MiR-130c-5p靶向乌鳢水泡病毒g基因抑制病毒增殖

MiR-130c-5p targets the snakehead vesicular virus g gene to inhibit viral proliferation

为了研究miR-130c-5p在乌鳢水泡病毒(snakehead vesiculovirus,SHVV)感染中潜在靶基因g的靶向关系以及对病毒复制的影响,本研究以斑点叉尾鮰卵巢(channel catfish ovary,CCO)为实验材料,通过实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和免疫印迹(Western blot)技术测定SHVV不同感染时间和感染剂量条件下,病毒基因水平和蛋白水平以及miR-130c-5p变化情况。此外,将SHVV的g基因上miR-130c-5p对应的靶序列克隆到质粒pmirGLO,构建质粒pmirGLO-G用于双荧光素酶报告实验进行靶基因验证。结果显示,随着SHVV感染时间及剂量的不断增加,miR-130c-5p和g基因的表达水平都显著上调。进一步实验证明,miR-130c-5p类似物和pmirGLO-G质粒共转染可显著抑制荧光素酶活性强度,而转染miR-130c-5p抑制剂则明显上调了pmirGLO-G报告载体的荧光信号。此外,miR-130c-5p的过表达显著降低了病毒g基因的mRNA及蛋白表达,而抑制miR-130c-5p的表达则上调了g基因的mRNA及蛋白的表达水平。研究结果表明,miR-130c-5p通过靶向SHVV的g基因,引起G蛋白的降解,从而抑制SHVV的增殖。本研究结果为理解microRNA调控SHVV的致病机制提供了重要基础,为抗SHVV疫苗等药物的研发提供了理论支持。

In order to investigate the targeting relationship of miR-130c-5p to the potential target gene g in snakehead vesiculovirus(SHVV)infection and its effect on viral replication,the changes of viral gene and protein levels and miR-130c-5p in SHVV were determined in this study by quantitative real-time PCR(qRT-PCR)and Western blot techniques using channel catfish ovary(CCO)as experimental materials.In addition,the target sequence corresponding to miR-130c-5p on the g gene of SHVV was cloned into the plasmid pmirGLO,and the plasmid pmir-GLO-G was constructed for dual luciferase reporter assay for target gene verification.The results showed that the expression levels of miR-130c-5p and g genes were significantly up-regulated with the increasing time and dose of SHVV infection.Further experiments showed that co-transfection of miR-130c-5p mimic and pmirGLO-G plasmid significantly inhibited luciferase activity,while transfection of miR-130c-5p inhibitor significantly up-regulated the fluorescence signal of pmirGLO-G reporter vector.In addition,overexpression of miR-130c-5p significantly reduced the mRNA and protein expression of the viral g gene,while inhibition of miR-130c-5p up-regulated the mRNA and protein expression levels of the g gene.The results showed that miR-130c-5p inhibited the proliferation of SHVV by targeting the g gene of SHVV and causing the degradation of G protein.The results of this study provide an important basis for understanding the pathogenic mechanism of microRNA regulation of SHVVV,and provide theoretical support for the development of anti-SHVV vaccines and other drugs.