生殖医学杂志2018，Vol.27Issue(2)：159-165,7.DOI:10.3969/j.issn.1004-3845.2018.02.012
Speedy A蛋白调控相关miRNA的筛选及miR-23b靶标的验证
Screening the microRNA associated with Speedy A and validation of miR-23b targets
摘要
Abstract
Objective:To screen and identify the miRNAs regulated the expression of speedy A.Methods:The potential candidate miRNAs associated with speedy A were screened by predicted the possible target miRNAs associated with speedy A based on online database,such as TargetScan,miRNA,miRwalk,and combined with utilizing the small RNA database of spermatogonia and spermatocyte,which was established in our laboratory.Real-time quantitative PCR was used to detect the expression of candidate miRNAs and Speedy gene in 7,14,21,35,64 days old mouse testis,and then the candidate miRNAs which were significantly negatively correlated with Speedy A were screened and identified.The 3'-untranslated regions (3'-UTR) of the wild type Speedy A and mutant Speedy A sequence were cloned into luciferase reporter vector pmirGLO.The HEK-293T cells were divided into four groups randomly:wild type experimental group(wild-type plasmid+miR-23b group);Wild type control group (wild-type plasmid +miRNA negative control group);Mutant type experimental group(mutant plasmid+ miR-23b group);Mutant type control group (mutant plasmid+miRNA negative control group).The relevant plasmids and miRNAs were transfected into the HEK-293T cells of each group.The luciferase activity was detected by dual luciferase reporter gene system.Results:Four miRNAs were screened by bioinformatics methods combined with utilizing the database of spermatogonia and spermatocyte established in our laboratory.The four miRNAs were miR-23b,mir-143,mir-345-3p and mir-881.The results of real-time quantitative PCR for different developmental stages testes showed that the expression of miR-23b was negatively correlated with speedy A (P＜0.01).The recombinant wild-type/mutant (WT/mut) Speedy A3'-UTR vector was successfully constructed.The luciferase activity of wild-type plasmid+ miR-23b group was significantly lower than that of wild-type plasmid+miRNA negative control group (P＜0.01).Conclusions:The wild type experimental group had obvious inhibitory effect on the fluorescence activity.It is confirmed that mir-23b can be used to regulate the expression of speedy A.关键词
Speedy A/miR-23b/生物信息学/双荧光素酶报告系统Key words
Speedy A gene/MiR-23b/Bioinformatics/Dual-luciferase reporting system引用本文复制引用
丁宁,张瑶楠,李媛媛,闫丽,贾孟春,刘美玲..Speedy A蛋白调控相关miRNA的筛选及miR-23b靶标的验证[J].生殖医学杂志,2018,27(2):159-165,7.基金项目
国家卫生计生委科学技术研究所中央级公益性科研院所基本科研业务费专项(2015GJZ07、2015GJM08) （2015GJZ07、2015GJM08）
