摘要
[目的]本文旨在预测和筛选靶向调控鸡糖皮质激素受体(glucocorticoid receptor,GR)表达的microRNA。[方法]用TargetScan、PicTar和miRDB软件分别预测靶向鸡GR 3′UTR microRNA并取交集;构建包含鸡GR 3′UTR的重组质粒及突变质粒,通过双荧光素酶试验鉴定microRNA和鸡GR 3′UTR的靶向性;用TargetScan和miRDB软件分别预测候选microRNA的靶基因,用DAVID软件对预测出的共同靶基因进行GO功能分析;用Western blot检测过表达候选microRNA对鸡DF1细胞GR蛋白表达的影响。[结果]miR124-3p、miR142-3p、miR204/211、miR183、miR18-5p和miR181a/181b是3种软件预测靶向GR 3′UTR的交集microRNA;双荧光素酶试验结果表明miR142-3p、miR204/211和miR18-5p靶向结合GR 3′UTR。2种软件预测3个候选microRNA的靶基因中都有GR且富集分子功能不同;在DF1细胞中过表达3个候选microRNA后,miR142-3p和miR204/211显著降低GR蛋白的表达水平(P<0.05)。[结论]miR142-3p、miR204/211和miR18-5p靶向结合鸡GR 3′UTR,且miR142-3p和miR204/21可以降低鸡DF1细胞中GR蛋白的表达水平。
[Objectives]This study aimed to predict and screen microRNA that targeted the expression of chicken glucocorticoid receptor(GR).[Methods]The microRNA targeting chicken GR 3′UTR was predicted by TargetScan,PicTar and miRDB software,respectively,and the overlapping microRNA was selected as candidates for further experiment.The recombinant plasmid and mutant plasmid containing chicken GR 3′UTR were constructed,and microRNA binding to chicken GR 3′UTR was verified by double luciferase assay.The target genes of candidate microRNA were predicted by TargetScan and miRDB software,respectively,and the GO function of the predicted common target genes was analyzed by DAVID software.Western blot was used to detect the expression of GR protein after overexpression of candidate microRNA in chicken DF1 cells.[Results]miR124-3p,miR142-3p,miR204/211,miR183,miR18-5p,and miR181a/181b were the intersection microRNA of 3 softwares prediction targeting GR 3′UTR.Dual luciferase assay showed that miR142-3p,miR204/211 and miR18-5p targeted GR 3′UTR.The two kinds of software predicted that the target genes of three candidate microRNA contained GR and the enriched molecular functions were different.After overexpression of the three candidate microRNA in DF1 cell,miR142-3p and miR204/211 significantly decreased the expression of GR protein level(P<0.05).[Conclusions]miR142-3p,miR204/211 and miR18-5p target chicken GR 3′UTR,and miR142-3p and miR204/21 can reduce the expression level of GR in chicken DF1 cell.
作者
赵敏蝶
刘杰
赵茹茜
ZHAO Mindie;LIU Jie;ZHAO Ruqian(College of Veterinary Medicine/Key Laboratory of Animal Physiology&Biochemistry,Ministry of Agriculture and Rural Affairs,Nanjing Agricultural University,Nanjing 210095,China)
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2024年第3期489-496,共8页
Journal of Nanjing Agricultural University
基金
国家自然科学基金项目(32272962,31972638)
“十四五”国家重点研发计划项目(2022YFD1300401)。
