-cn mcp基因在GCO中的表达。【结果】LCDV-cn感染GCO后3~6 d细胞聚集形成“疤痕”,之后细胞逐渐脱落、裂解,呈现空洞。cid-miR-146a的长度为23 bp,在LCDV-cn感染GCO过程中,cid-miR-146a的表达先上升(72 h前)后下降(72 h后)。用不同生物信息学方法共同预测到cid-miR-146a的靶基因为Flt1,在双荧光素酶系统验证实验中,cid-miR-146a靶向Flt1重组载体后荧光素酶活性降低(P<0.05),证实Flt1为cid-miR-146a的靶基因。对cid-miR-146a进行上调下调后,GCO中cid-miR-146a的表达差异显著(P<0.05)。在cid-miR-146a上调后,靶基因Flt1的表达显著下降(P<0.05),LCDV-cn mcp基因的表达显著上升(P<0.05);在cid-miR-146a下调后,靶基因Flt1的表达显著上升(P<0.05),LCDV-cn mcp基因的表达显著下降(P<0.05)。【结论】在LCDV-cn感染GCO过程中,CPE的变化与cid-miR-146a的表达变化时间点呈正相关。cid-miR-146a负调控其靶基因Flt1的表达,并对LCDV-cn的复制起着正调控作用。生物信息学方法预测说明cid-miR-146a参与调控的信号通路与免疫和肿瘤发生相关。【Objective】To analyze the expression characteristics, and to explore the regulation of cidmiR-146a in GCO cells infected with LCDV-cn. 【Method】 The method of stem-loop RT-PCR was used to acquire cid-miR-146a in GCO cells infected with LCDV-cn. During the infection of LCDV-cn,the expression of cid-miR-146a in GCO cells was detected by real-time quantitative PCR(qRT-PCR).Bioinformatics methods and dual-luciferase system were used to predict and verify the target gene of cid-miR-146a. After that, cid-miR-146a was up-regulated and down-regulated by transfection of cid-miR-146a mimic and cid-miR-146a inhibitor, and qRT-PCR was used to detect the expression of cid-miR-146a, the target gene Flt1 and the LCDV-cn mcp gene in GCO cells after up-regulation and down-regulation. 【Result】 GCO cells aggregated to form “scars” from the third day to the sixth day after infection of LCDV-cn, then gradually fell and lysed, and the cavities presented. The length of cidmiR-146a was 23 bp. During the infection of LCDV-cn, the expression of cid-miR-146a in GCO cells increased firstly(before 72 h) and then decreased(after 72 h). The target gene of cid-miR-146a was copredicted to be the Flt1 gene by different bioinformatics methods. The dual-luciferase system proved that the luciferase activity decreased(P < 0.05) after targeting of cid-miR-146a with the Flt1 recombinant vector, which confirmed that the Flt1 was the target gene of cid-miR-146a. The expression of cid-miR-146a in GCO cells was significantly different(P < 0.05) after up-regulation and down-regulation of cidmiR-146a. After cid-miR-146a was up-regulated, the expression of the target gene Flt1 decreased significantly(P < 0.05), and the expression of the LCDV-cn mcp gene increased significantly(P < 0.05).After cid-miR-146a was down-regulated, the expression of the target gene Flt1 increased significantly(P < 0.05), and the expression of the LCDV-cn mcp gene decreased significantly(P < 0.05).【Conclusion】 In GCO cells infected with LCDV-cn, the time of the cyt

关 键 词：鱼淋巴囊肿病毒中国株 草鱼卵巢细胞系 草鱼miR-146a Fms相关酪氨酸激酶1 定量PCR 

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